GENES AND GENE SIGNATURES FOR DIAGNOSIS AND TREATMENT OF MELANOMA

RELATED APPLICATIONS

[0001] This application claims priority to U.S. provisional application number 61/793,031 , filed March 15, 2013, and 61/889,609, filed October 1 1 , 2013 , the entire contents of which are hereby incorporated by reference

FIELD OF THE INVENTION

[0002] The invention generally relates to a molecular classification of disease and particularly to genes and gene signatures for diagnosis of melanoma and methods of use thereof.

BACKGROUND OF THE INVENTION

[0003] In the United States, over 76,000 new cases of melanoma will be diagnosed in 2013. American Cancer Society, FACTS AND FIGURES 2013.

Treatment of melanoma at an earlier stage is associated with higher survival rates in patients. There is therefore a great need for advances in methods of early diagnosis and treatment of melanoma.

BRIEF SUMMARY OF THE INVENTION

[0004] Panels of biomarkers, methods and systems are disclosed for

determining gene expression, and diagnosing and treating melanoma.

[0005] In a first aspect the disclosure is related to methods of diagnosing melanoma in a patient. In general, said methods comprise measuring in a sample obtained from the patient the expression of one or more genes, or a panel of genes. The genes may be cell cycle genes, immune genes or additional genes as defined herein. The genes may be selected from Table 1 , Table 3, or one of the many specifically defined panels (Panels A-I, or panels in Tables WW-ZZ). The method may also comprise comparing the measured expression levels of the one or more genes to the expression levels of the same one or more genes measured in a reference sample. Detecting a difference in the expression levels of the one or more genes indicates that the patient has melanoma.

[0006] In a second aspect, the disclosure is related to methods of detecting abnormal levels of gene expression in a skin lesion. In general, the methods comprise measuring in a skin lesion obtained from a patient the expression of one or more genes, or a panel of genes. The genes may be cell cycle genes, immune genes or additional genes as defined herein. The genes may be selected from Table 1 , Table 3, or one of the many specifically defined panels (Panels A-I, or panels in Tables WW-ZZ). The method may also comprise comparing the measured expression levels of the one or more genes to the expression levels of the same one or more genes measured in a reference sample. The method may also comprise detecting an abnormal level of gene expression of at least one of the one or more genes.

[0007] In a third aspect, the disclosure is related to treating a patient with melanoma. In general, the methods comprise measuring in a skin lesion obtained from a patient the expression of one or more genes, or a panel of genes. The genes may be cell cycle genes, immune genes or additional genes as defined herein. The genes may be selected from Table 1 , Table 3, or one of the many specifically defined panels (Panels A-I, or panels in Tables WW-ZZ). The method may also comprise comparing the measured expression levels of the one or more genes to the expression levels of the same one or more genes measured in a reference sample. The method may also comprise detecting an abnormal level of gene expression of at least one of the one or more genes, and altering the patient's treatment based at least in part on the difference.

[0008] Also disclosed are systems, compositions and kits to aid in detecting abnormal levels of gene expression, diagnosing melanoma or treating melanoma.

[0009] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains. Although methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention, suitable methods and materials are described below. In case of conflict, the present specification, including definitions, will control. In addition, the materials, methods, and examples are illustrative only and not intended to be limiting. [0010] Other features and advantages of the invention will be apparent from the following detailed description, and from the claims.

BRIEF DESCRIPTION OF THE DRAWINGS

[0011] Figure 1 shows a system for performing computer-assisted methods of diagnosing, detecting, screening and/or treating melanoma in a patient.

[0012] Figure 2 shows the distribution of the CCP scores from all 30 samples with a score and separated by clinical diagnosis. The melanoma samples have statistically different distributions when compared to the nevi samples.

[0013] Figure 3 shows the distributions of all 88 individual amplicon assays tested in Rounds 1 , 2, and 3 of biomarker discovery. The analysis was performed on 30 Group 1 samples (black circles) and 53 Group 2 samples (grey circles).

[0014] Figure 4 shows distributions of each individual amplicon tested in Round 1 and 2 of biomarker discovery. Samples are differentiated based on their pathological subtype on the Y-axis. The relative expression (Ct) of each gene

(compared to the expression of the housekeeper genes) is graphed on the X-axis. Each amplicon is identified by the gene name and the last three digits of the assay ID.

[0015] Figure 5 shows the normalized expression of PRAME in each sample, as differentiated by both site and histological subtype. Malignant samples are black, while benign samples are colored grey.

[0016] Figure 6 shows the expression of each of the best 8 immune genes. Each of the genes had a linear relationship with the average expression of all 8 of the immune genes (indicating that they all measure the same biological process). Furthermore, all immune genes can differentiate melanoma and nevi samples (black and grey colored, respectively).

[0017] Figure 7 shows graphs of the expression of each marker (of PRAME, the average of the 8 immune genes, and S 100A9) graphed against the other markers. Data from each site graphed separately. The lack of high correlation between each biomarker indicates that they each are likely measuring different biological processes and each has independent value. [0018] Figure 8 shows the score generated by the diagnostic model, using the expression of PRAME, the immune genes, and S 100A9. This score was used to differentiate malignant melanoma and benign nevi.

[0019] Figure 9 shows an AUROC curve generated from the dataset, using the score produced from the model. The AUC of the ROC curve is -0.96.

[0020] Figure 10 shows the distribution of scores (x-axis) from all tested samples. The data are differentiated by primary diagnosis. The top panel shows scores for malignant samples. The bottom panel shows scores of benign samples.

[0021] Figure 1 1 shows an AUROC curve generated from the dataset based on the ability of the model to differentiate melanoma and nevi samples. The AUC of the ROC is ~ 0.95. Sensitivity and specificity are shown.

[0022] Figure 12 shows the distribution of scores (x-axis) from all tested samples for the validation cohort. The data are differentiated by primary diagnosis. The top panel shows scores for malignant samples. The bottom panel shows scores of benign samples.

[0023] Figure 13 shows an AUROC curve generated based on the validation cohort. The AUC of the ROC is ~ 0.96. Sensitivity and specificity are shown.

DETAILED DESCRIPTION OF THE INVENTION

Genes and Panels

[0024] Disclosed herein are gene biomarkers and panels of biomarkers, methods and systems for determining gene expression, and methods for diagnosing and treating melanoma. It should be understood that the methods and systems disclosed are all intended to be utilized in conjunction with biomarkers as described herein. In particular, any panel disclosed may be used with any method or system of this disclosure. Furthermore, subpanels of any panel disclosed may furthermore be used, as described below.

[0025] The gene biomarkers and panels of biomarkers are useful, at least in part, for their predictive power in determining whether an individual has melanoma. It has been discovered that the predictive power of a panel or group of genes often ceases to increase significantly beyond a certain number. More specifically, the optimal number of genes in a panel, or used to generate a test value can be found wherever the following is true

(Pn+1 - Pn) < CO,

wherein P is the predictive power (i.e., Pn is the predictive power of a signature with n genes and Pn+1 is the predictive power of a signature with n genes plus one) and CO is some optimization constant. Predictive power can be defined in many ways known to those skilled in the art including, but not limited to, the signature's p-value. CO can be chosen by the artisan based on his or her specific constraints. For example, if cost is not a critical factor and extremely high levels of sensitivity and specificity are desired, CO can be set very low such that only trivial increases in predictive power are disregarded. On the other hand, if cost is decisive and moderate levels of sensitivity and specificity are acceptable, CO can be set higher such that only significant increases in predictive power warrant increasing the number of genes in the signature.

[0026] Additionally, a skilled person would recognize that individual panels may be combined to generate additional panels according to this disclosure, and that combining two panels with acceptable predictive power will result in a combined panel with acceptable predictive power. Additionally, a skilled person would recognize that while individual genes are described herein as belonging to certain groups (i.e. Cell Cycle Genes, immune genes, etc.), all panels and genes disclosed herein are unified by their common ability to aid in determining gene expression, and treating and diagnosing melanoma.

CCP Genes

[0027] The present invention is based in part on the discovery that the expression levels of CCP genes in a sample from a patient suspected of having melanoma predict whether the patient will be diagnosed with melanoma, and further that other genes, add significant prediction power when combined with CCP genes ("CCGs").

[0028] "Cell-cycle gene" and "CCG" herein refer to a gene whose expression level closely tracks the progression of the cell through the cell-cycle. See, e.g., Whitfield et al., Mol. Biol. Cell (2002) 13 : 1977-2000. The term "cell-cycle

progression" or "CCP" will also be used in this application and will generally be interchangeable with CCG (i.e., a CCP gene is a CCG; a CCP score is a CCG score). More specifically, CCGs show periodic increases and decreases in expression that coincide with certain phases of the cell cycle— e.g., STK15 and PLK show peak expression at G2/M. Id. Often CCGs have clear, recognized cell-cycle related function — e.g., in DNA synthesis or repair, in chromosome condensation, in cell-division, etc. However, some CCGs have expression levels that track the cell-cycle without having an obvious, direct role in the cell-cycle— e.g., UBE2S encodes a ubiquitin-conjugating enzyme, yet its expression closely tracks the cell-cycle. Thus a CCG according to the present invention need not have a recognized role in the cell-cycle. Exemplary CCGs are listed in Tables 1 , 2, 3, 5, 6, 7, 8 & 9. A fuller discussion of CCGs, including an extensive (though not exhaustive) list of CCGs, can be found in International

Application No. PCT /US2010/020397 (pub. no. WO/2010/080933) (see, e.g., Table 1 in WO/2010/080933). International Application No. PCT/US2010/020397 (pub. no. WO/2010/080933 (see also corresponding U.S. Application No. 13/177,887)) and International Application No. PCT/ US201 1/043228 (pub no. WO/2012/006447 (see also related U.S. Application No. 13/178,380)) and their contents are hereby

incorporated by reference in their entirety.

[0029] Whether a particular gene is a CCG may be determined by any technique known in the art, including those taught in Whitfield et al., Mol. Biol. Cell (2002) 13 : 1977-2000; Whitfield et al., Mol. Cell. Biol. (2000) 20:4188-4198; WO/2010/080933 (U [0039]). All of the CCGs in Table 1 below form a panel of CCGs ("Panel A").. As will be shown detail throughout this document, individual CCGs (e.g., CCGs in Table 1) and subsets of these genes can also be used.

Table 1

NM 001114121.1;

CHEK1 * 1111 Hs00967506_ml NM 001114122.1;

NM 001274.4

NM 018204.3;

CKAP2* 26586 Hs00217068_ml

NM_001098525.1

CKS1B* 1163 Hs01029137_gl NM_001826.2

CKS2* 1164 Hs01048812_gl NM_001827.1

CTPS* 1503 Hs01041851_ml NM 001905.2

CTSL2* 1515 Hs00952036_ml NM_001333.2

DBF 4* 10926 Hs00272696_ml NM_006716.3

DDX39* 10212 Hs00271794_ml NM_005804.2

DLGAP5/ DLG7*# 9787 Hs00207323_ml NM_014750.3

DONSON* 29980 Hs00375083_ml NM_017613.2

DSN1 * 79980 Hs00227760_ml NM_024918.2

DTL*# 51514 Hs00978565_ml NM_016448.2

E2F8* 79733 Hs00226635_ml NM_024680.2

ECT2* 1894 Hs00216455_ml NM_018098.4

ESPL1 * 9700 Hs00202246_ml NM_012291.4

NM 130398.2;

EXOl * 9156 Hs00243513_ml NM 003686.3;

NM 006027.3

NM 152998.1;

EZH2* 2146 Hs00544830_ml

NM_004456.3

NM 018193.2;

FANCI* 55215 Hs00289551_ml

NM_001113378.1

NM 001142522.1;

FBX05* 26271 Hs03070834_ml

NM_012177.3

NM 202003.1;

F0XM1 *# 2305 Hs01073586_ml NM 202002.1;

NM 021953.2

GINS1 * 9837 Hs00221421_ml NM_021067.3

GMPS* 8833 Hs00269500_ml NM_003875.2

GPSM2* 29899 Hs00203271_ml NM_013296.4

GTSE1 * 51512 Hs00212681_ml NM_016426.5

H2AFX* 3014 Hs00266783_sl NM_002105.2

HMMR* 3161 Hs00234864_ml NM_001142556.1; NM 001142557.1;

NM 012484.2; NM 012485.2

NM 001002033.1;

HN1 * 51155 Hs00602957_ml NM 001002032.1;

NM_016185.2

KIAA0101 * 9768 Hs00207134_ml NM_014736.4

KIF11 * 3832 Hs00189698_ml NM_004523.3

KIF15* 56992 Hs00173349_ml NM_020242.2

KIF18A * 81930 Hs01015428_ml NM_031217.3

KIF20A * 10112 Hs00993573_ml NM_005733.2

KIF20B/

9585 Hs01027505_ml NM_016195.2 MPHOSPH1 *

NM 138555.1;

KIF23* 9493 Hs00370852_ml

NM_004856.4

KIF2C* 11004 Hs00199232_ml NM_006845.3

KIF4A * 24137 Hs01020169_ml NM_012310.3

KIFC1 * 3833 Hs00954801_ml NM_002263.3

KPNA2 3838 Hs00818252_gl NM_002266.2

LMNB2* 84823 Hs00383326_ml NM_032737.2

MAD2L1 4085 Hs01554513_gl NM_002358.3

MCAM* 4162 Hs00174838_ml NM 006500.2

NM 018518.3;

MCM10*# 55388 Hs00960349_ml

NM 182751.1

MCM2* 4171 Hs00170472_ml NM_004526.2

NM 005914.2;

MCM4* 4173 Hs00381539_ml

NM 182746.1

MCM6* 4175 Hs00195504_ml NM_005915.4

NM 005916.3;

MCM7* 4176 Hs01097212_ml

NM 182776.1

MELK 9833 Hs00207681_ml NM_014791.2

MKI67* 4288 Hs00606991_ml NM_002417.3

MYBL2* 4605 Hs00231158_ml NM_002466.2

NCAPD2* 9918 Hs00274505_ml NM_014865.3 NCAPG* 64151 Hs00254617_ml NM_022346.3

NCAPG2* 54892 Hs00375141_ml NM_017760.5

NCAPH* 23397 Hs01010752_ml NM_015341.3

NDC80* 10403 Hs00196101_ml NM 006101.2

NEK2* 4751 Hs00601227_mH NM_002497.2

NM 018454.6;

NUSAP1 * 51203 Hs01006195_ml NM 001129897.1;

NM 016359.3

OIP5* 11339 Hs00299079_ml NM_007280.1

ORC6L* 23594 Hs00204876_ml NM_014321.2

NM 001079524.1;

PAICS* 10606 Hs00272390_ml NM 001079525.1;

NM 006452.3

PBK*# 55872 Hs00218544_ml NM_018492.2

NM 182649.1;

PCNA * 5111 Hs00427214_gl

NM 002592.2

PDSS1 * 23590 Hs00372008_ml NM_014317.3

PLK1 *# 5347 Hs00153444_ml NM 005030.3

PLK4* 10733 Hs00179514_ml NM_014264.3

POLE2* 5427 Hs00160277_ml NM_002692.2

NM 199413.1;

PRC1 * 9055 Hs00187740_ml NM 199414.1;

NM 003981.2

PSMA 7* 5688 Hs00895424_ml NM_002792.2

NM 032636.6; NM 001005290.2;

PSRC1 * 84722 Hs00364137_ml

NM 001032290.1; NM 001032291.1

PTTG1 * 9232 Hs00851754_ul NM_004219.2

RACGAP1 * 29127 Hs00374747_ml NM_013277.3

NM 133487.2;

RAD51 * 5888 Hs00153418_ml

NM 002875.3 NM 001130862.1;

RAD51AP1 * 10635 HsOl 54889 l ml

NM_006479.4

RAD54B* 25788 Hs00610716_ml NM_012415.2

NM 001142548.1;

RAD54L * 8438 Hs00269177_ml

NM 003579.3

NM 181471.1;

RFC2* 5982 Hs00945948_ml

NM_002914.3

NM 181573.2;

RFC4* 5984 Hs00427469_ml

NM_002916.3

NM 181578.2; NM 001130112.1;

RFC 5* 5985 Hs00738859_ml

NM 001130113.1; NM 007370.4

RNASEH2A * 10535 Hs00197370_ml NM_006397.2

RRM2*# 6241 Hs00357247_gl NM_001034.2

SHCBP1 * 79801 Hs00226915_ml NM_024745.4

NM 001042550.1;

SMC 2* 10592 Hs00197593_ml NM 001042551.1;

NM 006444.2

SPAG5* 10615 Hs00197708_ml NM_006461.3

SPC25* 57405 Hs00221100_ml NM_020675.3

NM 001048166.1;

STIL* 6491 Hs00161700_ml

NM 003035.2

Hs00606370 ml; NM 005563.3;

STMN1 * 3925

Hs01033129_ml NM_203399.1

TACC3* 10460 Hs00170751_ml NM_006342.1

TIMELESS* 8914 Hs01086966_ml NM_003920.2

TK1 * 7083 Hs01062125_ml NM_003258.4

TOP 2 A * 7153 Hs00172214_ml NM_001067.2

TPX2* 22974 Hs00201616_ml NM_012112.4

TRIP 13* 9319 Hs01020073_ml NM_004237.2

TTK* 7272 Hs00177412_ml NM_003318.3

TUBA1C* 84790 Hs00733770_ml NM_032704.3

TYMS* 7298 Hs00426591_ml NM_001071.2 NM 181799.1;

NM 181800.1;

NM 181801.1;

UBE2C 11065 Hs00964100_gl

NM 181802.1;

NM 181803.1;

NM 007019.2

UBE2S 27338 Hs00819350_ml NM_014501.2

VRK1 * 7443 Hs00177470_ml NM_003384.2

NM 017975.3;

ZWILCH* 55055 Hs01555249_ml

NR 003105.1

NM 032997.2;

ZWINT* 11130 Hs00199952_ml NM 001005413.1;

NM 007057.3

* 124-gene subset of CCGs ("Panel B"). #10-gene subset of CCGs (Panel C). ABI Assay ID means the catalogue ID number for the gene expression assay commercially available from Applied Biosystems Inc. (Foster City, CA) for the particular gene.

[0030] Accordingly, in a first aspect of the present disclosure, panels of genes comprising CCGs for use in determining gene expression, and for diagnosing and treating melanoma are disclosed. In some embodiments, use of panels comprising CCGs comprises determining expression of the CCGs in a sample from an individual or patient.

[0031] Gene expression can be determined either at the RNA level (i.e., mRNA or noncoding RNA (ncRNA)) (e.g., miRNA, tRNA, rRNA, snoRNA, siRNA and piRNA) or at the protein level. Measuring gene expression at the mRNA level includes measuring levels of cDNA corresponding to mRNA. Levels of proteins in a sample can be determined by any known techniques in the art, e.g., HPLC, mass spectrometry, or using antibodies specific to selected proteins (e.g., IHC, ELISA, etc.).

[0032] In one embodiment, the amount of RNA transcribed from the panel of genes including test genes is measured in the sample. In addition, the amount of RNA of one or more housekeeping genes in the sample is also measured, and used to normalize or calibrate the expression of the test genes. The terms "normalizing genes" and "housekeeping genes" are defined herein below.

[0033] In any embodiment of the invention involving a "plurality of test genes," the plurality of test genes may include at least 2, 3 or 4 cell-cycle genes, which constitute at least 50%, 75%) or 80%) of the plurality of test genes, and in some embodiments 100% of the plurality of test genes. In some embodiments, the plurality of test genes includes at least 5, 6, 7, or at least 8 cell-cycle genes, which constitute at least 20%, 25%, 30%, 40%, 50%, 60%, 70%, 75%, 80% or 90% of the plurality of test genes, and in some embodiments 100% of the plurality of test genes. As will be clear from the context of this document, a panel of genes is a plurality of genes. Typically these genes are assayed together in one or more samples from a patient.

[0034] In some other embodiments, the plurality of test genes includes at least 8, 10, 12, 15, 20, 25 or 30 cell-cycle genes, which constitute at least 20%, 25%, 30%, 40%, 50%, 60%, 70%, 75%, 80% or 90% of the plurality of test genes, and preferably 100% of the plurality of test genes.

[0035] As will be apparent to a skilled artisan apprised of the present invention and the disclosure herein, "sample" means any biological sample containing one or more suspected melanoma cells, or one or more R A or protein derived from suspected melanoma cells, and obtained from a patient. For example, a tissue sample obtained from a mole or nevus is a useful sample in the present invention. The tissue sample can be an FFPE sample, or fresh frozen sample, and preferably contain largely the suspect cells. A single cell from a patient's suspected melanoma is also a useful sample. Such a cell can be obtained directly from the patient's skin, or purified from the patient's bodily fluid (e.g., blood, urine). Thus, a bodily fluid such as blood, urine, sputum and saliva containing one or more suspected to be cancerous cells, or mole or nevus-derived RNA or proteins, can also be useful as a sample for purposes of practicing the present invention.

[0036] Those skilled in the art are familiar with various techniques for determining the status of a gene or protein in a tissue or cell sample including, but not limited to, microarray analysis (e.g., for assaying mRNA or microRNA expression, copy number, etc.), quantitative real-time PCR™ ("qRT-PCR™", e.g., TaqMan™), immunoanalysis (e.g., ELISA,

immunohistochemistry), etc. The activity level of a polypeptide encoded by a gene may be used in much the same way as the expression level of the gene or polypeptide. Often higher activity levels indicate higher expression levels and while lower activity levels indicate lower expression levels. Thus, in some embodiments, the invention provides any of the methods discussed above, wherein the activity level of a polypeptide encoded by the CCG is determined rather than or in adition to the expression level of the CCG. Those skilled in the art are familiar with techniques for measuring the activity of various such proteins, including those encoded by the genes listed in Table 1. The methods of the invention may be practiced independent of the particular technique used.

[0037] In some embodiments, the expression of one or more normalizing (often called "housekeeping" or "housekeeper") genes is also obtained for use in normalizing the expression of test genes. As used herein, "normalizing genes" referred to the genes whose expression is used to calibrate or normalize the measured expression of the gene of interest (e.g., test genes). Importantly, the expression of normalizing genes should be independent of cancer diagnosis, and the expression of the normalizing genes is very similar among all the samples. The

normalization ensures accurate comparison of expression of a test gene between different samples. For this purpose, housekeeping genes known in the art can be used. Housekeeping genes are well known in the art, with examples including, but are not limited to, GUSB

(glucuronidase, beta), HMBS (hydroxymethylbilane synthase), SDHA (succinate dehydrogenase complex, subunit A, flavoprotein), UBC (ubiquitin C), YWHAZ (tyrosine 3- monooxygenase/tryptophan 5-monooxygenase activation protein, zeta polypeptide), MRFAP1, PSMA1, RPL13A, TXNL1, SLC25A3, RPS29, RPL8, PSMC1 and RPL4. One or more housekeeping genes can be used. Preferably, at least 2, 5, 10 or 15 housekeeping genes are used to provide a combined normalizing gene set. The amount of gene expression of such

normalizing genes can be averaged, combined together by straight additions or by a defined algorithm. Some examples of particularly useful housekeeper genes for use in the methods and compositions of the invention include those listed in Table 2 below.

Table 2

HMBS 3145 Hs00609297_ml NM 000190.3

MMADHC 27249 Hs00739517_gl NM_015702.2

MRFAP1 * 93621 Hs00738144_gl NM_033296.1

PPP2CA 5515 Hs00427259_ml NM_002715.2

PSMA1 * 5682 Hs00267631_ml

PSMC1 * 5700 Hs02386942_gl NM_002802.2

RPL13A * 23521 Hs03043885_gl NM_012423.2

RPL37 6167 Hs02340038_gl NM 000997.4

RPL38 6169 Hs00605263_gl NM 000999.3

RPL4* 6124 Hs03044647_gl NM 000968.2

RPL8* 6132 Hs00361285_gl NM_033301.1; NM_000973.3

RPS29* 6235 Hs03004310_gl NM 001030001.1; NM OO 1032.3

SDHA 6389 Hs00188166_ml NM_004168.2

NM 213611.1; NM 002635.2;

SLC25A3* 6515 Hs00358082_ml

NM 005888.2

TXNL1 * 9352 Hs00355488_ml NR_024546.1; NM_004786.2

UBA52 7311 Hs03004332_gl NM_001033930.1; NM_003333.3

UBC 7316 Hs00824723_ml NM 021009.4

YWHAZ 7534 Hs00237047_ml NM_003406.3

* Subset of housekeeping genes used in, e.g., Example 3.

[0038] In the case of measuring RNA levels for the genes, one convenient and sensitive approach is real-time quantitative PCR™ (qPCR) assay, following a reverse transcription reaction. Typically, a cycle threshold (C _t ) is determined for each test gene and each normalizing gene, i.e., the number of cycles at which the fluorescence from a qPCR reaction above background is detectable.

[0039] The overall expression of the one or more normalizing genes can be represented by a "normalizing value" which can be generated by combining the expression of all normalizing genes, either weighted eaqually (straight addition or averaging) or by different predefined coefficients. For example, in a simplest manner, the normalizing value _H can be the cycle threshold (C _t ) of one single normalizing gene, or an average of the C _t values of 2 or more, preferably 10 or more, or 15 or more normalizing genes, in which case, the predefined coefficient is 1/N, where N is the total number of normalizing genes used. Thus, _H = (C _t m + C _t H2 + ^" C _t Hn) N. As will be apparent to skilled artisans, depending on the normalizing genes used, and the weight desired to be given to each normalizing gene, any coefficients (from 0/N to N/N) can be given to the normalizing genes in weighting the expression of such normalizing genes. That is, C _ffl = m + yC _tm + ^" zC _t Hn, wherein x + y + ^{" '} + z = 1.

[0040] As discussed above, the methods of the invention generally involve determining the level of expression of a panel of CCGs. With modern high-throughput techniques, it is often possible to determine the expression level of tens, hundreds or thousands of genes. Indeed, it is possible to determine the level of expression of the entire transcriptome (i.e., each transcribed sequence in the genome). Once such a global assay has been performed, one may then informatically analyze one or more subsets of transcripts (i.e., panels or, as often used herein, pluralities of test genes). After measuring the expression of hundreds or thousands of transcripts in a sample, for example, one may analyze (e.g., informatically) the expression of a panel or plurality of test genes comprising primarily CCGs according to the present invention by combining the expression level values of the individual test genes to obtain a test value.

[0041] As will be apparent to a skilled artisan, the test value provided in the present invention represents the overall expression level of the plurality of test genes composed substantially of cell-cycle genes. In one embodiment, to provide a test value in the methods of the invention, the normalized expression for a test gene can be obtained by normalizing the measured C _t for the test gene against the Cm, i.e., AC _t i = (C _t i - Cm)- Thus, the test value representing the overall expression of the plurality of test genes can be provided by combining the normalized expression of all test genes, either by straight addition or averaging (i.e., weighted eaqually) or by a different predefined coefficient. For example, the simplest approach is averaging the normalized expression of all test genes: test value = (AC _tl + AC _t2 + ^" + AC _ta )/n. As will be apparent to skilled artisans, depending on the test genes used, different weight can also be given to different test genes in the present invention. In each case where this document discloses using the expression of a plurality of genes (e.g., "determining [in a sample from the patient] the expression of a plurality of test genes" or "correlating increased expression of said plurality of test genes to an increased likelihood of having melanoma"), this includes in some embodiments using a test value representing, corresponding to or derived or calculated from the overall expression of this plurality of genes (e.g., "determining [in a sample from the patient] a test value representing the expression of a plurality of test genes" or "correlating an increased test value [or a test value above some reference value] (optionally representing the expression of said plurality of test genes) to an increased likelihood of response"). [0042] It has been determined that, once the CCP phenomenon reported herein is appreciated, the choice of individual CCGs for a test panel can often be somewhat arbitrary. In other words, many CCGs have been found to be very good surrogates for each other. Thus any CCG (or panel of CCGs) can be used in the various embodiments of the invention. In other embodiments of the invention, optimized CCGs are used. One way of assessing whether particular CCGs will serve well in the methods and compositions of the invention is by assessing their correlation with the mean expression of CCGs (e.g., all known CCGs , a specific set of CCGs , etc.). Those CCGs that correlate particularly well with the mean are expected to perform well in assays of the invention, e.g., because these will reduce noise in the assay.

[0043] Thus, in some embodiments of each of the various aspects of the invention the plurality of test genes comprises the top 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 20, 25, 30, 35, 40 or more CCGs from Panel A. In some embodiments of each of the various aspects of the invention the plurality of test genes comprises the top 2, 3, 4, 5, 6, 7, 8, 9 or 10 CCGs from Panel B. In some embodiments the plurality of test genes comprises at least some number of CCGs (e.g., at least 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, 50 or more CCGs) and this plurality of CCGs comprises at least 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 of the genes in Panel B. In some embodiments the plurality of test genes comprises at least some number of CCGs (e.g., at least 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, 50 or more CCGs) and this plurality of CCGs comprises any two, three, four, five, six, seven, eight, nine, or ten of gene numbers 1 & 2, 1 to 3, 1 to 4, 1 to 5, 1 to 6, 1 to 7, 1 to 8, 1 to 9, or 1 to 10 of any of the genes in Panel B (based on order of appearance in Table 1). In some embodiments the plurality of test genes comprises at least some number of CCGs (e.g., at least 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, 50 or more CCGs) and this plurality of CCGs comprises any one, two, three, four, five, six, seven, eight, or nine or all of gene numbers 2 & 3, 2 to 4, 2 to 5, 2 to 6, 2 to 7, 2 to 8, 2 to 9, or 2 to 10 of any of the genes in Panel B (based on order of appearance in Table 1). In some embodiments the plurality of test genes comprises at least some number of CCGs (e.g., at least 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, 50 or more CCGs) and this plurality of CCGs comprises any one, two, three, four, five, six, seven, or eight or all of gene numbers 3 & 4, 3 to 5, 3 to 6, 3 to 7, 3 to 8, 3 to 9, or 3 to 10 of any of the genes in Panel B (based on order of appearance in Table 1). In some embodiments the plurality of test genes comprises at least some number of CCGs (e.g., at least 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, 50 or more CCGs) and this plurality of CCGs comprises any one, two, three, four, five, six, or seven or all of gene numbers 4 & 5, 4 to 6, 4 to 7, 4 to 8, 4 to 9, or 4 to 10 of any of the genes in Panel B (based on order of appearance in Table 1).

[0044] In another embodiment, the plurality of CCGs or panel of CCGs comprises any set of genes from Table WW.

Immune and Additional Genes

[0045] It has additionally surprisingly been discovered that panels of immune genes are diagnostic for melanoma. Accordingly, in another aspect of the present disclosure, panels of genes comprising immune genes for use in determining gene expression, and for diagnosing and treating melanoma are disclosed. In some embodiments, use of panels comprising immune genes comprises determining expression of the immune genes in a sample from an individual or patient.

[0046] "Immune gene" herein refers to a gene associated with or expressed by one or more leukocytes. In particular embodiments, immune genes comprise genes associated with or expressed by lymphocytes. In some embodiments, the immune genes comprise genes expressed by activated lymphocytes. In some embodiments, immune genes comprise genes expressed by T cells. In some embodiments, immune genes comprise genes expressed by activated T cells. In some embodiments, immune genes comprise the immune genes identified in Table 3, below.

TABLE 3

CFH*# 3075 ENSG00000000971 NM 000186;

NM_001014975. 1

CXCL10*# ^A 3627 ENSG00000169245 NM_001565.2

CXCL12# 6387 ENSGOOOOO 107562|ENSG00000126214 NM 000609.4;

NM 001033886; NM 199168; NM 005552; NM_182923.3

CXCL13*# 10563 ENSG00000156234 NM_006419

CXCL9*# ^A 4283 ENSG00000138755 NM_002416

FABP7* 2173 ENSG000001 13805 NM_020872

FN1 * 2335 ENSG000001 15414|ENSG00000197721 NM 002026;

NM 054034.2; NM 212474; NM 212476. 1 ; NM 212482. 1 ; NM_175710. 1

GDF15 9518

HCLS1 *# 3059 ENSG000001 13070|ENSG00000180353 NM 001945;

NM_005335

HEY1 * 23462 ENSG00000164683 NM 001040708. 1 ;

NM_012258

HLA- 3108 ENSG00000204257|ENSG00000206229| NM_006120

DMA*# ENSG00000206293

HLA-DPA1# 31 13

HLA-DPB1# 31 15 ENSG000001 12242|ENSG00000168383 NM 001949;

NM_002121

HLA-DRA*# 3122 ENSG00000143768 |ENSG00000204287| NM 003240;

ENSG00000206243 NM_0191 1 1

HLA-E# 3133 ENSG00000204592 NM_005516

IFI6*# 2537 ENSG00000126709|ENSG00000135047 NM 002038;

NM 022872; NM 022873 ; NM 001912; NM 145918 IGHM# 3507

IGJ*# 3512 ENSG00000132465 |ENSG00000182197 NM 144646;

NM_000127

IGLL5/CKA 100423062

P2#

IRF1 *# ^A 3659

IRF4# 3662 ENSG00000137265 NM_002460

ITGB2*# 3689 ENSG00000160255 NM_00021 1

KRT15* 3866 ENSG00000171346 NM_002275

LCP2*# ^A 3937 ENSG00000043462 NM_005565.3

NCOA3 8202 ENSG00000124151 NM 006534;

NM_181659

NR4A1 3164 ENSG00000107223 |ENSG00000123358 NM 003792;

NM 153200; NM 002135; NM 173 157; NM_173 158

PECAM1 *# 5175 ENSG00000173744|ENSG00000198802 NM 004504;

NM_000442.3

PHACTR1 * 221692 ENSG000001 12137 NM_030948. 1

PHIP 55023 ENSG00000146247 NM_017934

POU5F1 5460 ENSG0000020453 1 |ENSG00000206349| NM 002701 ;

ENSG00000206454 NM_203289.3

PRAME* 23532 ENSG00000185686 NM 0061 15

;NM 206953 ; NM 206954; NM 206955; NM_206956

PTN* 5764

ΡΤΡΝ22*# ^Λ 26191 ENSG00000134242 NM 01241 1 ;

NM_015967 PTPRC*# ^A 5788 ENSG00000081237 NM 002838;

NM 080921 ;

NM 080922;

NM_080923.2

RGS1 * 5996 ENSG00000090104 NM_002922

S100A9* 6280 ENSG00000163220 NM_002965

SELL* 6402 ENSG00000188404 NM_000655.3

SERPINB4# 63 18 ENSG00000057149|ENSG00000068796 NM 002974;

NM_004520

SOCS3# 9021 ENSG00000184557 NM_003955

SPP1 * 6696

WIF1 1 1 197 ENSG00000125285 NM_007084

WNT2 7472 ENSG00000105989 NM_003391

*Panel of 30 mixed genes (Panel D); #panel of 28 immune genes (Panel E); ^A panel of 8 immune genes (Panel F)

[0047] Gene expression of immune genes can be determined as described above with respect to CCGs. In one embodiment, the amount of R A transcribed from the panel of genes including immune genes is measured in the sample. In addition, the amount of RNA of one or more housekeeping genes in the sample is also measured, and used to normalize or calibrate the expression of the test genes. The terms "normalizing genes" and "housekeeping genes" are defined above.

[0048] In any embodiment of the invention involving a "plurality of test genes," the plurality of test genes may include at least 2, 3 or 4 immune genes, which constitute at least 50%, 75% or 80% of the plurality of immune genes, and in some embodiments 100% of the plurality of immune genes. In some embodiments, the plurality of immune genes includes at least 5, 6, 7, or at least 8 cell-cycle genes, which constitute at least 20%, 25%, 30%, 40%, 50%, 60%, 70%, 75%), 80%) or 90%) of the plurality of immune genes, and in some embodiments 100% of the plurality of immune genes. As will be clear from the context of this document, a panel of genes is a plurality of genes. Typically these genes are assayed together in one or more samples from a patient. [0049] In some other embodiments, the plurality of immune genes includes at least 8, 10, 12, 15, 20, 25 or 30 immune, which constitute at least 20%, 25%, 30%, 40%, 50%, 60%, 70%), 75%), 80%) or 90%> of the plurality of immune genes, and preferably 100% of the plurality of immune genes.

[0050] The sample used to determine the expression of immune genes may be any sample as described above for CCGs.

[0051] In the case of measuring R A levels for the immune genes, real-time quantitative PCR™ (qPCR) assay with normalized values, as described above, may be used.

[0052] As discussed above, some embodiments of the methods disclosed generally involve determining the level of expression of a panel comprising immune genes. With modern high-throughput techniques, it is often possible to determine the expression level of tens, hundreds or thousands of genes. Indeed, it is possible to determine the level of expression of the entire transcriptome (i.e., each transcribed sequence in the genome). Once such a global assay has been performed, one may then informatically analyze one or more subsets of transcripts (i.e., panels or, as often used herein, pluralities of test genes). After measuring the expression of hundreds or thousands of transcripts in a sample, for example, one may analyze (e.g., informatically) the expression of a panel or plurality of test genes comprising primarily immune genes according to the present invention by combining the expression level values of the individual test genes to obtain a test value.

[0053] Thus, in some embodiments of each of the various aspects of the invention the plurality of test genes comprises any 2, 3, 4, 5, 6, 7, 8, 9, 10, 1 1 , 12, 13, 14, 15, 20, 25, or 28 Immune genes from Panel E. In some embodiments of each of the various aspects of the invention the plurality of test genes comprises 2, 3, 4, 5, 6, 7, 8 immune genes from Panel F. In some embodiments the plurality of test genes comprises at least some number of immune genes (e.g., at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, 50 or more immune genes) and this plurality of immune genes comprises at least 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, or 28 of the genes in Panel E, or 2, 3, 4, 5, 6, 7, 8, 9 or 10 immune genes from Panel F.

[0054] It has also been found that additional genes may be diagnostic for melanoma. Without being bound by theory, these additional genes are believed to be non-CCG and nonimmune genes, and comprise ARPC2, BCL2A1 , FABP7, FN1, GDF15, HEY1 , KRT15, NCOA3, NR4A1, PHACTR1, PHIP, POU5F1, PRAME, PTN, RGS1, S100A9, SELL, SPP1, WIF1, and WNT2 (Panel G). Accordingly, in another aspect of the present disclosure, panels of genes comprising these additional genes are disclosed for use in determining gene expression, and for diagnosing and treating melanoma.

[0055] In one embodiment, the panel comprising these additional genes: BCL2A1, FABP7, FN1, HEY1, KRT15, PHACTR1, PRAME, PTN, RGS1, S100A9, SELL, and SPP1 (Panel H). In another embodiment, the panel comprising additional genes comprises PRAME and S100A9.

[0056] In additional embodiments, this disclosure provides for mixed panels of genes which are useful in determining gene expression, and for diagnosing and treating melanoma. These mixed panels may comprise immune genes and CCGs, or immune genes and genes from Panel G or H, or CCGs and genes from panel G or H. In one embodiment, the mixed panel comprises one or more CCGs, one or more immune genes, and one or more additional genes from panel G. In one embodiment, the mixed panel comprises Panel D. In another embodiment, the mixed panel comprises PRAME, S100A9 and the genes of panel F.

[0057] In another embodiment, the mixed panel comprises one or more CCGs, one or more immune genes, and one or more additional genes from panel H. In one embodiment of a mixed panel, the mixed panel (Panel I) comprises the genes from Panel C and the genes from Panel D.

[0058] In one embodiment of a mixed panel, the mixed panel) comprises S 100A9 and/or S 100A9-related genes. The S 100A9 related genes can include genes that have highly correlated expression compared to S 100A9. These S 100A9-related genes may include genes that are closely clustered with S 100A9 on chromosome 1. These S 100A9-related genes may also include genes that have similar transcription control as S 100A9. The S 100A9-related proteins may also be part of the same biological pathway. The S 100A9-related genes may also code for proteins that interact with the protein coded by S 100A9. As a non-limiting example, the mixed panel may comprise S 100A9, S 100A7, S 100A8, S 100A12, PI3, S I OOAI O, and S 100A14 (Panel J). As a non-limiting example, the mixed panel may comprise S 100A9, S 100A7, S 100A8, S 100A12, and PI3 (Panel L). [0059] In an alternate embodiment, the mixed panel comprises PRAME. In another embodiment, the mixed panel comprises S100A9. In yet another embodiment, the mixed panel comprises CCL5, CD38, CXCLIO, CXCL9, IRF1, LCP2, PTPN22, or PTPRC. In other embodiments the mixed panel comprises S100A7, S100A8, S100A12, PI3, SIOOAIO, and S100A14. In some embodiments, the mixed panel comprises

S100A9, S100A7, S100A8, S100A12, andPB. Thus, in some embodiments of each of the various aspects of the invention the panel of mixed genes comprises any 2, 3, 4, 5, 6, or 7 S 100A9-related genes from Panel J. In other embodiments of each of the various aspects of the invention the panel of mixed genes comprises any 2, 3, 4, 5, 6, or 7 S 100A9-related genes from Panel L.

[0060] Thus, in some embodiments of each of the various aspects of the invention the panel of mixed genes comprises PRAME, at least one of the genes of Panel J, and at least one of the genes of panel F. In some embodiments, the mixed panel comprises PRAME, S100A9, S100A7, S100A8, S100A12, SIOOAIO, S100A14, PI3, CCL5, CD38, CXCLIO, CXCL9, IRF1, LCP2, PTPN22, and PTPRC.

[0061] Thus, in some embodiments of each of the various aspects of the invention the panel of mixed genes comprises PRAME, at least one of the genes of Panel L, and at least one of the genes of panel F. In some embodiments, the mixed panel comprises PRAME, S100A9, S100A7, S100A8, S100A12, PI3, CCL5, CD38, CXCLIO, CXCL9, IRF1, LCP2, PTPN22, and PTPRC.

[0062] Thus, in some embodiments of each of the various aspects of the invention the panel of mixed genes comprises PRAME, S100A9, and at least 1, 2, 3, 4, 5, 6, 7, or 8 genes of panel F. In some embodiments, the panel of mixed genes comprises PRAME and at least 1, 2, 3, 4, 5, 6, or 7 genes of Panel J, and at least 1, 2, 3, 4, 5, 6, 7, or 8 genes of panel F. In some embodiments, the panel of mixed genes comprises PRAME, S100A9, and at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 genes of panel E. In some embodiments, the panel of mixed genes comprises PRAME, and at least 1, 2, 3, 4, 5, 6, or 7 genes of Panel J, and at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 genes of Panel E. [0063] Thus, in some embodiments of each of the various aspects of the invention the panel of mixed genes comprises at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 genes of Panel D, and S100A9, and at least 1, 2, 3, 4, 5, 6, 7, or 8 genes of panel F. In some embodiments, the panel of mixed genes comprises at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 genes of Panel D and at least 1, 2, 3, 4, 5, 6, or 7 genes of Panel J, and at least 1, 2, 3, 4, 5, 6, 7, or 8 genes of panel F. In some embodiments, the panel of mixed genes comprises at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 genes of Panel D, S100A9, and at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 genes of panel E. In some embodiments, the panel of mixed genes comprises at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 genes of Panel D, and at least 1, 2, 3, 4, 5, 6, or 7 genes of Panel J, and at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 genes of Panel E.

[0064] Thus, in some embodiments of each of the various aspects of the invention the panel of mixed genes comprises at least at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 genes of Panel G, and S100A9, and at least 1, 2, 3, 4, 5, 6, 7, or 8 genes of panel F. In some embodiments, the panel of mixed genes comprises at least at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 genes of Panel G, and at least 1, 2, 3, 4, 5, 6, or 7 genes of Panel J, and at least 1, 2, 3, 4, 5, 6, 7, or 8 genes of panel F. In some embodiments, the panel of mixed genes comprises at least 1 at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 genes of Panel G, S100A9, and at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 genes of panel E. In some embodiments, the panel of mixed genes comprises at least at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 genes of Panel G, and at least 1, 2, 3, 4, 5, 6, or 7 genes of Panel J, and at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 genes of Panel E.

[0065] Thus, in some embodiments of each of the various aspects of the invention the panel of mixed genes comprises at least at least at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 genes of Panel H, and S100A9, and at least 1, 2, 3, 4, 5, 6, 7, or 8 genes of panel F. In some embodiments, the panel of mixed genes comprises at least at least at least 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, 1 1 , or 12 genes of Panel H, and at least 1 , 2, 3, 4, 5, 6, or 7 genes of Panel J, and at least 1 , 2, 3, 4, 5, 6, 7, or 8 genes of panel F. In some embodiments, the panel of mixed genes comprises at least 1 at least 1 at least 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, 1 1 , or 12 genes of Panel H, S 100A9, and at least 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, 1 1 , 12, 13, 14, 15, 16, 17, 18, 19, or 20 genes of panel E. In some embodiments, the panel of mixed genes comprises at least at least at least 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, 1 1 , or 12 genes of Panel H, and at least 1 , 2, 3, 4, 5, 6, or 7 genes of Panel J, and at least 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, 1 1 , 12, 13, 14, 15, 16, 17, 18, 19, or 20 genes of Panel E.

[0066] In one embodiment, the panel comprises any set of two genes from Table XX. In another embodiment, the panel comprises any set of three genes from Table YY. In another embodiment, the panel comprises any set of four genes from Table ZZ.

[0067] In one embodiment of a panel of housekeeper genes, the housekeeper panel (Panel K) comprises one or more genes for use in normalizing the expression of test genes. Panel K can be made up of any gene whose expression is used to calibrate or normalize measured expression of the gene or genes of interest. Panel K can be made up of any housekeeping or housekeeper genes known in the art. Examples of housekeeper genes that can be used in Panel K include CLTC, GUSB, HMBS,

MMADHC, MRFAP 1 , PPP2CA, PSMA1 , PSMC 1 , RPL13A, RPL37, RPL38, RPL4, RPL8, RPS29, SDHA, SLC25A3 , TXNL1 , UBA52, UBC, and YWHAZ. In some embodiments, the housekeeper genes used to normalize the expression of test genes can include at least 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, 1 1 , 12, 13, 14, 15, 16, 17, 18, 19, or 20 genes of Panel K.

Methods of Determining Gene Expression

[0068] Accordingly, in a first aspect of the present invention, a method is provided for determining gene expression in a sample from a patient (e.g., one suspected of containing melanoma). Generally, the method includes at least the following steps: (1) obtaining a sample from a patient (e.g., one suspected of containing melanoma); (2) determining the expression of a panel of genes in the sample; and (3) providing a test value by (a) weighting the determined expression of each gene from the panel of genes with a predefined coefficient, and (b) combining the weighted expression of each gene from the panel of genes to provide said test value.

[0069] Weighting the expression of each gene from the panel of genes may be performed individually for each gene, or genes may first be grouped and their normalized expression averaged or otherwise combined before weighting is performed. In some

embodiments, genes are grouped based on whether they provide independent information in separating nevi from melanoma. In some examples, CCGs are grouped before weighting. In other embodiments, immune genes are grouped before weighting. The skilled artisan will understand that in some embodiments, grouping may be conceptualized as a way of individually weighting each gene in the pre-defined group to arrive at an intermediate value, which intermediate value is weighted along with other individual gene expression values to obtain a final value. In some embodiments, multiple rounds of grouping may be performed, resulting in multiple intermediate values, which may be in turn grouped to obtain a final value.

[0070] In some embodiments, weighting coefficients are determined which optimize the contribution of each expression profile to the predictive value of any resulting test value. In some embodiments, genes whose expression is more highly correlated or anti-correlated with melanoma receive a larger weighting coefficient in order to maximize the overall predictive power of any resulting test value. In some embodiments, genes whose expression is correlated or anti-correlated with melanoma, but less correlated with the expression of other genes in the panel receive a larger weighting coefficient in order to maximize the overall predictive power of any resulting test value. In some embodiments, genes whose expression is significantly, moderately, or highly correlated may be grouped.

[0071] In some embodiments, regression analyses are utilized to obtain appropriate weighting coefficients to maximize the predictive power of a test value. In some embodiments, linear regression is used to fit expression levels to a model for providing test values which are diagnostic of melanoma. In other embodiments, logistic regression is used to determine weighting coefficients for expression levels of individual genes or groups of genes in a model for diagnosis of melanoma.

[0072] In some embodiments, weighting the expression of each gene comprises grouping immune genes, and then weighting the expression of immune genes, PRAME and S100A9 to arrive at a test value which is diagnostic for melanoma. In related embodiments, there are 8 immune genes. In related embodiments, the immune genes comprise Panel F. In some embodiments, the weighting to arrive at a test value is as follows: test value = (A x PRAME) + (B x grouped immune) + (C x S 100A9). In a related embodiment, A is 0.525, B is 0.677 and C is 0.357.

[0073] In some embodiments, weighting the expression of each gene comprises grouping immune genes, grouping S100A9-related genes and then weighting the expression of immune genes, PRAME and the S100A9-related genes to arrive at a test value which is diagnostic for melanoma. In related embodiments, there are 8 immune genes. In related embodiments, the immune genes comprise Panel F. In related embodiments, there are 7

S100A9-related genes. In related embodiments, the S100A9-related genes comprise Panel J. In related embodiments, the S100A9-related genes comprise Panel L. In some embodiments, the weighting to arrive at a test value is as follows: test value = (A x PRAME) + (B x grouped immune) + (C x grouped S 100A9-related). In a related embodiment, A is 1.149, B is 0.698 and C is 0.922.

[0074] In some embodiments, weighting the expression of each gene comprises grouping immune genes, grouping S100A9-related genes and then weighting the expression of immune genes, PRAME and the S100A9-related genes and then adjusting by a linear scale factor to arrive at a test value which is diagnostic for melanoma. The linear scale factor adjusts the cutoff value so that the cutoff value is centered about zero. In related embodiments, there are 8 immune genes. In related embodiments, the immune genes comprise Panel F. In related embodiments, there are 7 S100A9-related genes. In related embodiments, the S100A9-related genes comprise Panel J. In separate related embodiments, the S100A9-related genes comprise Panel L. In some embodiments, the weighting to arrive at a test value is as follows: test value = (A x PRAME) + (B x grouped immune) + (C x grouped S 100A9-related) + D. In a related embodiment, A is 1.149, B is 0.698, C is 0.922, and D is -0.334.

[0075] In some embodiments a test value derived from expression levels may be combined with non-expression parameters to arrive at a modified test value or score which is diagnostic for melanoma. In some embodiments, clinical factors may be combined with a test value derived from expression levels in order to provide a score which is diagnostic for melanoma. In related embodiments, clinical staging data may be weighted and combined with a test value based on expression to obtain a score which is diagnostic for melanoma.

Methods of Diagnosing Melanoma

[0076] Provided herein are also methods of diagnosing melanoma. Generally, a method is provided for diagnosing melanoma, which comprises a) determining in a sample from an individual the expression of a panel of genes; b) comparing the expression of the panel of genes in the sample to the expression of the panel of genes in one or more control samples; and c) diagnosing the individual with melanoma, or concluding that the individual is likely to have melanoma, based at least in part on a difference between the expression of one or more genes of the panel of genes in the sample versus the one or more control samples.

[0077] The step of comparing the expression of the panel of genes may be performed directly (i.e. obtaining an expression value for each gene in the panel of genes in the sample and in the one or more control sample, and determining on a gene by gene basis if there is a significant difference between the expression in the sample versus the one or more controls). Alternately, comparing the expression of the panel of genes in the sample to the expression in one or more control samples may be performed implicitly. In some embodiments, implicit comparison is achieved by building a model based on the one or more control samples and determining where the expression of the panel of genes in the individual sample fits within the model. In one embodiment, implicit comparison of the expression of the panel of genes in the sample to one or more control samples comprises utilizing a pre-determined set of weighting coefficients based on analysis of the one or more control samples to weight the expression of the panel of genes in the sample and arrive at a test value. In a related embodiment, the test value is compared to a pre-determined cutoff value based on analysis of the one or more control samples to achieve implicit comparison.

[0078] In some embodiments, the methods of diagnosis further comprise

communicating that the individual is likely to have melanoma.

[0079] As used herein, "communicating" a particular piece of information means to make such information known to another person or transfer such information to a thing (e.g., a computer). In some methods of the invention, a patient's diagnosis or likelihood of having melanoma is communicated. In some embodiments, the information used to arrive at such a diagnosis or likelihood prediction is communicated. This communication may be auditory (e.g., verbal), visual (e.g., written), electronic (e.g., data transferred from one computer system to another), etc. In some embodiments, communicating a diagnosis or likelihood of melanoma comprises generating a report that communicates the diagnosis or likelihood of melanoma. In some embodiments the report is a paper report, an auditory report, or an electronic record. In some embodiments the report is displayed and/or stored on a computing device (e.g., handheld device, desktop computer, smart device, website, etc.). In some embodiments the diagnosis or likelihood of melanoma is communicated to a physician (e.g., a report communicating the classification is provided to the physician). In some embodiments the diagnosis or likelihood of melanoma is communicated to a patient (e.g., a report communicating the classification is provided to the patient). Communicating a diagnosis or likelihood of melanoma can also be accomplished by transferring information (e.g., data) embodying the classification to a server computer and allowing an intermediary or end-user to access such information (e.g., by viewing the information as displayed from the server, by downloading the information in the form of one or more files transferred from the server to the intermediary or end-user's device, etc.).

[0080] Wherever an embodiment of the invention comprises concluding some fact (e.g., a patient's likelihood of having melanoma), this may include a computer program concluding such fact, typically after performing an algorithm that applies information on the expression of the panel of genes in the sample, as described above.

[0081] In some embodiments, the method of diagnosis includes (1) obtaining a sample from a patient suspected of having melanoma; (2) determining the expression of a panel of genes in the sample including at least 2, 4, 6, 8 or 10 cell-cycle genes, or at least 2, 4, 6 or 8 immune genes; and (3) providing a test value by (a) weighting the determined expression of each of a plurality of test genes selected from the panel of genes with a predefined coefficient, and (b) combining the weighted expression to provide said test value, wherein at least 20%, 50%>, at least 75%) or at least 90%> of said plurality of test genes are cell-cycle genes, and wherein high expression (or increased expression or overexpression) of the plurality of test genes indicates a increase likelihood of having melanoma. In some embodiments, the method comprises at least one of the following steps: (a) correlating high expression (or increased expression or overexpression) of the plurality of test genes to an increased likelihood of having melanoma; (b) concluding that the patient has an increased likelihood of having melanoma based at least in part on high expression (or increased expression or overexpression) of the plurality of test genes; or (c) communicating that the patient has an increased likelihood of having melanoma based at least in part on high expression (or increased expression or overexpression) of the plurality of test genes.

[0082] In some embodiments, the expression levels measured in a sample are used to derive or calculate a value or score, as described above. This value may be derived solely from expression levels or optionally derived from a combination of the expression value scores with other components (e.g., clinical staging, etc.) to give a potentially more comprehensive value/score. Thus, in every case where an embodiment of the invention described herein involves determining the status of a biomarker (e.g., CCGS, immune genes or additional genes, as defines), related embodiments involve deriving or calculating a value or score from the measured status (e.g., expression score, or combined score).

[0083] In some such embodiments, multiple scores (e.g., expression test value and clinical parameters, such as clinical staging) can be combined into a more comprehensive score. Single component (e.g., CCG) or combined test scores for a particular patient can be compared to single component or combined scores for reference populations, with differences between test and reference scores being correlated to or indicative of some clinical feature. Thus, in some embodiments the invention provides a method of determining a melanoma diagnosis comprising (1) obtaining the measured expression levels of a panel of genes in a sample from the patient, (2) calculating a test value from these measured expression levels, (3) comparing said test value to a reference value calculated from measured expression levels of the panel of genes in a reference population of patients, and (4)(a) correlating a test value greater than the reference value to a diagnosis of melanoma or (4)(b) correlating a test value equal to or less than the reference value to a benign diagnosis.

[0084] In some such embodiments the test value is calculated by averaging the measured expression of the panel genes (as discussed below). In some embodiments the test value is calculated by weighting each of the panel of genes in a particular way, as describe above.

[0085] In some embodiments the combined score includes CCP score as previously defined. In some embodiments, the combined score includes an immune score as demonstrated in the Examples. In some embodiments the immune score is an average of the expression of the genes in an immune gene panel. In some embodiments, the immune score is an average of the expression of the genes in Table 30. The immune score may be any value used to represent the expression of one or more immune genes as described herein. In some embodiments the immune score is an average of the expression of the genes in an immune gene panel. In some

embodiments, the immune score is an average of the expression of the genes in Panel D. In some embodiments the immune score is an average of the expression of the genes in an immune gene panel. In some embodiments, the immune score is an average of the expression of the genes in Panel E. In some embodiments the immune score is an average of the expression of the genes in an immune gene panel. In some embodiments, the immune score is an average of the expression of the genes in Panel F. A combined score may also include individual genes with independent predictive value, and other non-expression based clinical factors. CCP and immune scores can be a continuous numeric variable.

[0086] In some embodiments the combined score is calculated according to the following formula:

(1) Combined score = A * (CCP score) + B '(immune score) + {C* additional gene X

expression) +D ^additional gene Y expression ...}

Where X and Y represent any diagnostic additional gene as described herein, and the ellipsis indicates that extra additional genes, each with their own coefficient may be added.

[0087] Additionally, in some embodiments, the combined score is calculated according to the following formula:

(2) Combined score = B* (immune score) + {C* additional gene X expression) +D* additional gene Y expression...}

Where X and Y represent any diagnostic additional gene as described herein, and the ellipsis indicates that extra additional genes, each with their own coefficient may be added. In a related embodiment, additional gene X is PRAME and additional gene Y is S100A9.

[0088] Furthermore, in yet other embodiments, the combined score is calculated according the following formula:

(3) Combined score = B* (immune score) + {C* (additional gene X expression) +D* additional gene Y expression...} + adjustment factor Where X and Y represent any diagnostic additional gene as described herein, and the ellipsis indicates that extra additional genes, each with their own coefficient may be added. The adjustment factor represents a scalar factor that can be used to adjust the linear score. For example in some embodiments, the adjustment factor can adjust the score of a particular cutoff value such that the cutoff value is centered at zero. In a related embodiment, additional gene X is PRAME and additional gene Y is S100A9.

[0089] Furthermore, in yet other embodiments, the combined score is calculated according the following formula:

(4) Combined score = B* (immune score) +{C*(S100 Score) +D* additional gene Y expression...} + adjustment factor

Where Y represents any diagnostic additional gene as described herein, and the ellipsis indicates that extra additional genes, each with their own coefficient may be added. The adjustment factor represents a scalar factor that can be used to adjust the linear score. For example in some embodiments, the adjustment factor can adjust the score of a particular cutoff value such that the cutoff value is centered at zero. The SI 00 score may be any value used to represent the expression of one or more S100A9 and/or S100A9 related genes as described herein. In some embodiments the SI 00 score is an average of the expression of the genes in an S100A9 and/or S100A9 related gene panel. In a related embodiment, the SI 00 score is an average of the expression of the genes in Panel J. In a related embodiment, the SI 00 score is an average of the expression of the genes in Panel L. In a related embodiment, the SI 00 score is an average of the expression of SI 00A9, S 100A7, S 100A8, S 100A12, PI3, S 100A10, and S 100A14. In a related embodiment, the S100 score is an average of the expression of S 100A9, S 100A7, S 100A8, S 100A12, and PI3. In a related embodiment, additional gene Y is PRAME.

[0090] In some embodiments, formula (1) is used in the methods, systems, etc. of the invention to diagnose a patient with melanoma. In some embodiments, formula (2) is used in the methods, systems, etc. of the invention to diagnose a patient with melanoma. In some embodiments CCP score is the unweighted mean of CT values for expression of the CCP genes being analyzed, optionally normalized by the unweighted mean of the HK genes so that higher values indicate higher expression (in some embodiments one unit is equivalent to a two-fold change in expression). In some embodiments the CCP score ranges from -8 to 8 or from -1.6 to 3.7.

[0091] In some embodiments A = 0.95, B = 0.61, C = 0.90 (where applicable), and D = 1.00 (where applicable); A = 0.57 and B = 0.39; or A = 0.58 and B = 0.41. In some

embodiments, A, B, C, and/or D is within rounding of these values (e.g., A is between 0.945 and 0.954, etc.). In some cases a formula may not have all of the specified coefficients (and thus not incorporate the corresponding variable(s)). For example, the embodiment mentioned

immediately previously may be applied to formula (2) so that B in formula (2) is 0.61, C = 0.90 (where applicable), and D = 1.00 (where applicable). A would not be applicable as this coefficient and its corresponding variable is not found in formula (2). In some embodiments A is between 0.9 and 1, 0.9 and 0.99, 0.9 and 0.95, 0.85 and 0.95, 0.86 and 0.94, 0.87 and 0.93, 0.88 and 0.92, 0.89 and 0.91, 0.85 and 0.9, 0.8 and 0.95, 0.8 and 0.9, 0.8 and 0.85, 0.75 and 0.99, 0.75 and 0.95, 0.75 and 0.9, 0.75 and 0.85, or between 0.75 and 0.8. In some embodiments B is between 0.40 and 1, 0.45 and 0.99, 0.45 and 0.95, 0.55 and 0.8, 0.55 and 0.7, 0.55 and 0.65, 0.59 and 0.63, or between 0.6 and 0.62. In some embodiments C is, where applicable, between 0.9 and 1, 0.9 and 0.99, 0.9 and 0.95, 0.85 and 0.95, 0.86 and 0.94, 0.87 and 0.93, 0.88 and 0.92, 0.89 and 0.91, 0.85 and 0.9, 0.8 and 0.95, 0.8 and 0.9, 0.8 and 0.85, 0.75 and 0.99, 0.75 and 0.95, 0.75 and 0.9, 0.75 and 0.85, or between 0.75 and 0.8. In some embodiments D is, where applicable, between 0.9 and 1, 0.9 and 0.99, 0.9 and 0.95, 0.85 and 0.95, 0.86 and 0.94, 0.87 and 0.93, 0.88 and 0.92, 0.89 and 0.91, 0.85 and 0.9, 0.8 and 0.95, 0.8 and 0.9, 0.8 and 0.85, 0.75 and

0.99, 0.75 and 0.95, 0.75 and 0.9, 0.75 and 0.85, or between 0.75 and 0.8.

[0092] In some embodiments A is between 0.1 and 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9,

1, 1.5,2,2.5,3,3.5,4,4.5,5,6, 7, 8,9, 10, 11, 12, 13, 14, 15, or 20; or between 0.2 and 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1, 1.5, 2, 2.5, 3, 3.5, 4, 4.5, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, or 20; or between 0.3 and 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1, 1.5,2,2.5,3,3.5,4,4.5,5,6, 7, 8,9, 10, 11, 12, 13, 14, 15, or 20; or between 0.4 and 0.5, 0.6, 0.7, 0.8, 0.9, 1, 1.5, 2, 2.5, 3, 3.5, 4, 4.5, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, or 20; or between 0.5 and 0.6, 0.7, 0.8, 0.9, 1, 1.5,2,2.5,3,3.5,4,4.5, 5,6, 7, 8,9, 10, 11, 12, 13, 14, 15, or 20; or between 0.6 and 0.7, 0.8, 0.9, 1, 1.5,2,2.5,3,3.5,4, 4.5,5,6,7,8,9, 10, 11, 12, 13, 14, 15, or 20; or between 0.7 and 0.8, 0.9, 1, 1.5,2,2.5,3,3.5,4, 4.5,5,6,7,8,9, 10, 11, 12, 13, 14, 15, or 20; or between 0.8 and 0.9, 1, 1.5,2,2.5,3,3.5,4,4.5, 5,6, 7, 8,9, 10, 11, 12, 13, 14, 15, or 20; or between 0.9 and 1, 1.5,2,2.5,3,3.5,4,4.5,5,6, 7, 8, 9, 10, 11, 12, 13, 14, 15, or 20; or between 1 and 1.5, 2, 2.5, 3, 3.5, 4, 4.5, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, or 20; or between 1.5 and 2, 2.5, 3, 3.5, 4, 4.5, 5, 6, 7, 8,9, 10, 11, 12, 13, 14, 15, or 20; or between 2 and 2.5, 3, 3.5, 4, 4.5, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, or 20; or between 2.5 and 3, 3.5,4, 4.5, 5, 6, 7, 8,9, 10, 11, 12, 13, 14, 15, or 20; or between 3 and 3.5, 4, 4.5, 5, 6, 7, 8,9, 10, 11, 12, 13, 14, 15, or 20; or between 3.5 and 4, 4.5, 5, 6, 7, 8,9, 10, 11, 12, 13, 14, 15, or 20; or between 4 and 4.5, 5, 6, 7, 8,9, 10, 11, 12, 13, 14, 15, or 20; or between 4.5 and 5, 6, 7, 8,9, 10, 11, 12, 13, 14, 15, or 20; or between 5 and 6, 7, 8,9, 10, 11, 12, 13, 14, 15, or 20; or between 6 and 7, 8,9, 10, 11, 12, 13, 14, 15, or 20; or between 7 and 8, 9, 10, 11, 12, 13, 14, 15, or 20; or between 8 and 9, 10, 11, 12, 13, 14, 15, or 20; or between 9 and 10, 11, 12, 13, 14, 15, or 20; or between 10 and 11, 12, 13, 14, 15, or 20; or between 11 and 12, 13, 14, 15, or 20; or between 12 and 13, 14, 15, or 20; or between 13 and 14, 15, or 20; or between 14 and 15, or 20; or between 15 and 20; B is between 0.1 and 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8,0.9, 1, 1.5,2,2.5, 3, 3.5,4,4.5,5,6, 7, 8,9, 10, 11, 12, 13, 14, 15, or 20; or between 0.2 and 0.3, 0.4, 0.5, 0.6, 0.7, 0.8,0.9, 1, 1.5,2,2.5,3,3.5,4,4.5,5,6, 7, 8,9, 10, 11, 12, 13, 14, 15, or 20; or between 0.3 and 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1, 1.5, 2, 2.5, 3, 3.5, 4, 4.5, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, or 20; or between 0.4 and 0.5, 0.6, 0.7, 0.8, 0.9, 1, 1.5,2,2.5,3,3.5,4,4.5,5,6, 7, 8,9, 10, 11, 12, 13, 14, 15, or 20; or between 0.5 and 0.6, 0.7, 0.8, 0.9, 1, 1.5,2,2.5,3,3.5,4,4.5,5,6, 7, 8,9, 10, 11, 12, 13, 14, 15, or 20; or between 0.6 and 0.7, 0.8, 0.9, 1, 1.5,2,2.5,3,3.5,4,4.5,5,6, 7, 8,9, 10, 11, 12, 13, 14, 15, or 20; or between 0.7 and 0.8, 0.9, 1, 1.5,2,2.5,3,3.5,4,4.5,5,6, 7, 8,9, 10,

11, 12, 13, 14, 15, or 20; or between 0.8 and 0.9, 1, 1.5,2,2.5,3,3.5,4,4.5,5,6, 7, 8,9, 10, 11,

12, 13, 14, 15, or 20; or between 0.9 and 1, 1.5,2,2.5,3,3.5,4,4.5,5,6, 7, 8,9, 10, 11, 12, 13, 14, 15, or 20; or between 1 and 1.5,2,2.5, 3, 3.5,4,4.5, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, or 20; or between 1.5 and 2, 2.5, 3, 3.5,4, 4.5, 5, 6, 7, 8,9, 10, 11, 12, 13, 14, 15, or 20; or between 2 and 2.5, 3, 3.5,4, 4.5, 5, 6, 7, 8,9, 10, 11, 12, 13, 14, 15, or 20; or between 2.5 and 3, 3.5, 4, 4.5,5,6, 7, 8,9, 10, 11, 12, 13, 14, 15, or 20; or between 3 and 3.5, 4, 4.5, 5, 6, 7, 8,9, 10, 11, 12, 13, 14, 15, or 20; or between 3.5 and 4, 4.5, 5, 6, 7, 8,9, 10, 11, 12, 13, 14, 15, or 20; or between 4 and 4.5, 5, 6, 7, 8,9, 10, 11, 12, 13, 14, 15, or 20; or between 4.5 and 5, 6, 7, 8,9, 10, 11, 12, 13, 14, 15, or 20; or between 5 and 6, 7, 8,9, 10, 11, 12, 13, 14, 15, or 20; or between 6 and 7, 8,9, 10, 11, 12, 13, 14, 15, or 20; or between 7 and 8, 9, 10, 11, 12, 13, 14, 15, or 20; or between 8 and 9, 10, 11, 12, 13, 14, 15, or 20; or between 9 and 10, 11, 12, 13, 14, 15, or 20; or between 10 and 11, 12, 13, 14, 15, or 20; or between 11 and 12, 13, 14, 15, or 20; or between 12 and 13, 14, 15, or 20; or between 13 and 14, 15, or 20; or between 14 and 15, or 20; or between 15 and 20; C is, where applicable, between 0.1 and 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1, 1.5,2, 2.5,3,3.5,4,4.5,5,6, 7, 8,9, 10, 11, 12, 13, 14, 15, or 20; or between 0.2 and 0.3, 0.4, 0.5, 0.6, 0.7,0.8,0.9, 1, 1.5,2,2.5,3,3.5,4,4.5,5,6, 7, 8,9, 10, 11, 12, 13, 14, 15, or 20; or between 0.3 and 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1, 1.5, 2, 2.5, 3, 3.5, 4, 4.5, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, or 20; or between 0.4 and 0.5, 0.6, 0.7, 0.8, 0.9, 1, 1.5,2,2.5,3,3.5,4,4.5,5,6, 7, 8,9, 10, 11, 12, 13, 14, 15, or 20; or between 0.5 and 0.6, 0.7, 0.8, 0.9, 1, 1.5,2,2.5,3,3.5,4,4.5,5,6, 7, 8,9, 10, 11, 12, 13, 14, 15, or 20; or between 0.6 and 0.7, 0.8, 0.9, 1, 1.5,2,2.5,3,3.5,4,4.5,5,6, 7, 8,9, 10, 11, 12, 13, 14, 15, or 20; or between 0.7 and 0.8, 0.9, 1, 1.5,2,2.5,3,3.5,4,4.5,5,6, 7, 8,9, 10, 11, 12, 13, 14, 15, or 20; or between 0.8 and 0.9, 1, 1.5,2,2.5,3,3.5,4,4.5,5,6, 7, 8,9,

10, 11, 12, 13, 14, 15, or 20; or between 0.9 and 1, 1.5,2,2.5,3,3.5,4,4.5,5,6, 7, 8,9, 10, 11,

12, 13, 14, 15, or 20; or between 1 and 1.5, 2, 2.5, 3, 3.5, 4, 4.5, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, or 20; or between 1.5 and 2, 2.5, 3, 3.5, 4, 4.5, 5, 6, 7, 8,9, 10, 11, 12, 13, 14, 15, or 20; or between 2 and 2.5, 3, 3.5,4, 4.5, 5, 6, 7, 8,9, 10, 11, 12, 13, 14, 15, or 20; or between 2.5 and 3, 3.5,4,4.5,5,6, 7, 8,9, 10, 11, 12, 13, 14, 15, or 20; or between 3 and 3.5, 4, 4.5, 5, 6, 7, 8,9, 10,

11, 12, 13, 14, 15, or 20; or between 3.5 and 4, 4.5, 5, 6, 7, 8,9, 10, 11, 12, 13, 14, 15, or 20; or between 4 and 4.5, 5, 6, 7, 8,9, 10, 11, 12, 13, 14, 15, or 20; or between 4.5 and 5, 6, 7, 8,9, 10, 11, 12, 13, 14, 15, or 20; or between 5 and 6, 7, 8,9, 10, 11, 12, 13, 14, 15, or 20; or between 6 and 7, 8,9, 10, 11, 12, 13, 14, 15, or 20; or between 7 and 8, 9, 10, 11, 12, 13, 14, 15, or 20; or between 8 and 9, 10, 11, 12, 13, 14, 15, or 20; or between 9 and 10, 11, 12, 13, 14, 15, or 20; or between 10 and 11, 12, 13, 14, 15, or 20; or between 11 and 12, 13, 14, 15, or 20; or between 12 and 13, 14, 15, or 20; or between 13 and 14, 15, or 20; or between 14 and 15, or 20; or between 15 and 20; and D is, where applicable, between 0.1 and 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1, 1.5, 2,2.5,3,3.5,4,4.5,5,6, 7, 8,9, 10, 11, 12, 13, 14, 15, or 20; or between 0.2 and 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1, 1.5, 2, 2.5, 3, 3.5, 4, 4.5, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, or 20; or between 0.3 and 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1, 1.5,2,2.5,3,3.5,4,4.5,5,6, 7, 8,9, 10, 11, 12,

13, 14, 15, or 20; or between 0.4 and 0.5, 0.6, 0.7, 0.8, 0.9, 1, 1.5, 2, 2.5, 3, 3.5, 4, 4.5, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, or 20; or between 0.5 and 0.6, 0.7, 0.8, 0.9, 1, 1.5,2,2.5,3,3.5,4,4.5, 5,6, 7, 8,9, 10, 11, 12, 13, 14, 15, or 20; or between 0.6 and 0.7, 0.8, 0.9, 1, 1.5,2,2.5,3,3.5,4, 4.5,5,6,7,8,9, 10, 11, 12, 13, 14, 15, or 20; or between 0.7 and 0.8, 0.9, 1, 1.5,2,2.5,3,3.5,4, 4.5,5,6,7,8,9, 10, 11, 12, 13, 14, 15, or 20; or between 0.8 and 0.9, 1, 1.5,2,2.5,3,3.5,4,4.5, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, or 20; or between 0.9 and 1, 1.5, 2, 2.5, 3, 3.5, 4, 4.5, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, or 20; or between 1 and 1.5, 2, 2.5, 3, 3.5, 4, 4.5, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, or 20; or between 1.5 and 2, 2.5, 3, 3.5, 4, 4.5, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, or 20; or between 2 and 2.5, 3, 3.5, 4, 4.5, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, or 20; or between 2.5 and 3, 3.5, 4, 4.5, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, or 20; or between 3 and 3.5, 4, 4.5, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, or 20; or between 3.5 and 4, 4.5, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, or 20; or between 4 and 4.5, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, or 20; or between 4.5 and 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, or 20; or between 5 and 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, or 20; or between 6 and 7, 8, 9, 10, 11, 12, 13, 14, 15, or 20; or between 7 and 8, 9, 10, 11, 12, 13, 14, 15, or 20; or between 8 and 9, 10, 11, 12, 13, 14, 15, or 20; or between 9 and 10, 11, 12, 13, 14, 15, or 20; or between 10 and 11, 12, 13, 14, 15, or 20; or between 11 and 12, 13, 14, 15, or 20; or between 12 and 13, 14, 15, or 20; or between 13 and 14, 15, or 20; or between 14 and 15, or 20; or between 15 and 20. In some embodiments, A, B, and/or C is within rounding of any of these values (e.g., A is between 0.45 and 0.54, etc.).

[0093] As used herein, a patient has an "increased likelihood" of some clinical feature or outcome (e.g., having melanoma) if the probability of the patient having the feature or outcome exceeds some reference probability or value. The reference probability may be the probability of the feature or outcome across the general relevant patient population. For example, if the probability of having melanoma in the general population is X% and a particular patient has been determined by the methods of the present invention to have a probability of Y% of having melanoma, and if Y > X, then the patient has an "increased likelihood" of having melanoma. Alternatively, as discussed above, a threshold or reference value may be determined and a particular patient's probability of having melanoma may be compared to that threshold or reference.

[0094] In some embodiments the method correlates the patient's specific score (e.g., CCP score, combined score of CCP with clinical variables) to a specific probability (e.g., 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99%, 100%) or likelihood of having melanoma.

[0095] In some embodiments, the method of diagnosis includes (1) obtaining a sample from a patient suspected of having melanoma; (2) determining the expression of a panel of genes in the sample; (3) calculating test values or scores; and (4) providing a report communicating the test value or scores. In some embodiments the report is a paper report, an auditory report, or an electronic record. In some embodiments the report is displayed and/or stored on a computing device (e.g., handheld device, desktop computer, smart device, website, etc.). In some embodiments the report is

communicated to a physician (e.g., a report communicating the test values or scores is provided to the physician). In some embodiments the report is communicated to a patient (e.g., a report communicating the test values or scores is provided to the patient). Providing a report can also be accomplished by transferring information (e.g., data) embodying the test values or scores to a server computer and allowing an intermediary or end-user to access such information (e.g., by viewing the information as displayed from the server, by downloading the information in the form of one or more files transferred from the server to the intermediary or end-user's device, etc.).

[0096] In other embodiments, the report may communicate scores derived from different sources and other relevant patient information. For example, the report may communicate scores derived solely from expression levels. The report may also report the scores as calculated by formula (1) formula (2), formula (3), and/or formula (4). Alternately, the report may communicate scores derived from a combination of expression value scores with other components (e.g. clinical staging, personal/family history, dermatopathology results, etc.) to give a potentially more comprehensive score. In other cases, the report can communicate multiple scores (e.g. expression test value and clinical parameters, such as clinical staging) and/or a more comprehensive score. The report can also communicate scores for individual genes. In some instances, the report can communicate scores along with control or reference values. Some reports may communicate a specific probability (e.g., 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99%, 100%) or likelihood of having melanoma. Other reports may communicate classification of the sample as benign or malignant, predictions of melanoma risk, comparisons of melanoma risk, clinically actionable items, recommendations for cancer risk management and/or

recommendations for treatment. Yet other reports may include personal and family medical history. Methods of Treating Melanoma

[0097] In one aspect, the present invention provides methods of treating a patient comprising obtaining gene expression status information for a panel of genes (e.g., obtained by the method described herein), and recommending a treatment, prescribing a treatment, administering a treatment, creating a treatment plan, or modifying a treatment plan for the patient based on the gene expression status. In some embodiments, the method comprises obtaining CCG expression status. In some embodiments, the method comprises obtaining immune gene expression status. In some embodiments, the method comprises obtaining expression status for additional genes as described herein. For example, the invention provides a method of treating a patient comprising:

(1) determining the status of at least one CCG;

(2) determining the status of at least one immune gene;

(3) determining the status of at least one additional gene; and

(4) recommending, prescribing or administering either

(a) an active (including aggressive) treatment if the patient has at least one of increased expression of the CCG, increased expression of immune gene, or expression of an additional gene that differs significantly from expression in a control sample, or

(b) a passive (or less aggressive) treatment if the patient has none of increased expression of the CCG, increased expression of immune gene, or expression of an additional gene that differs significantly from expression in a control sample.

[0098] In a related embodiment, the invention provides a method of treating a patient comprising:

(1) determining the status of at least one immune gene;

(2) determining the status of at least one additional gene; and

(3) recommending, prescribing or administering either

(a) an active (including aggressive) treatment if the patient has at least one of increased expression of immune gene, or expression of an additional gene that differs significantly from expression in a control sample, or (b) a passive (or less aggressive) treatment if the patient has none of increased expression of immune gene, or expression of an additional gene that differs significantly from expression in a control sample.

[0099] In a related embodiment, the invention provides a method of treating a patient comprising:

(1) determining the status of at least one immune gene;

(2) determining the status of at least one additional gene; and

(3) creating a treatment plan comprising either

(a) more aggressive therapy components if the patient has at least one of increased expression of immune gene, or expression of an additional gene that differs significantly from expression in a control sample, or

(b) less aggressive therapy components if the patient has none of increased expression of immune gene, or expression of an additional gene that differs significantly from expression in a control sample; and

(4) implementing the treatment plan.

[00100] In some embodiments, the recommending, prescribing, or administering steps comprise receiving a report communicating the relevant expression status (e.g., CCG status). In some embodiments, the creating a treatment plan step comprises receiving a report

communicating the relevant expression status (e.g., CCG status). In some embodiments this report communicates such status in a qualitative manner (e.g., "high" or "increased" expression). In some embodiments this report communicates such status indirectly by communicating a test value or score (e.g., score reflecting likelihood of having melanoma, etc.) that incorporates such status.

[00101] Whether a treatment is aggressive or not will generally depend on the diagnosis or likelihood of having melanoma. For individuals diagnosed with melanoma, or having a high likelihood of having melanoma, aggressive treatment is preferred. Those skilled in the art are familiar with various other aggressive and less aggressive treatments for each type of cancer. On the other hand, if an individual has a low likelihood of having melanoma, a less aggressive therapy could be prescribed. Therefore, for an individual having a low risk of having melanoma, a medical provider could recommend a regime of "watchful- waiting." [00102] A range of melanoma treatments and/or therapies are known by those skilled in the art. This range of melanoma therapies can vary in their aggressiveness. In general, as the melanoma therapy increases in aggressiveness, the effectiveness of the treatment increases, but the adverse effects to the patient also increases. Skilled artisans can understand that the aggressiveness of the melanoma therapy that is used to treat the patient must be balanced to take into account both the effectiveness of the treatment and the adverse effects that will likely be experienced by the patient. Therefore, the skilled artisan will seek to maximize the effectiveness of the treatment while minimizing the adverse effects of the treatment by selecting an appropriate level of aggressiveness tailored to the individual patient. The appropriate level of treatment can be selected based at least in part on the report communicating the relevant expression status.

[00103] In some embodiments, a skilled artisan can incorporate the report

communicating the relevant expression status into the selection of aggressiveness of treatment. A report communicating a score indicating a high likelihood of melanoma would indicate a more aggressive treatment while a report communicating a score indicating a lower likelihood of melanoma would indicate a less aggressive treatment. In some embodiments, a less aggressive treatment may comprise the removal of the suspected melanoma during a biopsy. In some embodiments, a more aggressive treatment may include removal of the suspected melanoma as well as removal of a small border of normal skin and a layer of tissue beneath both the suspected melanoma and the small border of skin. In other embodiments, a more aggressive treatment may comprise a reexcision of the biopsy site to remove additional tissue that borders the removed biopsy sample. In yet other embodiments, a more aggressive treatment may comprise reexcision of additional tissue surrounding the biopsy site.

[00104] In other embodiments an even more aggressive treatment may include surgery to remove any affected lymph nodes or a lymph node dissection (lymphadenoectomy). In other embodiments an even more aggressive treatment may include surgery to remove any affected lymph nodes or a lymph node dissection (lymphadenoectomy) followed by adjuvant therapy with interferon. In other embodiments, an even more aggressive treatment may include surgery to remove any affected lymph nodes as well as additional treatments such as chemotherapy and/or radiation therapy. In other embodiments, an even more aggressive treatment can include surgery to remove any affected tissue or organs. In alternate embodiments, even more aggressive treatments can include chemotherapy. Some methods of administering chemotherapy include oral and intravenous treatments. Some methods of administering chemotherapy include isolated limb perfusion of chemotherapy drugs. In other embodiments, even more aggressive treatments can include radiation therapy. In yet other embodiments, even more aggressive treatments can include biological therapy. Some biological therapies can include interferon and/or interleukin- 2. Some biological therapies can include antibody-based therapies such as ipilimumab (Yervoy). In other embodiments, even more aggressive therapy can include immunotherapy. Some immunotherapies can include Interferon-alpha, Anti-CTLA-4, vaccines, Bacille Calmette-Guerin vaccine, Interleukin 2, and/or T-cell therapy. Some immunotherapies can also be combined with chemotherapy and/or radiation therapy. In alternate embodiments, even more aggressive therapies can include targeted therapy. Some targeted therapies can include drugs such as vemurafenib (Zelboraf) used to treat advanced melanoma. Other targeted therapies include B- RAF inhibitors and/or KIT inhibitors.

[00105] In some embodiments, the selection of the specific treatment can be based in part on the relative expression status of the tested genes. For example in some embodiments, the expression profile of the individual genes within the panel would be indicative of the selection of the type and/or the class of therapy. A certain expression profile of the individual genes within the panel may be indicative of melanoma that can be effectively treated with surgery alone. On the other hand, another expression profile may be indicative of melanoma that can be effectively treated with surgery combined with radiation therapy. In other cases, another expression profile may be indicative of melanoma that can be effectively treated with surgery combined with chemotherapy. In yet other cases, the expression profile may be indicative of melanoma that can be effectively treated with only careful monitoring and regular follow-up. In alternate embodiments, the expression profile might be indicative of melanoma that can be effectively treated with higher dosages of therapy administered at shorter intervals whereas other expression profiles might be indicative of lower dosages of therapy administered at longer intervals. In some embodiments, the expression profile may indicate certain combinations, dosages, and/or frequencies of therapies.

[00106] In other embodiments, a skilled artisan can utilize at least in part the report communicating the relevant expression status to guide the selection of appropriate melanoma drugs. For example in some embodiments, the expression profile of the individual genes within the panel may be indicative of the selection of particular melanoma drugs. In some embodiments melanoma drugs may be selected from Aldesleukin, Dabrafenib, Dacarbazine, DTIC-Dome (Dacarbazine), Intron A (Recombinant Interferon Alfa-2b), Ipilimumab, Mekinist (Trametinib), Peginterferon Alfa-2b, PEG-Intron (Peginterferon Alfa-2b), Proleukin (Aldesleukin),

Recombinant Interferon Alfa-2b, Sylatron (Peginterferon Alfa-2b), Tafmlar (Dabrafenib), Trametinib, Vemurafenib, Yervoy (Ipilimumab), and/or Zelboraf (Vemurafenib). In other embodiments, melanoma drugs may be selected from Bacille Calmette-Guerin (BCG) vaccine, interleukin-2, imiquimod, cytokines, dacarbazine (DTIC), temozolomide (Temodar), mitogen- activated protein kinase kinase (MEK) inhibitor (trametinib), and/or beta-adrenergic-blocking drugs.

[00107] In some embodiments, patients with melanoma can be treated by selecting the relative aggressiveness of the melanoma therapy based at least in part on the report

communicating the relevant expression status (or test values or scores) and then administering this selected therapy. In other embodiments, patients with melanoma can be treated by selecting the relative aggressiveness of the melanoma therapy based at least in part on the report communicating the relevant expression status, administering this selected therapy, measuring the relevant expression status again, comparing the latter expression status with the previous expression status, and continuing or modifying treatment based on the comparison of the previous and latter expression status. In alternate embodiments, the comparison of the relevant expression status can be used to monitor the efficacy of treatment. In some cases a change in the relevant expression status from a score indicative of a higher likelihood of melanoma to a score indicative of a lower likelihood of melanoma may indicate that the treatment is effective. In other cases a change in the relevant expression status from a score indicative of a lower likelihood of melanoma to a score indicative of a higher likelihood of melanoma may indicate that the treatment is less effective. Where a change in the expression status indicates an effective treatment the treatment may be continued or modified to comprise a less aggressive treatment. Where a change in the expression status indicates a less effective treatment the treatment may be continued or modified to comprise a more aggressive treatment.

[00108] In yet other embodiments, the skilled artisan can create or modify a treatment plan for the individual patient based at least in part on the report communicating the relevant expression status. In some embodiments, the selection of different therapy components that comprise the treatment plan can be based at least in part on the report communicating the relevant expression status (or test values or scores). For example, in some instances, the report will indicate a low likelihood of melanoma and the treatment plan may comprise less aggressive therapy components. The less aggressive therapy components can include removal of the suspected melanoma and follow up monitoring of the patient. In other cases, the report may indicate a high likelihood of melanoma and the treatment plan may comprise more aggressive therapy components. Components of a more aggressive treatment plan can include removal of the suspected melanoma and surrounding tissue, reexcision of the biopsy site to remove additional surrounding tissue, chemotherapy, radiation therapy and/or biological therapy. In alternate embodiments, the report communicating the relevant expression status can be used in part to select the different elements within each component of the treatment plan. As a non- limiting example, the report can be used to select individual melanoma drugs that comprise the chemotherapy component of the treatment plan. In other non-limiting examples, the report can be used to select the types of radiation, the amounts of radiation, and/or the dosing regime of the radiation component of the treatment plan. In some embodiments, the treatment plan can further comprise continued measurement of the relevant expression status to determine the efficacy of the treatment plan. In other embodiments, this continued measurement of the efficacy of treatment can be used to modify the treatment plan.

Systems for Diagnosing and Treating Melanoma

[00109] The results of any analyses according to the invention will often be

communicated to physicians, genetic counselors and/or patients (or other interested parties such as researchers) in a transmittable form that can be communicated or transmitted to any of the above parties. Such a form can vary and can be tangible or intangible. The results can be embodied in descriptive statements, diagrams, photographs, charts, images or any other visual forms. For example, graphs showing expression or activity level or sequence variation information for various genes can be used in explaining the results. Diagrams showing such information for additional target gene(s) are also useful in indicating some testing results. The statements and visual forms can be recorded on a tangible medium such as papers, computer readable media such as floppy disks, compact disks, etc., or on an intangible medium, e.g., an electronic medium in the form of email or website on internet or intranet. In addition, results can also be recorded in a sound form and transmitted through any suitable medium, e.g., analog or digital cable lines, fiber optic cables, etc., via telephone, facsimile, wireless mobile phone, internet phone and the like.

[00110] Thus, the information and data on a test result can be produced anywhere in the world and transmitted to a different location. As an illustrative example, when an expression level, activity level, or sequencing (or genotyping) assay is conducted outside the United States, the information and data on a test result may be generated, cast in a transmittable form as described above, and then imported into the United States. Accordingly, the present invention also encompasses a method for producing a transmittable form of information on at least one of (a) expression level or (b) activity level for at least one patient sample. The method comprises the steps of (1) determining at least one of (a) or (b) above according to methods of the present invention; and (2) embodying the result of the determining step in a transmittable form. The transmittable form is the product of such a method.

[00111] Techniques for analyzing such expression, activity, and/or sequence data (indeed any data obtained according to the invention) will often be implemented using hardware, software or a combination thereof in one or more computer systems or other processing systems capable of effectuating such analysis.

[00112] Thus, the present invention further provides a system for determining gene expression in a tumor sample, comprising: (1) a sample analyzer for determining the expression levels of a panel of genes in a patient sample, wherein the sample analyzer contains the patient sample, or cDNA molecules from mRNA expressed of the panel of genes derived from the sample; (2) a first computer program for (a) receiving gene expression data from the panel of genes, (b) weighting the determined expression of each of the test genes, and (c) combining the weighted expression to provide a test value; and optionally (3) a second computer program for comparing the test value to one or more reference values each associated with a predetermined degree of risk of melanoma.

[00113] In another embodiment, the amount of RNA transcribed from the panel of genes including test genes is measured in the sample. In addition, the amount of RNA of one or more housekeeping genes in the sample is also measured, and used to normalize or calibrate the expression of the test genes, as described above. [00114] The sample analyzer can be any instruments useful in determining gene expression, including, e.g., a sequencing machine, a real-time PCR machine, and a microarray instrument.

[00115] The computer-based analysis function can be implemented in any suitable language and/or browsers. For example, it may be implemented with C language and preferably using object-oriented high-level programming languages such as Visual Basic, SmallTalk, C++, and the like. The application can be written to suit environments such as the Microsoft

Windows™ environment including Windows™ 98, Windows™ 2000, Windows™ NT, and the like. In addition, the application can also be written for the Macintosh™, SUN™, UNIX or LINUX environment. In addition, the functional steps can also be implemented using a universal or platform-independent programming language. Examples of such multi-platform programming languages include, but are not limited to, hypertext markup language (HTML), JAVA™, JavaScript™, Flash programming language, common gateway interface/structured query language (CGI/SQL), practical extraction report language (PERL), AppleScript™ and other system script languages, programming language/structured query language (PL/SQL), and the like. Java™- or JavaScript™-enabled browsers such as HotJava™, Microsoft™ Explorer™, or Netscape™ can be used. When active content web pages are used, they may include Java™ applets or ActiveX™ controls or other active content technologies.

[00116] The analysis function can also be embodied in computer program products and used in the systems described above or other computer- or internet-based systems. Accordingly, another aspect of the present invention relates to a computer program product comprising a computer-usable medium having computer-readable program codes or instructions embodied thereon for enabling a processor to carry out gene status analysis. These computer program instructions may be loaded onto a computer or other programmable apparatus to produce a machine, such that the instructions which execute on the computer or other programmable apparatus create means for implementing the functions or steps described above. These computer program instructions may also be stored in a computer-readable memory or medium that can direct a computer or other programmable apparatus to function in a particular manner, such that the instructions stored in the computer-readable memory or medium produce an article of manufacture including instructions which implement the analysis. The computer program instructions may also be loaded onto a computer or other programmable apparatus to cause a series of operational steps to be performed on the computer or other programmable apparatus to produce a computer implemented process such that the instructions which execute on the computer or other programmable apparatus provide steps for implementing the functions or steps described above.

[00117] Thus one aspect of the present invention provides a system for determining whether a patient has increased likelihood of having melanoma. Generally speaking, the system comprises (1) computer program for receiving, storing, and/or retrieving a patient's gene status data (e.g., expression level, activity level, variants) and optionally clinical parameter data (e.g., clinical staging); (2) computer program for querying this patient data; (3) computer program for concluding whether there is an increased likelihood of having melanoma based on this patient data; and optionally (4) computer program for outputting/displaying this conclusion. In some embodiments this computer program for outputting the conclusion may comprise a computer program for informing a health care professional of the conclusion

[00118] The practice of the present invention may also employ conventional biology methods, software and systems. Computer software products of the invention typically include computer readable media having computer-executable Instructions for performing the logic steps of the method of the invention. Suitable computer readable medium include floppy disk, CD- ROM/DVD/DVD-ROM, hard-disk drive, flash memory, ROM/RAM, magnetic tapes and etc. Basic computational biology methods are described in, for example, Setubal et al, Introduction to Computational Biology Methods (PWS Publishing Company, Boston, 1997); Salzberg et al. (Ed.), Computational Methods in Molecular Biology, (Elsevier, Amsterdam, 1998); Rashidi & Buehler, Bioinformatics Basics: Application in Biological Science and Medicine (CRC Press, London, 2000); and Ouelette & Bzevanis, Bioinformatics: A Practical Guide for Analysis of Gene and Proteins (Wiley & Sons, Inc., 2nd ed., 2001); see also, U.S. Pat. No. 6,420,108.

[00119] The present invention may also make use of various computer program products and software for a variety of purposes, such as probe design, management of data, analysis, and instrument operation. See U.S. Pat. Nos. 5,593,839; 5,795,716; 5,733,729; 5,974,164;

6,066,454; 6,090,555; 6,185,561; 6,188,783; 6,223,127; 6,229,911 and 6,308,170. Additionally, the present invention may have embodiments that include methods for providing genetic information over networks such as the Internet as shown in U.S. Ser. Nos. 10/197,621 (U.S. Pub. No. 20030097222); 10/063,559 (U.S. Pub. No. 20020183936), 10/065,856 (U.S. Pub. No.

20030100995); 10/065,868 (U.S. Pub. No. 20030120432); 10/423,403 (U.S. Pub. No.

20040049354).

[00120] Techniques for analyzing such expression, activity, and/or sequence data (indeed any data obtained according to the invention) will often be implemented using hardware, software or a combination thereof in one or more computer systems or other processing systems capable of effectuating such analysis.

[00121] Thus one aspect of the present invention provides systems related to the above methods of the invention. In one embodiment the invention provides a system for determining gene expression in a sample, comprising:

(1) a sample analyzer for determining the expression levels in a sample of a panel of genes, wherein the sample analyzer contains the sample, RNA from the sample and expressed from the panel of genes, or DNA synthesized from said RNA;

(2) a first computer program for

(a) receiving gene expression data on one or more test genes selected from the panel of genes,

(b) weighting the determined expression of each of the one or more test genes with a predefined coefficient, and

(c) combining the weighted expression to provide a test value; and optionally

(3) a second computer program for comparing the test value to one or more reference values each associated with a predetermined degree of risk of having melanoma.

[00122] In another embodiment the invention provides a system for determining gene expression in a sample, comprising: (1) a sample analyzer for determining the expression levels of a panel of genes in a sample, wherein the sample analyzer contains the sample which is a nevus or mole suspected of having melanoma, RNA from the sample and expressed from the panel of genes, or DNA synthesized from said RNA; (2) a first computer program for (a) receiving gene expression data on one or more test genes selected from the panel of genes, (b) weighting the determined expression of each of the test genes with a predefined coefficient, and (c) combining the weighted expression to provide a test value, wherein the test genes comprise immune genes and additional genes; and optionally (3) a second computer program for comparing the test value to one or more reference values each associated with a predetermined degree of risk of having melanoma. In some embodiments, the system further comprises a display module displaying the comparison between the test value and the one or more reference values, or displaying a result of the comparing step, or displaying the patient's diagnosis and/or degree of risk of having melanoma.

[00123] In a preferred embodiment, the amount of R A transcribed from the panel of genes including test genes (and/or DNA reverse transcribed therefrom) is measured in the sample. In addition, the amount of RNA of one or more housekeeping genes in the sample (and/or DNA reverse transcribed therefrom) is also measured, and used to normalize or calibrate the expression of the test genes, as described above.

[00124] The sample analyzer can be any instrument useful in determining gene expression, including, e.g., a sequencing machine (e.g., Illumina HiSeq™, Ion Torrent PGM, ABI SOLiD™ sequencer, PacBio RS, Helicos Heliscope™, etc.), a real-time PCR machine (e.g., ABI 7900, Fluidigm BioMark™, etc.), a microarray instrument, etc.

[00125] Figure 1 illustrates a system 100 for performing computer-assisted methods of diagnosing, detecting, screening and/or treating melanoma in a patient.

[00126] System 100 comprises a patient/medical provider interface module 10 comprising a medical provider 1 1 and a patient 12. The medical provider 1 1 comprises a doctor and/or other medical staff that care for patient 12. The medical provider collects a complete medical history from patient 12 including but not limited to symptoms, past medical history, and/or family history. The medical provider 1 1 also conducts a physical examination of the patient 12 and obtains a sample of the patient 12.

[00127] System 100 further comprises a data processing device 20 comprising a sample analyzer module 21. The sample of the patient is conveyed from patient/medical provider interface module 10 to the sample analyzer device 20. The sample analyzer module 21 determines the gene expression levels of a panel of biomarkers in the patient sample. The panel of biomarkers may comprise biomarkers useful for determining the presence of melanoma in the patient 12. The panel of biomarkers may further comprise housekeeper genes useful for normalizing the levels of biomarker panel. The sample analyzer module 21 can comprise any instrument useful in determining gene expression levels including, e.g. a sequencing machine (e.g., Illumina HiSeq™, Ion Torrent PGM, ABI SOLiD™ sequencer, PacBio RS, Helicos Heliscope™, etc.), a real-time PCR machine (e.g., ABI 7900, Fluidigm BioMark™, etc.), a microarray instrument, etc.

[00128] System 100 further comprises a data processing device 30 comprising a medical history database module 31. The medical history database module 31 may comprise a complete medical history from patient 12 including family history

information comprising the number of family members of a patient diagnosed with cancer, including melanoma. The family history information may also comprise the degree of relationship to a patient of each family member diagnosed with cancer, including melanoma. The medical history database module 31 may be in communication with patient/medical provider interface module 10. The medical history database module 31 may be configured to receive the patient's medical history from

patient/medical provider interface module 10 either as a physical record or as an electronic transmission.

[00129] The system 100 further comprises a data processing device 40 comprising a patient information database module 41. The patient information database module 41 comprises patient information comprising gene expression levels of a panel of biomarkers of the patient 12. The patient information database module 41 may be in communication with sample analyzer module 41. The patient information database module 41 may be configured to receive the patient's gene expression levels from the sample analyzer module 21 either as a physical record or as an electronic transmission.

[00130] System 100 further comprises a data processing device 50 and a data processing device 60. Data processing device 50 comprises a scoring module 51. Data processing device 60 comprises a biomarker information database module 61. The biomarker information database module 61 comprises biomarker information

comprising threshold level information for each biomarker of a panel of biomarkers, wherein the panel of biomarkers comprises positive biomarkers, negative biomarkers, or both, wherein a level statistically significantly above a threshold level for each particular positive biomarker is indicative of melanoma in a patient and a level statistically significantly below a threshold level for each particular negative biomarker is indicative of the presence of melanoma in a patient.

[00131] The scoring module 51 may be in communication with the biomarker information database module 61 and the patient information database module 41. The scoring module 51 may be configured to compare biomarker information and patient information to generate a score representing the comparison between the biomarker information and the patient information. The scoring module 51 may be further configured to normalize, average, and apply weighting of sub-groups of biomarkers during the generation of the score. The scoring module 51 may be further configured to algebraically add and/or subtract subgroups of biomarkers during the generation of the score.

[00132] The data processing device 50 may further comprise an evaluation module 52 in communication with the scoring module 51. The evaluation module 22 may further be in communication with the biomarker information database module 61. The evaluation module 52 may further be in communication the medical history database module 31. The evaluation module 52 may be configured to determine a probability of the presence of melanoma in the patient based on the patient score as compared to scores of groups of patients diagnosed with melanoma and scores of groups of patients that were not diagnosed with melanoma. The evaluation module 52 may be further configured to determine a probability of the presence of melanoma in the patient based on the patient score and the patient information. The data processing device 50 may further comprise a diagnostic module 53 in communication with the evaluation module 52. The diagnostic module 53 may be configured to determine additional suggested diagnostic procedures based on a patient's probability of melanoma. The data processing device 50 may further comprise a report generation module 54. The report generation module 54 can comprise any device that aggregates the data and process of data processing device 50 into a report. The report produced by report generation module 54 can comprise the score. The report can further comprise the probability of the presence of melanoma. The report can further comprise additional suggested diagnostic procedures. The report can further comprise suggested treatments.

[00133] System 100 further comprises a data processing device 70. Data processing device 70 comprises communication means 71. Communication means 71 is in communication with report generation module 54 and patient/medical provider interface module 10. Communication means 71 is configured to transmit the report generated by report generation module 54 to the patient/medical provider interface module 10. The report can further be transmitted by electronic means to the

patient/medical provider interface module 10. Communication means 71 can also transmit the report by printing on a tangible medium such as paper and conveying the report to patient/medical provider interface module 10. Upon receiving the transmitted report, the medical provider 1 1 can treat the patient 12 according to the information in the report. The medical provider 1 1 can further diagnose the patient 12 based on the report. The medical provider 1 1 can further create or modify a treatment plan for the patient 12 based on the report. The medical provider 1 1 can further follow the suggested additional diagnostic test from the report and/or follow the suggested treatments in the report.

[00134] Accordingly, the various components, modules, systems, and/or features disclosed herein may be embodied as modules within a system. Such a system may be implemented in software, firmware, hardware, and/or physical infrastructure. Although not always explicitly named herein, a module may be identified (named) based on a function it performs. For example, a module that is configured to calculate something may comprise specific hardware, software, or firmware and be properly referred to as a "calculation module."

[00135] Embodiments may also be provided as a computer program product including a non-transitory machine-readable medium having stored thereon instructions that may be used to program, or be executed on, a computer (or other electronic device) to perform processes described herein. The machine-readable medium may include, but is not limited to, hard drives, floppy diskettes, optical disks, CD-ROMs, DVD-ROMs, ROMs, RAMs, EPROMs, EEPROMs, magnetic or optical cards, solid-state memory devices, or other types of media/machine-readable media suitable for storing electronic instructions. Moreover, a computer program product may be run, executed, downloaded, and/or otherwise used locally or remotely via a network.

[00136] It should be understood that references to "a data processing device" may refer to the same device or one or more different devices. For example, certain steps of the computer-assisted methods may be performed on a device controlled by a diagnostic service provider and other steps may be performed on a device controlled by a medical practitioner. Likewise, the data processing devices 10, 20, 30, 40, 50, 60, and 70 may be a single device or, for example, the data processing device 50 may be multiple data processing devices.

[00137] In certain embodiments, the computer-implemented method may be configured to identify a patient as having or not having pancreatic cancer. For example, the computer-implemented method may be configured to inform a physician that a particular patient has pancreatic cancer. Alternatively or additionally, the computer- implemented method may be configured to actually suggest a particular course of treatment based on the answers to/results for various queries.

Probes and Kits

[00138] In some embodiments the invention provides a probe comprising an isolated oligonucleotide capable of selectively hybridizing to at least one of the genes in Table 1 , 3 or Panels A through I. The terms "probe" and "oligonucleotide" (also "oligo"), when used in the context of nucleic acids, interchangeably refer to a relatively short nucleic acid fragment or sequence. The invention also provides primers useful in the methods of the invention.

"Primers" are probes capable, under the right conditions and with the right companion reagents, of selectively amplifying a target nucleic acid (e.g., a target gene). In the context of nucleic acids, "probe" is used herein to encompass "primer" since primers can generally also serve as probes.

[00139] The probe can generally be of any suitable size/length. In some embodiments the probe has a length from about 8 to 200, 15 to 150, 15 to 100, 15 to 75, 15 to 60, or 20 to 55 bases in length. They can be labeled with detectable markers with any suitable 7etection marker including but not limited to, radioactive isotopes, fluorophores, biotin, enzymes (e.g., alkaline phosphatase), enzyme substrates, ligands and antibodies, etc. See Jablonski et al, Nucleic Acids Res. (1986) 14:6115-6128; Nguyen et al, Biotechniques (1992) 13: 116-123; Rigby et al, J. Mol. Biol. (1977) 113:237-251. Indeed, probes may be modified in any conventional manner for various molecular biological applications. Techniques for producing and using such

oligonucleotide probes are conventional in the art.

[00140] Probes according to the invention can be used in the

hybridization/amplification/detection techniques discussed above. Thus, some embodiments of the invention comprise probe sets suitable for use in a microarray. In some embodiments the probe sets have a certain proportion of their probes directed to CCGs— e.g., a probe set consisting of 10%, 20%, 30%, 40%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%o, 97%), 98%), 99%), or 100% probes specific for CCGs. In some embodiments the probe set comprises probes directed to at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 40, 45, 50, 60, 70, 80, 90, 100, 125, 150, or more, or all, of the genes in Table 1, 3 or Panels A through I, or any of the panels disclosed in Tables WW-ZZ. Such probe sets can be incorporated into high-density arrays comprising 5,000, 10,000, 20,000, 50,000, 100,000, 200,000, 300,000, 400,000, 500,000, 600,000, 700,000, 800,000, 900,000, or 1,000,000 or more different probes.

[00141] In another aspect of the present invention, a kit is provided for practicing the diagnosis of the present invention. The kit may include a carrier for the various components of the kit. The carrier can be a container or support, in the form of, e.g., bag, box, tube, rack, and is optionally compartmentalized. The carrier may define an enclosed confinement for safety purposes during shipment and storage. The kit includes various components useful in

determining the status of one or more panels of genes as described herein, and one or more housekeeping gene markers, using the above-discussed detection techniques. For example, the kit many include oligonucleotides specifically hybridizing under high stringency to mRNA or cDNA of the genes in Table 1, 3 or Panels A through I, or any of the panels disclosed in Tables WW-ZZ. Such oligonucleotides can be used as PCR primers in RT-PCR reactions, or hybridization probes. In some embodiments the kit comprises reagents (e.g., probes, primers, and or antibodies) for determining the expression level of a panel of genes, where said panel comprises at least 25%, 30%, 40%, 50%, 60%, 75%, 80%, 90%, 95%, 99%, or 100% CCGs. In some embodiments the kit consists of reagents (e.g., probes, primers, and or antibodies) for determining the expression level of no more than 2500 genes, wherein at least 5, 10, 15, 20, 30, 40, 50, 60, 70, 80, 90, 100, 120, 150, 200, 250, or more of these genes are CCGs.

[00142] The oligonucleotides in the detection kit can be labeled with any suitable detection marker including but not limited to, radioactive isotopes, fluorophores, biotin, enzymes (e.g., alkaline phosphatase), enzyme substrates, ligands and antibodies, etc. See Jablonski et al, Nucleic Acids Res., 14:6115-6128 (1986); Nguyen et al, Biotechniques, 13:116-123 (1992); Rigby et al, J. Mol. Biol, 113:237-251 (1977). Alternatively, the oligonucleotides included in the kit are not labeled, and instead, one or more markers are provided in the kit so that users may label the oligonucleotides at the time of use.

[00143] In another embodiment of the invention, the detection kit contains one or more antibodies selectively immunoreactive with one or more proteins encoded by any of the genes in Table 1, 3 or Panels A through I, or any of the panels disclosed in Tables WW-ZZ.

[00144] Various other components useful in the detection techniques may also be included in the detection kit of this invention. Examples of such components include, but are not limited to, Taq polymerase, deoxyribonucleotides, dideoxyribonucleotides, other primers suitable for the amplification of a target DNA sequence, RNase A, and the like. In addition, the detection kit preferably includes instructions on using the kit for practice the diagnosis method of the present invention using human samples.

Examples

Example 1

[00145] In this example we determined whether cell cycle progression genes can differentiate between malignant melanoma and non-malignant nevi. Specifically, this example assesses whether melanoma can be differentiated from benign nevi, as well as dysplastic nevi.

Materials and Methods

[00146] Samples: 31 FFPE skin samples, with 3 x 10 mm slides for each sample, and a 4 mm slide for H&E review between the ages of 10-11 years old were obtained from an academic institution. The samples consisted of 11 benign nevi, 10 dysplastic nevi and 10 melanoma samples. Table 4 lists samples and corresponding clinical details.

Table 4. Skin samples with diagnosis, clinical data, and CCP score.

Junctional

01095554- MG12- Very

dysplastic 0.70 21.11 0.02 2/21 2001 M 45 Chest BLD 38 Good

nevus

Junctional

01095562- MG12- Very

dysplastic 1.45 21.86 0.04 2/14/2001 F 40 Back BLD 37 Good

nevus

Junctional

01095578- MG12- Very

dysplastic 1.28 20.47 0.05 1/17/2001 F 69 Thigh BLD 43 Good

nevus

01095621- MG12- Nodular 11/13/200

NA 30.34 NA Rejected F 9 Back BLD 46 melanoma 1

01095551- MG12- Very

SSM 1.50 21.13 0.01 9/26/2001 M 51 Arm BLD 50 Good

01095555- MG12- Very

SSM 1.28 19.73 0.07 7/1/2002 M 61 Back BLD 53 Good

01095557- MG12- Very

SSM 2.20 21.07 0.06 6/24/2001 F 36 Chest BLD 52 Good

01095558- MG12- Very

SSM 1.67 19.19 0.03 1/4/2001 F 68 Arm BLD 44 Good

01095561- MG12- Very

SSM 1.76 20.20 0.03 3/1/2001 M 36 Back BLD 51 Good

01095573- MG12- Very

SSM 1.67 20.02 0.05 3/14/2001 F 36 Knee BLD 45 Good

01095575- MG12- Very

SSM 2.39 19.54 0.04 8/16/2001 F 38 Back BLD 49 Good

01095623- MG12- Very

SSM 2.12 19.82 0.02 7/21/2001 M 38 Shoulder BLD 48 Good

01095625- MG12- Very

SSM 2.01 20.46 0.05 8/7/2001 M 43 shoulder BLD 47 Good

SSM = superficial spreading melanoma

[00147] Sample Processing, CCP score generation, and analysis: 4 mm slides were stained to make H&E slides and reviewed by a pathologist who circled the lesion (either the nevus or the melanoma). Using the H&E slide, the lesions were dissected and removed from each of the three 10 μΜ slides. All three dissected lesions from a single patient were pooled.

[00148] R A was extracted from samples and RNA expression levels were determined using standard qPCR techniques. Of the 31 samples, 30 were successfully run and generated a CCP score (see Table 4 for data).

[00149] CCP scores of the melanoma samples were compared to the other two groups, benign nevi and dysplastic nevi, as well as to dysplastic nevi alone, using the Student's t-test, to determine if the CCP scores of the groups were different in a statistically significant manner.

Results [00150] We observed the melanoma samples had different CCP scores than both nevi subgroups combined, in a very statistically significant manner (p-value, 1.4 x 10-6, see Figure 2). When using this data in a diagnostic model, the melanoma samples could be identified with an AUC of 0.97. On average, melanoma CCP scores were 1.08 higher than nevi CCP scores. See Table 4 for a list of all data. Figure 2 shows the distribution of the CCP scores from all 30 samples with a score, separated by the clinical diagnosis.

[00151] We also observed the melanoma samples had different CCP scores compared to just dysplastic nevi, in a statistically significant manner (p-value, 3.9 x 10-5, see Figure 2). When using this data in a diagnostic model, the melanoma samples could be identified, compared to just the dysplastic nevi, with an AUC of 0.97. On average, melanoma CCP scores were 0.94 higher than nevi CCP scores.

[00152] Finally, we observed the benign nevi were not statistically different than the dysplastic nevi (p-value, 0.17), indicating that when nevi become dysplastic they are not replicating at a faster rate, and only upon transitioning to a melanoma do the cells begin to replicate faster. The data indicates that the melanoma samples are replicating at a 2-fold higher rate than nevi.

Discussion

[00153] These data show the measurement of CCP scores can differentiate between malignant melanoma and nonmalignant nevi. More specifically these data show that a CCP score can differentiate between melanoma and dysplastic nevi. While the average difference between the two groups is moderate (~1 CCP unit), precision of the measurements allows for good separation of the two datasets.

Example 2

[00154] In this example we determined whether certain CCP genes differentiated nevi and melanoma more effectively (Table 6). We indeed observed that there were certain CCP genes that had much better AUC values than others, even though all but one gene had an AUC >0.8, which was still quite impressive. We decided to move forward and selected ten CCP genes whose AUC scores were >0.95 (see Table 6). Ten CCP genes were sufficient to produce a robust and reliable CCP score.

Table 6.

Example 3 [00155] This example assesses a variety of potential biomarkers to determine if their altered expression can differentiate between malignant melanoma and non-malignant nevi.

Materials and Methods

[00156] Samples: Biomarker discovery was performed using two independent datasets. The first dataset consisted of 31 samples (Group 1). See Table 7 for a list of these samples and their clinical details. The second dataset consisted of 53 samples (Group 2). See Table 8 for a list of these samples and their clinical details.

Table 7. Group 1 samples.

Table 8. Group 2 Samples

42 CPP-12- 14855 Melanoma Acral 5/25/2012 71 M Leg

Dysplastic low/

43 CPP-1 1 - 19854 Nevus 6/27/201 1 31 M Torso

Compound

44 CPP-1 1 -24358 Melanoma Lentigo Maligna 8/9/201 1 89 F Head

Dysplastic low/

45 CPP- 10-9477 B Nevus 4/1 /2010 40 M Torso

Junctional

46 CPP-10- 16714 Melanoma Lentigo Maligna 6/10/2010 58 M Head

47 CPP-12- 17349 Melanoma Desmoplastic 6/20/2012 62 M Leg

Dysplastic low/

48 CPP- 10-8105 Nevus 3/22/2010 48 F Torso

Junctional

49 CPP-12- 17027 Nevus Spitz 6/18/2012 25 M Arm

Superficial

50 CPP-1 1 - 17106 Melanoma 6/2/201 1 52 M Head

Spreading

51 CPP-12- 14808 Melanoma Desmoplastic 5/24/2012 66 F Head

Superficial

52 CPP-1 1 -32490 Melanoma 10/27/201 1 58 F Arm

Spreading

53 CPP-1 1 - 17723 Nevus Dysplastic low 6/8/201 1 36 F Torso

[00157] RNA extraction: The 31 group 1 samples were anonymized with a qPCRID and a 4 mm H&E slide was reviewed by a pathologist who circled the lesion of interest (either the nevus or the melanoma). The lesions were then macro-dissected and removed from three 10 μΜ slides. All three dissected lesions from a single patient were pooled into a single tube. RNA was extracted from samples, RNA expression levels were determined using standard qPCR techniques, and a standard CCP score was generated.

[00158] RNA from 30 of the samples was then used in the first two rounds of biomarker discovery. RNA was DNased and quantified. RNA with concentrations >40ng/uL were normalized to 40 ng/uL.

[00159] The 53 group 2 samples were anonymized with a qPCR ID, and a H&E slide was reviewed by a pathologist who circled the lesion of interest. The lesions were then macro-dissected from five 4mm slides. All five dissected lesions from a single patient were pooled into a single tube. The RNA from each tube was extracted using standard RNA extraction techniques. RNA was DNased and quantified. All RNA samples with concentrations >40ng/uL were normalized to 40 ng/uL.

[00160] Measurement of gene expression: RNA expression was measured using standard qPCR techniques. Ct values were determined, and the expression of each gene was normalized to the expression of housekeeper genes. [00161] Biomarkers were assessed in three rounds of testing. Only samples from group 1 were analyzed in rounds 1 and 2 of testing. Samples from groups 1 and 2 were analyzed in round 3 of testing. Data from each round of testing was analyzed separately and aggregated. The housekeeping genes tested for normalization in all three rounds included MRFAP l , PSMAl , RPL13A, and TXNLl . In addition, housekeeping genes SLC25A3, RPS29, RPL8, PSMC l and RPL4 were included in the first round of testing.

Results

[00162] Table 9 lists all amplicons used in biomarker discovery and lists p- values for separate rounds of analysis, and aggregated analysis as indicated in the round tested column. P-values are Wilcoxon Rank Sum P-values, and p-values and AUC indicate differentiation all melanomas subtypes from all nevi subtypes.

Table 9. All amplicons used during biomarker discovery

Hs00959010 ml 5.3E-09 0.89 1

Hs00960641 ml 4.0E-01 1.00 1, 3

Hs00608222 ml 3.2E-01 0.92 1

WNT2 Hs00608224 ml 4.5E-01 0.61

Hs01128652 ml 8.3E-01 0.55 1

Hs00183662 ml 5.3E-02 0.63 1, 3

WIF1

Hs01548029 ml 7.6E-01 0.54 1

Hs00374226 ml 2.9E-01 0.57 1, 3

NR4A1

Hs00926542 gl l.OE-01 0.72 1

Hs00269575 si 9.3E-09 0.87 1, 3

SOCS3 Hs01000485 gl 9.4E-08 0.85 2, 3

Hs02330328 si 5.4E-03 0.84 1

Hs00196132 ml 2.0E-02 0.88 2

Hs01022299 ml l.OE+0 0.50 2

PRAME

Hs01022301 ml 1.3E-09 0.89 2,3

Hs04186846 ml 3.8E-06 0.84 2,3

Hs00267035 ml l.OE-01 0.71 2

Hs00951967 gl 1.3E-08 0.89 2,3

KRT15

Hs00951968 gH l.OE-08 0.89 2,3

Hs02558897 si 8.3E-02 0.74 2

Hs00361424 gl 1.2E-05 0.80 2,3

FABP7 Hs00361426 ml 3.5E-02 0.77 2

Hs00953719 gl 1.7E-03 0.72 2,3

Hs00962362 gl 1.4E-03 0.72 2,3

CFH Hs00962365 gl 5.0E-01 0.59 2

Hs00962373 ml 6.3E-03 0.69 2,3

Hs00383235 ml 1.5E-02 0.67 2,3

PTN Hs01085690 ml 5.5E-02 0.75 2

Hs01085691 ml 1.4E-03 0.72 2,3

HsOl 114111 gl 1.0E-04 0.76 2,3

HEY1 Hs01114112 gl 4.5E-04 0.74 2,3

Hs01114113 ml 6.9E-01 0.56 2

GDF15 Hs00171132 ml 5.0E-02 0.64 2,3

Hs01116208 ml 9.4E-02 0.72 2

Hs01116210 ml 2.1E-01 0.59 2,3

PHACTR1

Hs01116212 ml 4.8E-02 0.76 2

Hs01116214 ml 2.1E-07 0.86 2,3

Hs00175501 ml 2.2E-07 0.89 3

LCP2

Hs01092638 ml 2.4E-06 0.86 3

Hs00171065 ml 4.7E-11 0.97 3

CXCL9

Hs00970537 ml 3.6E-11 0.97 3

Hs01124251 gl 8.9E-07 0.92 3

CXCL10

HsOl 124252 gl 8.1E-12 0.98 3

Hs00757930 ml 4.0E-10 0.96 3

CXCL13

Hs99999094 ml 4.5E-09 0.95 3 S 100A9 Hs00610058 ml 6.1E-05 0.82 3

SERPINB4 Hs00741313 gl 1.6E-01 0.65 3

CCL 19 Hs00171 149 ml 8.3E-02 0.64 3

CCL5 Hs00174575 ml 1.5E- 10 0.96 3

CD38 Hs01 120071 ml 2.2E-09 0.95 3

CXCL 12 Hs00171022 ml 3.5E-01 0.58 3

HCLS 1 Hs00945386 ml 1.1 E-03 0.76 3

HLA-DMA Hs00185435 ml 3.7E-04 0.78 3

HLA-DPA1 Hs01072899 ml 3.8E-02 0.67 3

HLA-DPB 1 Hs00157955 ml 2.9E-01 0.59 3

HLA-DRA Hs00219575 ml 6.4E-04 0.77 3

HLA-E Hs03045171 ml l . l E-01 0.63 3

IGHM Hs00378435 ml 1.8E-02 0.76 3

IGJ Ha00950678 gl 5.2E-03 0.73 3

IGLL5;CKAP2 Hs00382306 ml 1.8E-01 0.64 3

IRF1 Hs00971966 gl 2.0E-08 0.92 3

IRF4 Hs00180031 ml 2.4E-02 0.69 3

ITGB2 Hs01051739 ml 1.7E-05 0.84 3

PECAM 1 Hs00169777 ml 1.8E-03 0.75 3

PTPN22 Hs00249262 ml 1.4E-05 0.84 3

PTPRC Hs00894732 ml 1.5E-04 0.80 3

SELL Hs01046459 ml 9.0E-09 0.93 3

[00163] Figure 3 shows the distribution of all amplicons tested in all three rounds of biomarker discovery. Samples are differentiated based on diagnosis on the Y- axis. The relative expression (Ct) of each gene (compared to the expression of the housekeeper genes) is graphed on the X axis

[00164] Figure 4 shows the distributions of each individual amplicon tested in rounds one and two of biomarker discovery. Samples are differentiated based on their pathological subtype on the Y axis. The relative expression (Ct) of each gene

(compared to the expression of the housekeeper genes) is graphed on the X axis. Each amplicon is identified by the gene and the last three digits of the assay ID

Discussion

[00165] These data strongly indicate that expression of specific biomarker genes can be used to differentiate malignant melanoma from non-malignant nevi.

Nonetheless, a larger dataset is needed to determine with higher confidence how effective each individual biomarker is in differentiating melanoma, as well as to determine how heavily each biomarker would need to be weighted in a diagnostic model.

[00166] Table 10 lists the best performing assay/amplicon of each biomarkers determined by its A UC, P-value, and lack of missing data. P-values are Wilcoxon Rank Sum P-values. P-values and A UC indicate differentiation of all melanomas subtypes from all nevi subtypes.

Table 10.

Example 4 [00167] P-values were calculated for distinguishing melanoma from nevi for all combinations of two, three, and four genes from Panel I with data from the same samples used above. Firth's logistic regression was used to assign the best weights to each of the genes in each combination. The p-values were calculated using a likelihood ratio test comparing a model containing all genes in each combination with a reduced model containing no predictor variables. The number of samples with data for all genes in each combination and whether the combination contains CCP genes, other genes, or a mix of CCP and other genes are included in the results.

[00168] Additionally, p-values were calculated for average CCP expression of all combinations of one to four of the CCP genes in panel C as continuing to the next stage for use in a potential training set. The average of each combination of CCP genes was calculated and a t-test was performed to test for a difference in average expression between melanomas and nevi.

[00169] Table WW contains the results for the combinations of between one and 4 CCP gene averages. Table XX contains the results for all combinations of two genes. Table YY contains the results for the top 500 combinations of three genes.

Table ZZ contains the results for the top 500 combinations of four genes.

Table WW

PBK DLGAP5 3.93E-07

PBK FOXMl 3.73E-07

PBK MCMIO 3.39E-07

PBK CEP55 1.09E-07

PBK RRM2 1.67E-06

PBK DTL 2.68E-07

PBK CENPF 5.39E-06

PLKl DLGAP5 1.17E-07

PLKl FOXMl 2.12E-07

PLKl MCMIO 1.10E-07

PLKl CEP55 1.02E-07

PLKl RRM2 8.79E-07

PLKl DTL 9.73E-08

PLKl CENPF 1.17E-06

DLGAP5 FOXMl 9.55E-08

DLGAP5 MCMIO 7.54E-08

DLGAP5 CEP55 4.09E-07

DLGAP5 RRM2 1.61E-06

DLGAP5 DTL 1.12E-07

DLGAP5 CENPF 1.86E-06

FOXMl MCMIO 1.24E-07

FOXMl CEP55 6.29E-08

FOXMl RRM2 3.72E-07

FOXMl DTL 3.19E-08

FOXMl CENPF 1.22E-06

MCMIO CEP55 4.51E-08

MCMIO RRM2 7.02E-07

MCMIO DTL 1.11E-07

MCMIO CENPF 1.06E-06

CEP55 RRM2 1.29E-06

CEP55 DTL 8.42E-08

CEP55 CENPF 1.23E-06

RRM2 DTL 1.10E-06

RRM2 CENPF 1.24E-05

DTL CENPF 1.44E-06

C18orf24 PBK PLKl 6.42E-08

C18orf24 PBK DLGAP5 3.31E-08

C18orf24 PBK FOXMl 8.71E-08

C18orf24 PBK MCMIO 3.72E-08

C18orf24 PBK CEP55 2.45E-08

C18orf24 PBK RRM2 2.18E-07

C18orf24 PBK DTL 3.29E-08

C18orf24 PBK CENPF 5.36E-07

C18orf24 PLKl DLGAP5 1.15E-08

C18orf24 PLKl FOXMl 4.55E-08

C18orf24 PLKl MCMIO 1.24E-08

C18orf24 PLKl CEP55 1.12E-08

C18orf24 PLKl RRM2 1.10E-07

C18orf24 PLKl DTL 1.18E-08

C18orf24 PLKl CENPF 1.28E-07

C18orf24 DLGAP5 FOXMl 1.38E-08

C18orf24 DLGAP5 MCMIO 3.59E-09

C18orf24 DLGAP5 CEP55 6.49E-09 C18orf24 DLGAP5 RRM2 6.24E-08

C18orf24 DLGAP5 DTL 3.98E-09

C18orf24 DLGAP5 CENPF 1.01E-07

C18orf24 FOXMl MCMIO 2.06E-08

C18orf24 FOXMl CEP55 1.68E-08

C18orf24 FOXMl RRM2 9.04E-08

C18orf24 FOXMl DTL 9.34E-09

C18orf24 FOXMl CENPF 2.06E-07

C18orf24 MCMIO CEP55 4.69E-09

C18orf24 MCMIO RRM2 5.26E-08

C18orf24 MCMIO DTL 4.24E-09

C18orf24 MCMIO CENPF 7.72E-08

C18orf24 CEP55 RRM2 6.49E-08

C18orf24 CEP55 DTL 3.18E-09

C18orf24 CEP55 CENPF 5.69E-08

C18orf24 RRM2 DTL 6.20E-08

C18orf24 RRM2 CENPF 7.48E-07

C18orf24 DTL CENPF 7.84E-08

PBK PLK1 DLGAP5 1.43E-07

PBK PLK1 FOXMl 2.22E-07

PBK PLK1 MCMIO 1.37E-07

PBK PLK1 CEP55 9.21E-08

PBK PLK1 RRM2 4.62E-07

PBK PLK1 DTL 1.54E-07

PBK PLK1 CENPF 1.19E-06

PBK DLGAP5 FOXMl 1.61E-07

PBK DLGAP5 MCMIO 1.29E-07

PBK DLGAP5 CEP55 9.22E-08

PBK DLGAP5 RRM2 4.94E-07

PBK DLGAP5 DTL 1.22E-07

PBK DLGAP5 CENPF 1.02E-06

PBK FOXMl MCMIO 1.67E-07

PBK FOXMl CEP55 1.00E-07

PBK FOXMl RRM2 3.36E-07

PBK FOXMl DTL 1.06E-07

PBK FOXMl CENPF 1.15E-06

PBK MCMIO CEP55 5.92E-08

PBK MCMIO RRM2 3.66E-07

PBK MCMIO DTL 1.10E-07

PBK MCMIO CENPF 1.06E-06

PBK CEP55 RRM2 2.83E-07

PBK CEP55 DTL 5.70E-08

PBK CEP55 CENPF 6.18E-07

PBK RRM2 DTL 4.40E-07

PBK RRM2 CENPF 3.26E-06

PBK DTL CENPF 1.13E-06

PLK1 DLGAP5 FOXMl 8.52E-08

PLK1 DLGAP5 MCMIO 4.43E-08

PLK1 DLGAP5 CEP55 9.78E-08

PLK1 DLGAP5 RRM2 3.26E-07

PLK1 DLGAP5 DTL 7.28E-08

PLK1 DLGAP5 CENPF 6.07E-07

PLK1 FOXMl MCMIO 1.02E-07 PLKl FOXMl CEP55 8.13E-08

PLKl FOXMl RRM2 2.37E-07

PLKl FOXMl DTL 6.80E-08

PLKl FOXMl CENPF 5.69E-07

PLKl MCMIO CEP55 4.23E-08

PLKl MCMIO RRM2 2.06E-07

PLKl MCMIO DTL 6.45E-08

PLKl MCMIO CENPF 3.81E-07

PLKl CEP55 RRM2 3.20E-07

PLKl CEP55 DTL 5.89E-08

PLKl CEP55 CENPF 3.85E-07

PLKl RRM2 DTL 3.02E-07

PLKl RRM2 CENPF 1.92E-06

PLKl DTL CENPF 4.81E-07

DLGAP5 FOXMl MCMIO 5.28E-08

DLGAP5 FOXMl CEP55 5.47E-08

DLGAP5 FOXMl RRM2 1.71E-07

DLGAP5 FOXMl DTL 3.33E-08

DLGAP5 FOXMl CENPF 3.81E-07

DLGAP5 MCMIO CEP55 3.16E-08

DLGAP5 MCMIO RRM2 1.85E-07

DLGAP5 MCMIO DTL 3.39E-08

DLGAP5 MCMIO CENPF 2.95E-07

DLGAP5 CEP55 RRM2 5.67E-07

DLGAP5 CEP55 DTL 9.06E-08

DLGAP5 CEP55 CENPF 6.16E-07

DLGAP5 RRM2 DTL 3.77E-07

DLGAP5 RRM2 CENPF 2.36E-06

DLGAP5 DTL CENPF 5.59E-07

FOXMl MCMIO CEP55 4.29E-08

FOXMl MCMIO RRM2 1.31E-07

FOXMl MCMIO DTL 3.20E-08

FOXMl MCMIO CENPF 3.91E-07

FOXMl CEP55 RRM2 1.49E-07

FOXMl CEP55 DTL 2.40E-08

FOXMl CEP55 CENPF 2.83E-07

FOXMl RRM2 DTL 1.14E-07

FOXMl RRM2 CENPF 1.17E-06

FOXMl DTL CENPF 3.00E-07

MCMIO CEP55 RRM2 1.25E-07

MCMIO CEP55 DTL 2.08E-08

MCMIO CEP55 CENPF 1.94E-07

MCMIO RRM2 DTL 2.08E-07

MCMIO RRM2 CENPF 1.29E-06

MCMIO DTL CENPF 3.12E-07

CEP55 RRM2 DTL 3.03E-07

CEP55 RRM2 CENPF 1.91E-06

CEP55 DTL CENPF 3.68E-07

RRM2 DTL CENPF 2.18E-06

C18orf24 PBK PLKl DLGAP5 3.34E-08

C18orf24 PBK PLKl FOXMl 7.43E-08

C18orf24 PBK PLKl MCMIO 3.44E-08

C18orf24 PBK PLKl CEP55 2.85E-08 C18orf24 PBK PLK1 RRM2 1.24E-07

C18orf24 PBK PLK1 DTL 3.58E-08

C18orf24 PBK PLK1 CENPF 2.47E-07

C18orf24 PBK DLGAP5 FOXMl 3.79E-08

C18orf24 PBK DLGAP5 MCMIO 1.94E-08

C18orf24 PBK DLGAP5 CEP55 2.06E-08

C18orf24 PBK DLGAP5 RRM2 8.84E-08

C18orf24 PBK DLGAP5 DTL 2.07E-08

C18orf24 PBK DLGAP5 CENPF 1.83E-07

C18orf24 PBK FOXMl MCMIO 4.65E-08

C18orf24 PBK FOXMl CEP55 3.71E-08

C18orf24 PBK FOXMl RRM2 1.12E-07

C18orf24 PBK FOXMl DTL 3.40E-08

C18orf24 PBK FOXMl CENPF 2.99E-07

C18orf24 PBK MCMIO CEP55 1.61E-08

C18orf24 PBK MCMIO RRM2 7.76E-08

C18orf24 PBK MCMIO DTL 1.92E-08

C18orf24 PBK MCMIO CENPF 1.85E-07

C18orf24 PBK CEP55 RRM2 7.78E-08

C18orf24 PBK CEP55 DTL 1.55E-08

C18orf24 PBK CEP55 CENPF 1.35E-07

C18orf24 PBK RRM2 DTL 8.96E-08

C18orf24 PBK RRM2 CENPF 6.27E-07

C18orf24 PBK DTL CENPF 1.93E-07

C18orf24 PLK1 DLGAP5 FOXMl 2.31E-08

C18orf24 PLK1 DLGAP5 MCMIO 8.15E-09

C18orf24 PLK1 DLGAP5 CEP55 1.21E-08

C18orf24 PLK1 DLGAP5 RRM2 5.15E-08

C18orf24 PLK1 DLGAP5 DTL 1.01E-08

C18orf24 PLK1 DLGAP5 CENPF 8.24E-08

C18orf24 PLK1 FOXMl MCMIO 2.72E-08

C18orf24 PLK1 FOXMl CEP55 2.47E-08

C18orf24 PLK1 FOXMl RRM2 7.73E-08

C18orf24 PLK1 FOXMl DTL 1.96E-08

C18orf24 PLK1 FOXMl CENPF 1.43E-07

C18orf24 PLK1 MCMIO CEP55 9.51E-09

C18orf24 PLK1 MCMIO RRM2 4.38E-08

C18orf24 PLK1 MCMIO DTL 9.86E-09

C18orf24 PLK1 MCMIO CENPF 6.72E-08

C18orf24 PLK1 CEP55 RRM2 5.21E-08

C18orf24 PLK1 CEP55 DTL 9.01E-09

C18orf24 PLK1 CEP55 CENPF 5.77E-08

C18orf24 PLK1 RRM2 DTL 5.41E-08

C18orf24 PLK1 RRM2 CENPF 3.11E-07

C18orf24 PLK1 DTL CENPF 7.33E-08

C18orf24 DLGAP5 FOXMl MCMIO 1.04E-08

C18orf24 DLGAP5 FOXMl CEP55 1.41E-08

C18orf24 DLGAP5 FOXMl RRM2 4.18E-08

C18orf24 DLGAP5 FOXMl DTL 8.19E-09

C18orf24 DLGAP5 FOXMl CENPF 8.90E-08

C18orf24 DLGAP5 MCMIO CEP55 5.09E-09

C18orf24 DLGAP5 MCMIO RRM2 2.53E-08

C18orf24 DLGAP5 MCMIO DTL 3.79E-09 C18orf24 DLGAP5 MCMIO CENPF 4.23E-08

C18orf24 DLGAP5 CEP55 RRM2 4.01E-08

C18orf24 DLGAP5 CEP55 DTL 5.40E-09

C18orf24 DLGAP5 CEP55 CENPF 5.34E-08

C18orf24 DLGAP5 RRM2 DTL 3.30E-08

C18orf24 DLGAP5 RRM2 CENPF 2.57E-07

C18orf24 DLGAP5 DTL CENPF 5.41E-08

C18orf24 FOXMl MCMIO CEP55 1.24E-08

C18orf24 FOXMl MCMIO RRM2 3.92E-08

C18orf24 FOXMl MCMIO DTL 8.06E-09

C18orf24 FOXMl MCMIO CENPF 9.12E-08

C18orf24 FOXMl CEP55 RRM2 4.91E-08

C18orf24 FOXMl CEP55 DTL 8.24E-09

C18orf24 FOXMl CEP55 CENPF 7.57E-08

C18orf24 FOXMl RRM2 DTL 3.49E-08

C18orf24 FOXMl RRM2 CENPF 2.98E-07

C18orf24 FOXMl DTL CENPF 7.30E-08

C18orf24 MCMIO CEP55 RRM2 2.65E-08

C18orf24 MCMIO CEP55 DTL 3.76E-09

C18orf24 MCMIO CEP55 CENPF 3.47E-08

C18orf24 MCMIO RRM2 DTL 2.74E-08

C18orf24 MCMIO RRM2 CENPF 2.03E-07

C18orf24 MCMIO DTL CENPF 4.00E-08

C18orf24 CEP55 RRM2 DTL 3.00E-08

C18orf24 CEP55 RRM2 CENPF 2.15E-07

C18orf24 CEP55 DTL CENPF 3.57E-08

C18orf24 RRM2 DTL CENPF 2.44E-07

PBK PLK1 DLGAP5 FOXMl 1.14E-07

PBK PLK1 DLGAP5 MCMIO 7.66E-08

PBK PLK1 DLGAP5 CEP55 7.68E-08

PBK PLK1 DLGAP5 RRM2 2.27E-07

PBK PLK1 DLGAP5 DTL 9.18E-08

PBK PLK1 DLGAP5 CENPF 4.87E-07

PBK PLK1 FOXMl MCMIO 1.25E-07

PBK PLK1 FOXMl CEP55 1.00E-07

PBK PLK1 FOXMl RRM2 2.25E-07

PBK PLK1 FOXMl DTL 1.07E-07

PBK PLK1 FOXMl CENPF 6.01E-07

PBK PLK1 MCMIO CEP55 5.77E-08

PBK PLK1 MCMIO RRM2 1.86E-07

PBK PLK1 MCMIO DTL 8.38E-08

PBK PLK1 MCMIO CENPF 4.59E-07

PBK PLK1 CEP55 RRM2 1.80E-07

PBK PLK1 CEP55 DTL 6.39E-08

PBK PLK1 CEP55 CENPF 3.40E-07

PBK PLK1 RRM2 DTL 2.40E-07

PBK PLK1 RRM2 CENPF 1.14E-06

PBK PLK1 DTL CENPF 5.16E-07

PBK DLGAP5 FOXMl MCMIO 9.06E-08

PBK DLGAP5 FOXMl CEP55 7.63E-08

PBK DLGAP5 FOXMl RRM2 1.94E-07

PBK DLGAP5 FOXMl DTL 7.27E-08

PBK DLGAP5 FOXMl CENPF 4.29E-07 PBK DLGAP5 MCMIO CEP55 5.18E-08

PBK DLGAP5 MCMIO RRM2 1.87E-07

PBK DLGAP5 MCMIO DTL 6.58E-08

PBK DLGAP5 MCMIO CENPF 3.75E-07

PBK DLGAP5 CEP55 RRM2 1.76E-07

PBK DLGAP5 CEP55 DTL 5.50E-08

PBK DLGAP5 CEP55 CENPF 3.43E-07

PBK DLGAP5 RRM2 DTL 2.16E-07

PBK DLGAP5 RRM2 CENPF 1.03E-06

PBK DLGAP5 DTL CENPF 4.40E-07

PBK FOXMl MCMIO CEP55 6.51E-08

PBK FOXMl MCMIO RRM2 1.60E-07

PBK FOXMl MCMIO DTL 6.86E-08

PBK FOXMl MCMIO CENPF 4.74E-07

PBK FOXMl CEP55 RRM2 1.47E-07

PBK FOXMl CEP55 DTL 5.24E-08

PBK FOXMl CEP55 CENPF 3.57E-07

PBK FOXMl RRM2 DTL 1.53E-07

PBK FOXMl RRM2 CENPF 9.03E-07

PBK FOXMl DTL CENPF 4.25E-07

PBK MCMIO CEP55 RRM2 1.17E-07

PBK MCMIO CEP55 DTL 3.76E-08

PBK MCMIO CEP55 CENPF 2.62E-07

PBK MCMIO RRM2 DTL 1.76E-07

PBK MCMIO RRM2 CENPF 9.20E-07

PBK MCMIO DTL CENPF 3.94E-07

PBK CEP55 RRM2 DTL 1.42E-07

PBK CEP55 RRM2 CENPF 8.19E-07

PBK CEP55 DTL CENPF 2.93E-07

PBK RRM2 DTL CENPF 1.11E-06

PLKl DLGAP5 FOXMl MCMIO 5.28E-08

PLKl DLGAP5 FOXMl CEP55 6.35E-08

PLKl DLGAP5 FOXMl RRM2 1.29E-07

PLKl DLGAP5 FOXMl DTL 4.89E-08

PLKl DLGAP5 FOXMl CENPF 2.84E-07

PLKl DLGAP5 MCMIO CEP55 3.45E-08

PLKl DLGAP5 MCMIO RRM2 1.02E-07

PLKl DLGAP5 MCMIO DTL 3.53E-08

PLKl DLGAP5 MCMIO CENPF 1.92E-07

PLKl DLGAP5 CEP55 RRM2 2.17E-07

PLKl DLGAP5 CEP55 DTL 6.29E-08

PLKl DLGAP5 CEP55 CENPF 2.99E-07

PLKl DLGAP5 RRM2 DTL 1.79E-07

PLKl DLGAP5 RRM2 CENPF 8.59E-07

PLKl DLGAP5 DTL CENPF 3.16E-07

PLKl FOXMl MCMIO CEP55 5.33E-08

PLKl FOXMl MCMIO RRM2 1.13E-07

PLKl FOXMl MCMIO DTL 4.70E-08

PLKl FOXMl MCMIO CENPF 2.71E-07

PLKl FOXMl CEP55 RRM2 1.32E-07

PLKl FOXMl CEP55 DTL 4.30E-08

PLKl FOXMl CEP55 CENPF 2.23E-07

PLKl FOXMl RRM2 DTL 1.16E-07 4 PLK1 FOXMl RRM2 CENPF 6.11E-07

4 PLK1 FOXMl DTL CENPF 2.44E-07

4 PLK1 MCMIO CEP55 RRM2 9.39E-08

4 PLK1 MCMIO CEP55 DTL 3.03E-08

4 PLK1 MCMIO CEP55 CENPF 1.47E-07

4 PLK1 MCMIO RRM2 DTL 1.23E-07

4 PLK1 MCMIO RRM2 CENPF 5.31E-07

4 PLK1 MCMIO DTL CENPF 2.05E-07

4 PLK1 CEP55 RRM2 DTL 1.66E-07

4 PLK1 CEP55 RRM2 CENPF 7.23E-07

4 PLK1 CEP55 DTL CENPF 2.26E-07

4 PLK1 RRM2 DTL CENPF 7.86E-07

4 DLGAP5 FOXMl MCMIO CEP55 3.54E-08

4 DLGAP5 FOXMl MCMIO RRM2 8.38E-08

4 DLGAP5 FOXMl MCMIO DTL 2.63E-08

4 DLGAP5 FOXMl MCMIO CENPF 1.78E-07

4 DLGAP5 FOXMl CEP55 RRM2 1.03E-07

4 DLGAP5 FOXMl CEP55 DTL 2.93E-08

4 DLGAP5 FOXMl CEP55 CENPF 1.99E-07

4 DLGAP5 FOXMl RRM2 DTL 8.20E-08

4 DLGAP5 FOXMl RRM2 CENPF 4.74E-07

4 DLGAP5 FOXMl DTL CENPF 1.77E-07

4 DLGAP5 MCMIO CEP55 RRM2 8.27E-08

4 DLGAP5 MCMIO CEP55 DTL 2.04E-08

4 DLGAP5 MCMIO CEP55 CENPF 1.36E-07

4 DLGAP5 MCMIO RRM2 DTL 9.42E-08

4 DLGAP5 MCMIO RRM2 CENPF 4.58E-07

4 DLGAP5 MCMIO DTL CENPF 1.49E-07

4 DLGAP5 CEP55 RRM2 DTL 2.18E-07

4 DLGAP5 CEP55 RRM2 CENPF 9.29E-07

4 DLGAP5 CEP55 DTL CENPF 2.76E-07

4 DLGAP5 RRM2 DTL CENPF 8.77E-07

4 FOXMl MCMIO CEP55 RRM2 7.16E-08

4 FOXMl MCMIO CEP55 DTL 2.05E-08

4 FOXMl MCMIO CEP55 CENPF 1.51E-07

4 FOXMl MCMIO RRM2 DTL 6.53E-08

4 FOXMl MCMIO RRM2 CENPF 4.06E-07

4 FOXMl MCMIO DTL CENPF 1.48E-07

4 FOXMl CEP55 RRM2 DTL 6.90E-08

4 FOXMl CEP55 RRM2 CENPF 4.43E-07

4 FOXMl CEP55 DTL CENPF 1.34E-07

4 FOXMl RRM2 DTL CENPF 4.26E-07

4 MCMIO CEP55 RRM2 DTL 6.63E-08

4 MCMIO CEP55 RRM2 CENPF 3.72E-07

4 MCMIO CEP55 DTL CENPF 1.05E-07

4 MCMIO RRM2 DTL CENPF 4.85E-07

4 CEP55 RRM2 DTL CENPF 7.19E-07

Table XX

CCP C18orf24 PLK1 3.88E-06 29

CCP C18orf24 DLGAP5 1.84E-06 29

CCP C18orf24 FOXM1 1.79E-06 29

CCP C18orf24 MCM10 4.03E-06 29

CCP C18orf24 CEP55 6.28E-06 29

CCP C18orf24 RRM2 4.31E-06 29

CCP C18orf24 DTL 3.78E-06 29

CCP C18orf24 CENPF 4.40E-06 29

CCP PBK PLK1 3.85E-06 29

CCP PBK DLGAP5 3.79E-05 29

CCP PBK FOXM1 2.45E-05 29

CCP PBK MCM10 9.54E-06 29

CCP PBK CEP55 2.84E-06 29

CCP PBK RRM2 3.20E-05 29

CCP PBK DTL 7.93E-06 29

CCP PBK CENPF 3.10E-06 29

CCP PLK1 DLGAP5 3.12E-06 30

CCP PLK1 FOXM1 3.67E-06 29

CCP PLK1 MCM10 1.94E-05 29

CCP PLK1 CEP55 5.98E-06 30

CCP PLK1 RRM2 1.16E-05 30

CCP PLK1 DTL 5.99E-06 30

CCP PLK1 CENPF 1.72E-05 30

CCP DLGAP5 FOXM1 2.45E-05 29

CCP DLGAP5 MCM10 1.95E-05 29

CCP DLGAP5 CEP55 0.00013 30

CCP DLGAP5 RRM2 0.000132 30

CCP DLGAP5 DTL 3.80E-06 30

CCP DLGAP5 CENPF 1.04E-05 30

CCP FOXM1 MCM10 1.28E-05 29

CCP FOXM1 CEP55 2.57E-06 29

CCP FOXM1 RRM2 1.88E-05 29

CCP FOXM1 DTL 4.61E-06 29

CCP FOXM1 CENPF 2.39E-06 29

CCP MCM10 CEP55 1.51E-05 29

CCP MCM10 RRM2 4.52E-05 29

CCP MCM10 DTL 5.91E-06 29

CCP MCM10 CENPF 2.54E-05 29

CCP CEP55 RRM2 7.82E-05 30

CCP CEP55 DTL 3.90E-06 30

CCP CEP55 CENPF 2.18E-05 30

CCP RRM2 DTL 4.43E-06 30

CCP RRM2 CENPF 2.10E-05 30

CCP DTL CENPF 7.11E-06 30

Immune/Additional HEY1 CXCL10 1.54E-10 50

Immune/Additional HEY1 CCL3 1.22E-09 78

Immune/Additional HEY1 FABP7 2.58E-07 79

Immune/Additional HEY1 FN1 1.49E-06 79

Immune/Additional HEY1 PTN 0.000108 79

Immune/Additional HEY1 IGJ 2.86E-06 50

Immune/Additional HEY1 PRAME 6.21E-09 76

Immune/Additional HEY1 BCL2A1 2.06E-06 78

Immune/Additional HEY1 IFI6 6.07E-06 73 Immune/Additional HEY1 CFH 6.34E-05 79

Immune/Additional HEY1 HLA 1.91E-06 52

Immune/Additional HEY1 PECAM1 2.94E-05 52

Immune/Additional HEY1 PTPN22 2.52E-07 52

Immune/Additional HEY1 RGS1 1.44E-14 79

Immune/Additional HEY1 IRF1 5.61E-09 52

Immune/Additional HEY1 CCL5 5.43E-11 52

Immune/Additional HEY1 CD38 9.00E-11 49

Immune/Additional HEY1 SELL 1.27E-10 52

Immune/Additional HEY1 HLA 6.11E-06 52

Immune/Additional HEY1 S100A9 1.05E-08 52

Immune/Additional HEY1 ITGB2 4.91E-06 52

Immune/Additional HEY1 PTPRC 4.07E-07 52

Immune/Additional HEY1 CXCL13 1.61E-09 48

Immune/Additional HEY1 PHACTRl 1.05E-06 78

Immune/Additional HEY1 SPP1 5.19E-10 78

Immune/Additional HEY1 LCP2 9.82E-09 52

Immune/Additional HEY1 KRT15 1.05E-11 79

Immune/Additional HEY1 CXCL9 1.84E-10 52

Immune/Additional HEY1 HCLS1 2.40E-05 52

Immune/Additional CXCLIO CCL3 2.51E-10 50

Immune/Additional CXCLIO FABP7 6.14E-11 50

Immune/Additional CXCLIO FN1 4.37E-11 50

Immune/Additional CXCLIO PTN 3.58E-10 50

Immune/Additional CXCLIO IGJ 2.34E-10 48

Immune/Additional CXCLIO PRAME 2.26E-10 50

Immune/Additional CXCLIO BCL2A1 2.33E-10 50

Immune/Additional CXCLIO IFI6 2.64E-10 47

Immune/Additional CXCLIO CFH 2.54E-10 50

Immune/Additional CXCLIO HLA 1.60E-10 50

Immune/Additional CXCLIO PECAM1 2.46E-10 50

Immune/Additional CXCLIO PTPN22 2.16E-10 50

Immune/Additional CXCLIO RGS1 2.49E-11 50

Immune/Additional CXCLIO IRF1 2.39E-10 50

Immune/Additional CXCLIO CCL5 2.29E-10 50

Immune/Additional CXCLIO CD38 4.47E-10 48

Immune/Additional CXCLIO SELL 1.30E-10 50

Immune/Additional CXCLIO HLA 2.71E-10 50

Immune/Additional CXCLIO S100A9 1.39E-10 50

Immune/Additional CXCLIO ITGB2 3.34E-10 50

Immune/Additional CXCLIO PTPRC 1.25E-10 50

Immune/Additional CXCLIO CXCL13 8.57E-10 46

Immune/Additional CXCLIO PHACTRl 5.53E-11 50

Immune/Additional CXCLIO SPP1 1.29E-10 49

Immune/Additional CXCLIO LCP2 3.18E-10 50

Immune/Additional CXCLIO KRT15 1.94E-12 50

Immune/Additional CXCLIO CXCL9 1.32E-10 50

Immune/Additional CXCLIO HCLS1 2.82E-10 50

Immune/Additional CCL3 FABP7 7.30E-09 78

Immune/Additional CCL3 FN1 3.23E-10 78

Immune/Additional CCL3 PTN 5.96E-08 78

Immune/Additional CCL3 IGJ 5.68E-06 50

Immune/Additional CCL3 PRAME 1.42E-10 76 Immune/Additional CCL3 BCL2A1 2.24E-09 78

Immune/Additional CCL3 IFI6 5.70E- 10 73

Immune/Additional CCL3 CFH 4.01E-09 78

Immune/Additional CCL3 HLA 4.15E-05 52

Immune/Additional CCL3 PECAM1 3.80E-05 52

Immune/Additional CCL3 PTPN22 2.20E-05 52

Immune/Additional CCL3 RGS 1 2.1 1E- 13 78

Immune/Additional CCL3 IRF 1 1.96E-07 52

Immune/Additional CCL3 CCL5 3.60E-09 52

Immune/Additional CCL3 CD38 4.84E-09 49

Immune/Additional CCL3 SELL 2.17E-08 52

Immune/Additional CCL3 HLA 1.73E-05 52

Immune/Additional CCL3 S 100A9 3.86E-06 52

Immune/Additional CCL3 ITGB2 4.55E-06 52

Immune/Additional CCL3 PTPRC 8.63E-05 52

Immune/ Additional CCL3 CXCL13 8.90E-09 48

Immune/Additional CCL3 PHACTR1 1.33E-09 78

Immune/Additional CCL3 SPP1 2.68E-1 1 77

Immune/Additional CCL3 LCP2 1.00E-06 52

Immune/Additional CCL3 KRT15 8.96E- 14 78

Immune/ Additional CCL3 CXCL9 1.07E-09 52

Immune/Additional CCL3 HCLS 1 6.08E-05 52

Immune/Additional FABP7 FN1 1.78E-07 79

Immune/Additional FABP7 PTN 2.27E-05 79

Immune/Additional FABP7 IGJ 5.08E-05 50

Immune/Additional FABP7 PRAME 1.28E-09 76

Immune/Additional FABP7 BCL2A1 8.49E-08 78

Immune/Additional FABP7 IFI6 1.86E-06 73

Immune/Additional FABP7 CFH 3.20E-06 79

Immune/Additional FABP7 HLA 5.35E-05 52

Immune/Additional FABP7 PECAM1 0.000245 52

Immune/Additional FABP7 PTPN22 1.22E-05 52

Immune/Additional FABP7 RGS 1 8.23E- 14 79

Immune/Additional FABP7 IRF 1 2.27E-07 52

Immune/Additional FABP7 CCL5 8.87E- 10 52

Immune/Additional FABP7 CD38 1.05E-09 49

Immune/Additional FABP7 SELL 2.67E-09 52

Immune/Additional FABP7 HLA 0.0001 1 52

Immune/Additional FABP7 S 100A9 1.1 1E-05 52

Immune/Additional FABP7 ITGB2 3.71E-05 52

Immune/Additional FABP7 PTPRC 8.1 1E-05 52

Immune/Additional FABP7 CXCL13 2.09E-09 48

Immune/Additional FABP7 PHACTR1 7.46E-08 78

Immune/Additional FABP7 SPP 1 3.72E- 10 78

Immune/Additional FABP7 LCP2 3.95E-07 52

Immune/Additional FABP7 KRT 15 2.19E- 1 1 79

Immune/Additional FABP7 CXCL9 3.66E- 10 52

Immune/Additional FABP7 HCLS 1 0.000618 52

Immune/Additional FN1 PTN 2.19E-06 79

Immune/Additional FN1 IGJ 1.24E-05 50

Immune/Additional FN1 PRAME 8.56E- 1 1 76

Immune/Additional FN1 BCL2A1 6.94E- 10 80

Immune/Additional FN1 IFI6 1.56E-07 75 Immune/Additional FN1 CFH 1.53E-07 79

Immune/Additional FN1 HLA 1.75E-05 52

Immune/Additional FN1 PEC AMI 2.43E-05 52

Immune/Additional FN1 PTPN22 1.91E-06 52

Immune/Additional FN1 RGS 1 1.86E- 14 81

Immune/Additional FN1 IRF 1 3.87E-08 52

Immune/Additional FN1 CCL5 2.24E- 10 52

Immune/Additional FN1 CD38 1.18E-09 49

Immune/Additional FN1 SELL 1.60E-09 52

Immune/Additional FN1 HLA 2.03E-05 52

Immune/Additional FN1 S 100A9 1.57E-08 52

Immune/Additional FN1 ITGB2 7.52E-06 52

Immune/Additional FN1 PTPRC 2.23E-06 52

Immune/Additional FN1 CXCL13 2.70E- 10 48

Immune/Additional FN1 PHACTR1 1.20E-08 78

Immune/Additional FN1 SPP 1 1.19E- 10 80

Immune/Additional FN1 LCP2 1.06E-07 52

Immune/Additional FN1 KRT 15 5.41E- 1 1 79

Immune/Additional FN1 CXCL9 5.73E- 1 1 52

Immune/Additional FN1 HCLS 1 3.39E-05 52

Immune/Additional PTN IGJ 0.000612 50

Immune/Additional PTN PRAME 3.23E-08 76

Immune/Additional PTN BCL2A1 5.21E-06 78

Immune/Additional PTN IFI6 3.65E-06 73

Immune/Additional PTN CFH 0.000585 79

Immune/Additional PTN HLA 0.000518 52

Immune/Additional PTN PEC AMI 0.001185 52

Immune/Additional PTN PTPN22 0.000136 52

Immune/Additional PTN RGS 1 6.74E- 13 79

Immune/Additional PTN IRF 1 4.83E-07 52

Immune/Additional PTN CCL5 3.78E-09 52

Immune/Additional PTN CD38 7.93E-09 49

Immune/Additional PTN SELL 1.39E-08 52

Immune/Additional PTN HLA 0.000275 52

Immune/Additional PTN S 100A9 2.00E-05 52

Immune/Additional PTN ITGB2 8.87E-05 52

Immune/Additional PTN PTPRC 0.00121 52

Immune/Additional PTN CXCL13 1.26E-08 48

Immune/Additional PTN PHACTR1 9.62E-07 78

Immune/Additional PTN SPP1 2.59E-09 78

Immune/Additional PTN LCP2 1.54E-06 52

Immune/Additional PTN KRT15 4.49E- 1 1 79

Immune/Additional PTN CXCL9 1.22E-09 52

Immune/Additional PTN HCLS 1 0.004588 52

Immune/Additional IGJ PRAME 7.28E-07 50

Immune/Additional IGJ BCL2A1 5.28E-06 50

Immune/Additional IGJ IFI6 8.23E-05 47

Immune/Additional IGJ CFH 0.000244 50

Immune/Additional IGJ HLA 0.000484 50

Immune/Additional IGJ PEC AMI 0.000469 50

Immune/Additional IGJ PTPN22 1.66E-05 50

Immune/Additional IGJ RGS 1 7.60E-09 50

Immune/Additional IGJ IRF 1 8.66E-08 50 Immune/Additional IGJ CCL5 2.21E- 10 50

Immune/Additional IGJ CD38 4.38E-08 47

Immune/Additional IGJ SELL 2.63E-08 50

Immune/Additional IGJ HLA 0.000321 50

Immune/Additional IGJ S 100A9 6.51E-05 50

Immune/Additional IGJ ITGB2 9.60E-05 50

Immune/Additional IGJ PTPRC 0.000199 50

Immune/Additional IGJ CXCL13 2.02E-09 46

Immune/Additional IGJ PHACTRl 5.37E-06 50

Immune/Additional IGJ SPP 1 2.34E-06 49

Immune/Additional IGJ LCP2 2.62E-06 50

Immune/Additional IGJ KRT 15 1.27E- 10 50

Immune/Additional IGJ CXCL9 5.41E- 1 1 50

Immune/Additional IGJ HCLS 1 0.001058 50

Immune/Additional PRAME BCL2A1 1.49E-08 76

Immune/Additional PRAME IFI6 1.53E-09 71

Immune/Additional PRAME CFH 2.22E-08 76

Immune/Additional PRAME HLA 4.16E-07 52

Immune/Additional PRAME PEC AMI 3.62E-07 52

Immune/Additional PRAME PTPN22 6.26E-08 52

Immune/Additional PRAME RGS 1 1.59E- 14 76

Immune/Additional PRAME IRF 1 1.69E-08 52

Immune/Additional PRAME CCL5 4.98E- 10 52

Immune/Additional PRAME CD38 3.68E-09 49

Immune/Additional PRAME SELL 2.91E- 10 52

Immune/Additional PRAME HLA 1.01E-07 52

Immune/Additional PRAME S 100A9 4.66E-07 52

Immune/Additional PRAME ITGB2 4.59E-08 52

Immune/Additional PRAME PTPRC 6.65E-07 52

Immune/Additional PRAME CXCL13 6.08E-09 48

Immune/Additional PRAME PHACTRl 1.41E-09 76

Immune/Additional PRAME SPP1 2.72E- 12 75

Immune/Additional PRAME LCP2 1.40E-08 52

Immune/Additional PRAME KRT15 1.24E- 13 76

Immune/Additional PRAME CXCL9 1.41E- 10 52

Immune/Additional PRAME HCLS 1 2.34E-07 52

Immune/Additional BCL2A1 IFI6 2.60E-07 75

Immune/Additional BCL2A1 CFH 1.70E-06 78

Immune/Additional BCL2A1 HLA 4.86E-06 52

Immune/Additional BCL2A1 PECAM1 9.68E-06 52

Immune/Additional BCL2A1 PTPN22 8.54E-07 52

Immune/Additional BCL2A1 RGS 1 1.44E- 15 80

Immune/Additional BCL2A1 IRF 1 8.10E-08 52

Immune/Additional BCL2A1 CCL5 1.04E-09 52

Immune/Additional BCL2A1 CD38 2.63E-09 49

Immune/Additional BCL2A1 SELL 6.78E- 10 52

Immune/Additional BCL2A1 HLA 7.02E-06 52

Immune/Additional BCL2A1 S 100A9 1.46E-06 52

Immune/Additional BCL2A1 ITGB2 5.56E-06 52

Immune/Additional BCL2A1 PTPRC 9.48E-06 52

Immune/Additional BCL2A1 CXCL13 8.21E-09 48

Immune/Additional BCL2A1 PHACTRl 1.47E-07 78

Immune/Additional BCL2A1 SPP 1 5.1 1E-1 1 79 Immune/Additional BCL2A1 LCP2 1.96E-07 52

Immune/Additional BCL2A1 KRT15 1.03E- 10 78

Immune/Additional BCL2A1 CXCL9 7.62E- 10 52

Immune/Additional BCL2A1 HCLS 1 2.07E-05 52

Immune/Additional IFI6 CFH 3.68E-06 73

Immune/Additional IFI6 HLA 7.56E-05 48

Immune/Additional IFI6 PECAM1 5.43E-05 48

Immune/Additional IFI6 PTPN22 2.22E-06 48

Immune/Additional IFI6 RGS 1 2.29E- 14 75

Immune/Additional IFI6 IRF 1 1.67E-07 48

Immune/Additional IFI6 CCL5 1.41E- 10 48

Immune/Additional IFI6 CD38 8.58E-09 47

Immune/Additional IFI6 SELL 5.75E-09 48

Immune/Additional IFI6 HLA 0.000195 48

Immune/Additional IFI6 S 100A9 8.00E-07 48

Immune/Additional IFI6 ITGB2 0.00015 48

Immune/Additional IFI6 PTPRC 2.48E-06 48

Immune/Additional IFI6 CXCL13 5.65E- 10 44

Immune/Additional IFI6 PHACTR1 6.48E-07 73

Immune/Additional IFI6 SPP1 4.87E- 1 1 74

Immune/Additional IFI6 LCP2 1.34E-06 48

Immune/Additional IFI6 KRT15 4.94E- 10 73

Immune/Additional IFI6 CXCL9 4.76E- 1 1 48

Immune/Additional IFI6 HCLS 1 0.00025 48

Immune/Additional CFH HLA 8.66E-06 52

Immune/Additional CFH PECAM1 7.69E-05 52

Immune/Additional CFH PTPN22 9.54E-06 52

Immune/Additional CFH RGS 1 1.04E- 13 79

Immune/Additional CFH IRF 1 5.53E-08 52

Immune/Additional CFH CCL5 2.63E- 10 52

Immune/Additional CFH CD38 3.05E-09 49

Immune/Additional CFH SELL 6.80E- 1 1 52

Immune/Additional CFH HLA 2.71E-05 52

Immune/Additional CFH S 100A9 4.14E-05 52

Immune/Additional CFH ITGB2 8.68E-06 52

Immune/Additional CFH PTPRC 4.04E-05 52

Immune/Additional CFH CXCL13 3.56E-09 48

Immune/Additional CFH PHACTR1 6.52E-07 78

Immune/Additional CFH SPP1 3.73E- 10 78

Immune/Additional CFH LCP2 2.72E-07 52

Immune/Additional CFH KRT15 2.57E- 1 1 79

Immune/Additional CFH CXCL9 1.90E- 10 52

Immune/Additional CFH HCLS 1 0.000201 52

Immune/Additional HLA PECAM1 0.00018 52

Immune/Additional HLA PTPN22 0.000137 52

Immune/Additional HLA RGS 1 1.18E-08 52

Immune/Additional HLA IRF 1 5.15E-07 52

Immune/Additional HLA CCL5 1.15E-09 52

Immune/Additional HLA CD38 4.54E-08 49

Immune/Additional HLA SELL 2.06E-08 52

Immune/Additional HLA HLA 0.000606 52

Immune/Additional HLA S 100A9 5.76E-06 52

Immune/Additional HLA ITGB2 0.000104 52 Immune/Additional HLA PTPRC 0.000677 52

Immune/Additional HLA CXCL13 1.20E-08 48

Immune/Additional HLA PHACTRl 4.78E-07 52

Immune/Additional HLA SPP1 2.02E-06 51

Immune/Additional HLA LCP2 2.60E-06 52

Immune/Additional HLA KRT15 1.30E- 10 52

Immune/Additional HLA CXCL9 6.15E- 10 52

Immune/Additional HLA HCLS 1 0.000831 52

Immune/Additional PECAMl PTPN22 5.46E-05 52

Immune/Additional PECAMl RGS 1 2.18E-09 52

Immune/Additional PECAMl IRF 1 3.88E-07 52

Immune/Additional PECAMl CCL5 3.68E-09 52

Immune/Additional PECAMl CD38 6.53E-09 49

Immune/Additional PECAMl SELL 2.58E-08 52

Immune/Additional PECAMl HLA 0.000172 52

Immune/Additional PECAMl S 100A9 1.05E-05 52

Immune/Additional PECAMl ITGB2 0.000148 52

Immune/Additional PECAMl PTPRC 0.000492 52

Immune/Additional PECAMl CXCL13 3.31E-09 48

Immune/Additional PECAMl PHACTRl 1.99E-05 52

Immune/Additional PECAMl SPP1 4.86E-06 51

Immune/Additional PECAMl LCP2 2.21E-06 52

Immune/Additional PECAMl KRT15 2.02E- 10 52

Immune/Additional PECAMl CXCL9 1.00E-09 52

Immune/Additional PECAMl HCLS 1 0.00209 52

Immune/Additional PTPN22 RGS 1 9.07E-09 52

Immune/Additional PTPN22 IRF 1 4.83E-07 52

Immune/Additional PTPN22 CCL5 4.80E- 10 52

Immune/Additional PTPN22 CD38 3.32E-08 49

Immune/Additional PTPN22 SELL 3.20E-08 52

Immune/Additional PTPN22 HLA 7.86E-05 52

Immune/Additional PTPN22 S 100A9 4.52E-06 52

Immune/Additional PTPN22 ITGB2 1.27E-05 52

Immune/Additional PTPN22 PTPRC 0.000269 52

Immune/Additional PTPN22 CXCL13 1.22E-08 48

Immune/Additional PTPN22 PHACTRl 9.01E-08 52

Immune/Additional PTPN22 SPP1 1.15E-07 51

Immune/Additional PTPN22 LCP2 2.41E-06 52

Immune/Additional PTPN22 KRT15 1.39E- 1 1 52

Immune/Additional PTPN22 CXCL9 1.05E-09 52

Immune/Additional PTPN22 HCLS 1 0.000267 52

Immune/Additional RGS 1 IRF 1 3.00E-09 52

Immune/Additional RGS 1 CCL5 5.00E- 10 52

Immune/Additional RGS 1 CD38 8.59E- 10 49

Immune/Additional RGS 1 SELL 7.08E- 10 52

Immune/Additional RGS 1 HLA 1.24E-08 52

Immune/Additional RGS 1 S 100A9 8.85E- 10 52

Immune/Additional RGS 1 ITGB2 2.49E-09 52

Immune/Additional RGS 1 PTPRC 9.02E-09 52

Immune/Additional RGS 1 CXCL13 9.90E- 10 48

Immune/Additional RGS 1 PHACTRl 4.53E- 15 78

Immune/Additional RGS 1 SPP1 7.14E- 16 80

Immune/Additional RGS 1 LCP2 1.81E-09 52 Immune/Additional RGS 1 KRT15 9.63E- 17 79

Immune/Additional RGS 1 CXCL9 1.28E- 10 52

Immune/Additional RGS 1 HCLS 1 7.83E-09 52

Immune/Additional IRF 1 CCL5 2.15E-09 52

Immune/Additional IRF 1 CD38 1.45E-08 49

Immune/Additional IRF 1 SELL 1.31E-08 52

Immune/Additional IRF 1 HLA 5.22E-07 52

Immune/Additional IRF 1 S 100A9 5.05E-08 52

Immune/Additional IRF 1 ITGB2 5.80E-07 52

Immune/Additional IRF 1 PTPRC 4.43E-07 52

Immune/Additional IRF 1 CXCL13 5.13E-09 48

Immune/Additional IRF 1 PHACTR1 1.09E-08 52

Immune/Additional IRF 1 SPP1 1.90E-08 51

Immune/Additional IRF 1 LCP2 2.48E-07 52

Immune/Additional IRF 1 KRT15 1.40E- 12 52

Immune/Additional IRF 1 CXCL9 1.16E-09 52

Immune/Additional IRF 1 HCLS 1 2.76E-07 52

Immune/Additional CCL5 CD38 1.06E-09 49

Immune/Additional CCL5 SELL 1.69E-09 52

Immune/Additional CCL5 HLA 3.02E-09 52

Immune/Additional CCL5 S 100A9 1.40E-09 52

Immune/Additional CCL5 ITGB2 2.93E-09 52

Immune/Additional CCL5 PTPRC 3.10E- 13 52

Immune/ Additional CCL5 CXCL13 2.12E-09 48

Immune/Additional CCL5 PHACTR1 1.49E- 10 52

Immune/Additional CCL5 SPP1 3.63E-10 51

Immune/Additional CCL5 LCP2 2.51E-09 52

Immune/Additional CCL5 KRT15 1.41E- 12 52

Immune/ Additional CCL5 CXCL9 6.43E- 10 52

Immune/Additional CCL5 HCLS 1 1.36E-09 52

Immune/Additional CD38 SELL 6.31E-09 49

Immune/Additional CD38 HLA 3.70E-08 49

Immune/Additional CD38 S 100A9 1.02E-08 49

Immune/Additional CD38 ITGB2 1.99E-08 49

Immune/Additional CD38 PTPRC 5.17E-08 49

Immune/Additional CD38 CXCL13 3.25E-09 46

Immune/Additional CD38 PHACTR1 4.58E- 10 49

Immune/Additional CD38 SPP1 1.43E-09 48

Immune/Additional CD38 LCP2 3.38E-08 49

Immune/Additional CD38 KRT15 1.17E- 1 1 49

Immune/Additional CD38 CXCL9 2.23E- 10 49

Immune/Additional CD38 HCLS 1 4.45E-08 49

Immune/Additional SELL HLA 2.63E-08 52

Immune/Additional SELL S 100A9 1.25E-08 52

Immune/Additional SELL ITGB2 3.37E-08 52

Immune/Additional SELL PTPRC 1.01E-08 52

Immune/Additional SELL CXCL13 2.54E-09 48

Immune/Additional SELL PHACTR1 8.28E- 12 52

Immune/Additional SELL SPP1 1.07E-09 51

Immune/Additional SELL LCP2 1.86E-08 52

Immune/Additional SELL KRT 15 1.15E- 12 52

Immune/Additional SELL CXCL9 1.03E-09 52

Immune/Additional SELL HCLS 1 9.23E-09 52 Immune/Additional HLA S100A9 6.82E-07 52

Immune/Additional HLA ITGB2 0.000111 52

Immune/Additional HLA PTPRC 0.000408 52

Immune/Additional HLA CXCL13 5.87E-09 48

Immune/Additional HLA PHACTRl 3.24E-06 52

Immune/Additional HLA SPP1 2.32E-06 51

Immune/Additional HLA LCP2 2.63E-06 52

Immune/Additional HLA KRT15 1.04E-09 52

Immune/Additional HLA CXCL9 1.24E-09 52

Immune/Additional HLA HCLS1 0.000741 52

Immune/Additional S100A9 ITGB2 6.11E-07 52

Immune/Additional S100A9 PTPRC 2.82E-05 52

Immune/Additional S100A9 CXCL13 1.20E-08 48

Immune/Additional S100A9 PHACTRl 7.76E-08 52

Immune/Additional S100A9 SPP1 1.35E-09 51

Immune/Additional S100A9 LCP2 6.68E-08 52

Immune/Additional S100A9 KRT15 1.39E-11 52

Immune/Additional S100A9 CXCL9 3.73E-10 52

Immune/Additional S100A9 HCLS1 2.40E-05 52

Immune/Additional ITGB2 PTPRC 5.57E-05 52

Immune/Additional ITGB2 CXCL13 3.27E-09 48

Immune/Additional ITGB2 PHACTRl 1.34E-06 52

Immune/Additional ITGB2 SPP1 1.83E-06 51

Immune/Additional ITGB2 LCP2 1.74E-06 52

Immune/Additional ITGB2 KRT15 1.83E-10 52

Immune/Additional ITGB2 CXCL9 1.27E-09 52

Immune/Additional ITGB2 HCLS1 0.000143 52

Immune/Additional PTPRC CXCL13 1.28E-08 48

Immune/Additional PTPRC PHACTRl 9.30E-07 52

Immune/Additional PTPRC SPP1 1.52E-06 51

Immune/Additional PTPRC LCP2 1.72E-06 52

Immune/Additional PTPRC KRT15 1.38E-11 52

Immune/Additional PTPRC CXCL9 1.30E-10 52

Immune/Additional PTPRC HCLS1 0.001982 52

Immune/Additional CXCL13 PHACTRl 1.04E-09 48

Immune/Additional CXCL13 SPP1 1.24E-09 47

Immune/Additional CXCL13 LCP2 2.17E-09 48

Immune/Additional CXCL13 KRT15 4.98E-11 48

Immune/Additional CXCL13 CXCL9 1.44E-09 48

Immune/Additional CXCL13 HCLS1 8.81E-09 48

Immune/Additional PHACTRl SPP1 1.08E-10 77

Immune/Additional PHACTRl LCP2 5.75E-08 52

Immune/Additional PHACTRl KRT15 1.82E-11 78

Immune/Additional PHACTRl CXCL9 2.74E-11 52

Immune/Additional PHACTRl HCLS1 1.34E-05 52

Immune/Additional SPP1 LCP2 8.37E-08 51

Immune/Additional SPP1 KRT15 8.66E-13 78

Immune/Additional SPP1 CXCL9 1.60E-10 51

Immune/Additional SPP1 HCLS1 5.76E-06 51

Immune/Additional LCP2 KRT15 1.70E-11 52

Immune/Additional LCP2 CXCL9 1.13E-09 52

Immune/Additional LCP2 HCLS1 2.91E-06 52

Immune/Additional KRT15 CXCL9 6.42E-13 52 Immune/Additional KRT 15 HCLS 1 2.51E- 10 52

Immune/Additional CXCL9 HCLS 1 5.62E- 10 52

CCP/Immune/Additional C 18orf24 HEY1 5.85E-05 26

CCP/Immune/Additional C 18orf24 CCL3 3.94E-05 26

CCP/Immune/Additional C 18orf24 FABP7 1.80E-05 26

CCP/Immune/Additional C 18orf24 FN1 5.36E-06 28

CCP/Immune/Additional C 18orf24 PTN 5.99E-05 26

CCP/Immune/Additional C 18orf24 PRAME 3.08E-05 24

CCP/Immune/Additional C 18orf24 BCL2A1 8.35E-06 28

CCP/Immune/Additional C 18orf24 IFI6 8.84E-06 27

CCP/Immune/Additional C 18orf24 CFH 4.86E-05 26

CCP/Immune/Additional C 18orf24 RGS 1 3.63E-06 28

CCP/Immune/Additional C 18orf24 PHACTR1 4.73E-05 26

CCP/Immune/Additional C 18orf24 SPP1 4.43E-06 28

CCP/Immune/Additional C 18orf24 KRT 15 3.32E-05 26

CCP/Immune/Additional PBK HEY1 0.000325 26

CCP/Immune/Additional PBK CCL3 0.00014 26

CCP/Immune/Additional PBK FABP7 0.000157 26

CCP/Immune/Additional PBK FN1 3.72E-05 28

CCP/Immune/Additional PBK PTN 0.000215 26

CCP/Immune/Additional PBK PRAME 0.000425 24

CCP/Immune/Additional PBK BCL2A1 5.82E-05 28

CCP/Immune/Additional PBK IFI6 6.75E-05 27

CCP/Immune/Additional PBK CFH 0.000292 26

CCP/Immune/Additional PBK RGS 1 9.29E-06 28

CCP/Immune/Additional PBK PHACTR1 0.000384 26

CCP/Immune/Additional PBK SPP1 7.27E-06 28

CCP/Immune/Additional PBK KRT15 9.68E-05 26

CCP/Immune/Additional PLK1 HEY1 5.62E-05 27

CCP/Immune/Additional PLK1 CCL3 0.000102 26

CCP/Immune/Additional PLK1 FABP7 5.48E-05 27

CCP/Immune/Additional PLK1 FN1 2.02E-05 29

CCP/Immune/Additional PLK1 PTN 4.72E-05 27

CCP/Immune/Additional PLK1 PRAME 9.46E-05 24

CCP/Immune/Additional PLK1 BCL2A1 1.04E-05 28

CCP/Immune/Additional PLK1 IFI6 2.14E-05 27

CCP/Immune/Additional PLK1 CFH 0.000128 27

CCP/Immune/Additional PLK1 RGS 1 8.92E-06 29

CCP/Immune/Additional PLK1 PHACTR1 0.000164 26

CCP/Immune/Additional PLK1 SPP1 1.30E-05 29

CCP/Immune/Additional PLK1 KRT15 2.78E-05 27

CCP/Immune/Additional DLGAP5 HEY1 0.00312 27

CCP/Immune/Additional DLGAP5 CCL3 9.72E-05 26

CCP/Immune/Additional DLGAP5 FABP7 0.001317 27

CCP/Immune/Additional DLGAP5 FN1 0.000619 29

CCP/Immune/Additional DLGAP5 PTN 0.003429 27

CCP/Immune/Additional DLGAP5 PRAME 0.000944 24

CCP/Immune/Additional DLGAP5 BCL2A1 8.89E-05 28

CCP/Immune/Additional DLGAP5 IFI6 0.000124 27

CCP/Immune/Additional DLGAP5 CFH 0.003343 27

CCP/Immune/Additional DLGAP5 RGS 1 2.08E-05 29

CCP/Immune/Additional DLGAP5 PHACTR1 0.001083 26

CCP/Immune/Additional DLGAP5 SPP 1 1.65E-05 29 CCP/Immune/Additional DLGAP5 KRT 15 0.000588 27

CCP/Immune/Additional FOXM1 HEY1 0.00017 26

CCP/Immune/Additional FOXM1 CCL3 5.45E-05 26

CCP/Immune/Additional FOXM1 FABP7 9.52E-05 26

CCP/Immune/Additional FOXM1 FN1 2.60E-05 28

CCP/Immune/Additional FOXM1 PTN 0.000135 26

CCP/Immune/Additional FOXM1 PRAME 0.000248 24

CCP/Immune/Additional FOXM1 BCL2A1 2.31E-05 28

CCP/Immune/Additional FOXM1 IFI6 2.42E-05 27

CCP/Immune/Additional FOXM1 CFH 0.000167 26

CCP/Immune/Additional FOXM1 RGS 1 5.89E-06 28

CCP/Immune/Additional FOXM1 PHACTR1 0.00015 26

CCP/Immune/Additional FOXM1 SPP1 1.1 1E-05 28

CCP/Immune/Additional FOXM1 KRT 15 6.62E-05 26

CCP/Immune/Additional MCM10 HEY1 0.000797 26

CCP/Immune/Additional MCM10 CCL3 6.76E-05 26

CCP/Immune/Additional MCM10 FABP7 0.000278 26

CCP/Immune/Additional MCM10 FN1 0.000123 28

CCP/Immune/Additional MCM10 PTN 0.000778 26

CCP/Immune/Additional MCM10 PRAME 0.000394 24

CCP/Immune/Additional MCM10 BCL2A1 4.19E-05 28

CCP/Immune/Additional MCM10 IFI6 2.26E-05 27

CCP/Immune/Additional MCM10 CFH 0.000605 26

CCP/Immune/Additional MCM10 RGS 1 1.91E-05 28

CCP/Immune/Additional MCM10 PHACTR1 0.000376 26

CCP/Immune/Additional MCM10 SPP1 2.95E-05 28

CCP/Immune/Additional MCM10 KRT15 0.000253 26

CCP/Immune/Additional CEP55 HEY1 0.006628 27

CCP/Immune/Additional CEP55 CCL3 7.18E-05 26

CCP/Immune/Additional CEP55 FABP7 0.001276 27

CCP/Immune/Additional CEP55 FN1 0.001029 29

CCP/Immune/Additional CEP55 PTN 0.006228 27

CCP/Immune/Additional CEP55 PRAME 6.91E-05 24

CCP/Immune/Additional CEP55 BCL2A1 1.67E-05 28

CCP/Immune/Additional CEP55 IFI6 1.61E-05 27

CCP/Immune/Additional CEP55 CFH 0.004256 27

CCP/Immune/Additional CEP55 RGS 1 6.16E-05 29

CCP/Immune/Additional CEP55 PHACTR1 8.03E-05 26

CCP/Immune/Additional CEP55 SPP1 2.23E-05 29

CCP/Immune/Additional CEP55 KRT 15 0.00161 27

CCP/Immune/Additional RRM2 HEY1 0.002325 27

CCP/Immune/Additional RRM2 CCL3 0.000657 26

CCP/Immune/Additional RRM2 FABP7 0.000572 27

CCP/Immune/Additional RRM2 FN1 0.000235 29

CCP/Immune/Additional RRM2 PTN 0.001985 27

CCP/Immune/Additional RRM2 PRAME 0.001068 24

CCP/Immune/Additional RRM2 BCL2A1 9.16E-05 28

CCP/Immune/Additional RRM2 IFI6 5.58E-05 27

CCP/Immune/Additional RRM2 CFH 0.001764 27

CCP/Immune/Additional RRM2 RGS 1 2.32E-05 29

CCP/Immune/Additional RRM2 PHACTR1 0.001505 26

CCP/Immune/Additional RRM2 SPP1 1.44E-05 29

CCP/Immune/Additional RRM2 KRT 15 7.47E-05 27 CCP/Immune/Additional DTL HEY1 3.43E-05 27

CCP/Immune/Additional DTL CCL3 4.30E-05 26

CCP/Immune/Additional DTL FABP7 4.43E-05 27

CCP/Immune/Additional DTL FN1 6.55E-06 29

CCP/Immune/Additional DTL PTN 3.14E-05 27

CCP/Immune/Additional DTL PRAME 8.49E-05 24

CCP/Immune/Additional DTL BCL2A1 7.58E-06 28

CCP/Immune/Additional DTL IFI6 1.61E-05 27

CCP/Immune/Additional DTL CFH 3.93E-05 27

CCP/Immune/Additional DTL RGS l 6.10E-06 29

CCP/Immune/Additional DTL PHACTRl 7.43E-05 26

CCP/Immune/Additional DTL SPP1 9.41E-06 29

CCP/Immune/Additional DTL KRT 15 1.93E-05 27

CCP/Immune/Additional CENPF HEY1 0.000139 27

CCP/Immune/Additional CENPF CCL3 0.000159 26

CCP/Immune/Additional CENPF FABP7 5.14E-05 27

CCP/Immune/Additional CENPF FN1 3.21E-05 29

CCP/Immune/Additional CENPF PTN 0.000175 27

CCP/Immune/Additional CENPF PRAME 6.06E-05 24

CCP/Immune/Additional CENPF BCL2A1 1.62E-05 28

CCP/Immune/Additional CENPF IFI6 3.12E-05 27

CCP/Immune/Additional CENPF CFH 0.000172 27

CCP/Immune/Additional CENPF RGS l 7.57E-06 29

CCP/Immune/Additional CENPF PHACTRl 0.000169 26

CCP/Immune/Additional CENPF SPP1 1.05E-05 29

CCP/Immune/Additional CENPF KRT15 2.96E-05 27

Table YY

Immune/Additional FABP7 RGSl PHACTRl 3.40E-14 78

Immune/Additional HEY1 FABP7 RGSl 3.41E-14 79

Immune/Additional PRAME RGSl SPP1 3.64E-14 75

Immune/Additional CFH RGSl SPP1 3.74E-14 78

Immune/Additional HEY1 BCL2A1 RGSl 3.86E-14 78

Immune/Additional PTN RGSl PHACTRl 3.95E-14 78

Immune/Additional HEY1 PRAME RGSl 4.02E-14 76

Immune/Additional PTN RGSl SPP1 4.11E-14 78

Immune/Additional FN1 PRAME RGSl 4.64E-14 76

Immune/Additional CFH RGSl PHACTRl 4.94E-14 78

Immune/Additional FABP7 RGSl SPP1 5.15E-14 78

Immune/Additional PRAME SPP1 KRT15 5.41E-14 75

Immune/Additional CCL3 RGSl PHACTRl 5.56E-14 78

Immune/Additional CCL3 RGSl SPP1 6.03E-14 77

Immune/Additional HEY1 CCL3 RGSl 6.22E-14 78

Immune/Additional FABP7 BCL2A1 RGSl 7.77E-14 78

Immune/Additional PTN BCL2A1 RGSl 8.10E-14 78

Immune/Additional PRAME BCL2A1 RGSl 9.11E-14 76

Immune/Additional PTN PRAME RGSl 9.99E-14 76

Immune/Additional BCL2A1 CFH RGSl 1.07E-13 78

Immune/Additional PRAME IFI6 RGSl 1.10E-13 71

Immune/Additional CCL3 BCL2A1 RGSl 1.12E-13 78

Immune/Additional FN1 IFI6 RGSl 1.18E-13 75

Immune/Additional FABP7 PRAME RGSl 1.22E-13 76

Immune/Additional HEY1 FN1 RGSl 1.31E-13 79

Immune/Additional HEY1 PTN RGSl 1.34E-13 79

Immune/Additional IFI6 RGSl PHACTRl 1.49E-13 73

Immune/Additional HEY1 IFI6 RGSl 1.65E-13 73

Immune/Additional HEY1 CFH RGSl 1.86E-13 79

Immune/Additional CCL3 PRAME RGSl 1.91E-13 76

Immune/Additional PRAME CFH RGSl 1.97E-13 76

Immune/Additional FABP7 FN1 RGSl 2.40E-13 79

Immune/Additional CCL3 FN1 RGSl 2.50E-13 78

Immune/Additional FABP7 PRAME KRT15 2.70E-13 76

Immune/Additional PRAME PHACTRl KRT15 2.94E-13 76

Immune/Additional FN1 CFH RGSl 2.99E-13 79

Immune/Additional FABP7 CFH RGSl 3.62E-13 79

Immune/Additional CCL3 SPP1 KRT15 3.97E-13 77

Immune/Additional PRAME CFH KRT15 4.09E-13 76

Immune/Additional HEY1 PRAME KRT15 4.25E-13 76

Immune/Additional FABP7 IFI6 RGSl 4.97E-13 73

Immune/Additional FN1 PRAME KRT15 5.45E-13 76

Immune/Additional IFI6 CFH RGSl 5.48E-13 73

Immune/Additional FABP7 PTN RGSl 5.52E-13 79

Immune/Additional HEY1 CCL3 KRT15 5.81E-13 78

Immune/Additional PTN CFH RGSl 5.84E-13 79

Immune/Additional CCL3 FN1 KRT15 6.03E-13 78

Immune/Additional CCL3 CFH RGSl 6.37E-13 78

Immune/Additional CCL3 FABP7 RGSl 6.69E-13 78

Immune/Additional CCL3 PTN KRT15 7.34E-13 78

Immune/Additional CCL3 CFH KRT15 7.78E-13 78

Immune/Additional PTN PRAME KRT15 8.20E-13 76

Immune/Additional CCL3 PHACTRl KRT15 8.53E-13 78 Immune/Additional CCL3 FABP7 KRT15 9.58E-13 78

Immune/Additional CCL3 IFI6 RGS1 9.78E-13 73

Immune/Additional CCL3 BCL2A1 KRT15 9.85E-13 78

Immune/Additional PRAME BCL2A1 KRT15 1.25E-12 76

Immune/Additional CFH SPP1 KRT15 1.35E-12 78

Immune/Additional FN1 PTN RGS1 1.55E-12 79

Immune/Additional PTN IFI6 RGS1 1.70E-12 73

Immune/Additional HEY1 SPP1 KRT15 1.86E-12 78

Immune/Additional CCL3 PTN RGS1 2.20E-12 78

Immune/Additional CCL3 IFI6 KRT15 2.24E-12 73

Immune/Additional PRAME IFI6 KRT15 2.87E-12 71

Immune/Additional RGS1 CCL5 PTPRC 3.53E-12 52

Immune/Additional CCL3 PRAME SPP1 3.65E-12 75

Immune/Additional BCL2A1 CCL5 PTPRC 3.67E-12 52

Immune/Additional PTPN22 CCL5 PTPRC 3.92E-12 52

Immune/Additional PHACTR1 SPP1 KRT15 4.11E-12 77

Immune/Additional PRAME CCL5 PTPRC 4.36E-12 52

Immune/Additional CCL5 PTPRC PHACTR1 4.48E-12 52

Immune/Additional FABP7 CCL5 PTPRC 4.71E-12 52

Immune/Additional CCL5 S100A9 KRT15 4.88E-12 52

Immune/Additional HEY1 CCL5 PTPRC 4.91E-12 52

Immune/Additional CCL5 PTPRC KRT15 4.92E-12 52

Immune/Additional PRAME IFI6 SPP1 4.93E-12 70

Immune/Additional PRAME SELL KRT15 4.93E-12 52

Immune/Additional CCL5 HLA PTPRC 5.28E-12 52

Immune/Additional CCL3 CCL5 PTPRC 5.34E-12 52

Immune/Additional CCL5 S100A9 PTPRC 5.46E-12 52

Immune/Additional PTN SPP1 KRT15 5.53E-12 78

Immune/Additional PTN KRT15 CXCL9 5.57E-12 52

Immune/Additional FABP7 SPP1 KRT15 5.61E-12 78

Immune/Additional CCL5 PTPRC LCP2 5.87E-12 52

Immune/Additional CCL5 SELL KRT15 5.98E-12 52

Immune/Additional CCL5 SELL PTPRC 6.10E-12 52

Immune/Additional IRF1 CCL5 PTPRC 6.13E-12 52

Immune/Additional S100A9 PTPRC KRT15 6.37E-12 52

Immune/Additional SELL S100A9 KRT15 6.38E-12 52

Immune/Additional S100A9 LCP2 KRT15 6.50E-12 52

Immune/Additional PTN CCL5 PTPRC 6.54E-12 52

Immune/Additional HLA CCL5 PTPRC 6.65E-12 52

Immune/Additional PRAME PHACTR1 SPP1 6.68E-12 75

Immune/Additional FN1 CCL5 PTPRC 6.74E-12 52

Immune/Additional CFH CCL5 PTPRC 6.79E-12 52

Immune/Additional FN1 SPP1 KRT15 6.83E-12 78

Immune/Additional RGS1 SELL KRT15 6.83E-12 52

Immune/Additional PECAMl CCL5 PTPRC 6.86E-12 52

Immune/Additional BCL2A1 SPP1 KRT15 6.88E-12 77

Immune/Additional S100A9 KRT15 CXCL9 6.94E-12 52

Immune/Additional RGS1 KRT15 CXCL9 7.07E-12 52

Immune/Additional PRAME KRT15 CXCL9 7.25E-12 52

Immune/Additional CCL5 PTPRC HCLS1 7.35E-12 52

Immune/Additional SELL ITGB2 KRT15 7.68E-12 52

Immune/Additional SELL KRT15 CXCL9 7.73E-12 52

Immune/Additional PECAMl S100A9 KRT15 7.83E-12 52 Immune/Additional CCL3 KRT15 CXCL9 7.90E-12 52

Immune/Additional CCL5 PTPRC CXCL9 7.97E-12 52

Immune/Additional LCP2 KRT15 CXCL9 8.01E-12 52

Immune/Additional PEC AMI KRT15 CXCL9 8.05E-12 52

Immune/Additional S100A9 KRT15 HCLS1 8.43E-12 52

Immune/Additional CCL5 ITGB2 PTPRC 8.51E-12 52

Immune/Additional PHACTRl KRT15 CXCL9 8.70E-12 52

Immune/Additional FABP7 PRAME SPP1 8.72E-12 75

Immune/Additional IRF1 S100A9 KRT15 8.92E-12 52

Immune/Additional FN1 PRAME SPP1 8.99E-12 75

Immune/Additional SELL PHACTRl HCLS1 9.11E-12 52

Immune/Additional HLA KRT15 CXCL9 9.25E-12 52

Immune/Additional HLA KRT15 CXCL9 9.70E-12 52

Immune/Additional PRAME IRF1 KRT15 1.03E-11 52

Immune/Additional ITGB2 KRT15 CXCL9 1.03E-11 52

Immune/Additional FABP7 KRT15 CXCL9 1.05E-11 52

Immune/Additional IRF1 KRT15 CXCL9 1.05E-11 52

Immune/Additional KRT15 CXCL9 HCLS1 1.05E-11 52

Immune/Additional HEY1 KRT15 CXCL9 1.06E-11 52

Immune/Additional CCL3 IRF1 KRT15 1.09E-11 52

Immune/Additional CCL5 PTPRC SPP1 l.lOE-11 51

Immune/Additional SELL KRT15 HCLS1 l.lOE-11 52

Immune/Additional PRAME CCL5 KRT15 1.12E-11 52

Immune/Additional CCL5 PHACTRl KRT15 1.12E-11 52

Immune/Additional BCL2A1 KRT15 CXCL9 1.14E-11 52

Immune/Additional PTPN22 S100A9 KRT15 1.17E-11 52

Immune/Additional CFH SELL KRT15 1.20E-11 52

Immune/Additional CCL3 CCL5 KRT15 1.24E-11 52

Immune/Additional PTPN22 KRT15 CXCL9 1.24E-11 52

Immune/Additional RGS1 CCL5 KRT15 1.24E-11 52

Immune/Additional PTPRC KRT15 CXCL9 1.26E-11 52

Immune/Additional IRF1 HLA KRT15 1.27E-11 52

Immune/Additional SELL PTPRC KRT15 1.30E-11 52

Immune/Additional S100A9 ITGB2 KRT15 1.31E-11 52

Immune/Additional CCL5 KRT15 CXCL9 1.32E-11 52

Immune/Additional PRAME PTPRC KRT15 1.33E-11 52

Immune/Additional IRF1 ITGB2 KRT15 1.35E-11 52

Immune/Additional IGJ PHACTRl CXCL9 1.36E-11 50

Immune/Additional PEC AMI CCL5 KRT15 1.36E-11 52

Immune/Additional SELL PHACTRl KRT15 1.41E-11 52

Immune/Additional CCL5 HLA KRT15 1.42E-11 52

Immune/Additional FN1 KRT15 CXCL9 1.45E-11 52

Immune/Additional PTPN22 SELL KRT15 1.48E-11 52

Immune/Additional IRF1 CCL5 KRT15 1.48E-11 52

Immune/Additional IRF1 KRT15 HCLS1 1.49E-11 52

Immune/Additional IGJ CCL5 PTPRC 1.50E-11 50

Immune/Additional BCL2A1 SELL KRT15 1.50E-11 52

Immune/Additional HLA S100A9 KRT15 1.51E-11 52

Immune/Additional HLA SELL KRT15 1.53E-11 52

Immune/Additional BCL2A1 CCL5 KRT15 1.57E-11 52

Immune/Additional CCL5 LCP2 KRT15 1.57E-11 52

Immune/Additional IGJ KRT15 CXCL9 1.59E-11 50

Immune/Additional PRAME PTPN22 KRT15 1.62E-11 52 Immune/Additional RGS1 S100A9 KRT15 1.62E-11 52

Immune/Additional IRF1 SELL KRT15 1.63E-11 52

Immune/Additional CCL3 SELL KRT15 1.65E-11 52

Immune/Additional HEY1 CCL5 KRT15 1.66E-11 52

Immune/Additional SELL HLA KRT15 1.66E-11 52

Immune/Additional SELL LCP2 KRT15 1.70E-11 52

Immune/Additional CFH KRT15 CXCL9 1.71E-11 52

Immune/Additional IGJ CCL5 KRT15 1.75E-11 50

Immune/Additional PRAME KRT15 HCLS1 1.77E-11 52

Immune/Additional CFH IRF1 KRT15 1.79E-11 52

Immune/Additional CXCLIO RGS1 KRT15 1.83E-11 50

Immune/Additional PRAME LCP2 KRT15 1.85E-11 52

Immune/Additional FN1 SELL KRT15 1.87E-11 52

Immune/Additional HLA IRF1 KRT15 1.87E-11 52

Immune/Additional PRAME PECAMl KRT15 1.92E-11 52

Immune/Additional IRF1 LCP2 KRT15 1.93E-11 52

Immune/Additional CD38 KRT15 CXCL9 1.94E-11 49

Immune/Additional HEY1 PRAME SPP1 1.95E-11 75

Immune/Additional SPP1 KRT15 CXCL9 1.97E-11 51

Immune/Additional FABP7 SELL KRT15 1.98E-11 52

Immune/Additional HLA S100A9 KRT15 1.98E-11 52

Immune/Additional SELL ITGB2 PHACTRl 1.99E-11 52

Immune/Additional PTN CCL5 KRT15 2.00E-11 52

Immune/Additional HEY1 FABP7 KRT15 2.02E-11 79

Immune/Additional FABP7 IRF1 KRT15 2.02E-11 52

Immune/Additional PTN SELL KRT15 2.02E-11 52

Immune/Additional HEY1 RGS1 SELL 2.04E-11 52

Immune/Additional IGJ PRAME CXCL9 2.04E-11 50

Immune/Additional IRF1 PTPRC KRT15 2.04E-11 52

Immune/Additional CFH SELL PHACTRl 2.05E-11 52

Immune/Additional IRF1 PHACTRl KRT15 2.10E-11 52

Immune/Additional HLA CCL5 KRT15 2.12E-11 52

Immune/Additional PTN IRF1 KRT15 2.16E-11 52

Immune/Additional PRAME BCL2A1 SPP1 2.16E-11 75

Immune/Additional PECAMl SELL KRT15 2.19E-11 52

Immune/Additional CCL5 KRT15 HCLS1 2.21E-11 52

Immune/Additional HEY1 SELL KRT15 2.22E-11 52

Immune/Additional CXCLIO SELL KRT15 2.24E-11 50

Immune/Additional CCL5 ITGB2 KRT15 2.27E-11 52

Immune/Additional CXCLIO IRF1 KRT15 2.28E-11 50

Immune/Additional CXCLIO S100A9 KRT15 2.30E-11 50

Immune/Additional FN1 CCL5 KRT15 2.33E-11 52

Immune/Additional PTPN22 CCL5 KRT15 2.33E-11 52

Immune/Additional RGS1 IRF1 KRT15 2.35E-11 52

Immune/Additional CXCLIO CCL5 PTPRC 2.45E-11 50

Immune/Additional FABP7 CCL5 KRT15 2.45E-11 52

Immune/Additional PTN PRAME SPP1 2.45E-11 75

Immune/Additional CCL5 CD38 PTPRC 2.45E-11 49

Immune/Additional CCL5 CD38 KRT15 2.51E-11 49

Immune/Additional SELL SPP1 KRT15 2.53E-11 51

Immune/Additional BCL2A1 IRF1 KRT15 2.56E-11 52

Immune/Additional IRF1 CD38 KRT15 2.57E-11 49

Immune/Additional CXCLIO PRAME KRT15 2.68E-11 50 Immune/Additional CXCLIO PHACTRl KRT15 2.71E-11 50

Immune/Additional CCL3 IFI6 SPP1 2.73E-11 72

Immune/Additional PRAME S100A9 KRT15 2.74E-11 52

Immune/Additional FN1 CXCL9 HCLS1 2.76E-11 52

Immune/Additional IFI6 KRT15 CXCL9 2.78E-11 48

Immune/Additional HEY1 IRF1 KRT15 2.79E-11 52

Immune/Additional HEY1 S100A9 KRT15 2.79E-11 52

Immune/Additional HEY1 CXCLIO KRT15 2.80E-11 50

Immune/Additional CXCLIO FABP7 KRT15 2.80E-11 50

Immune/Additional CFH CCL5 KRT15 2.82E-11 52

Immune/Additional CXCLIO KRT15 CXCL9 2.87E-11 50

Immune/Additional CXCLIO HLA KRT15 2.88E-11 50

Immune/Additional CXCLIO RGS1 PTPRC 2.90E-11 50

Immune/Additional RGS1 LCP2 KRT15 2.93E-11 52

Immune/Additional PECAMl IRF1 KRT15 2.95E-11 52

Immune/Additional CXCLIO BCL2A1 KRT15 2.96E-11 50

Immune/Additional PTPN22 IRF1 KRT15 2.96E-11 52

Immune/Additional CXCLIO HLA KRT15 2.97E-11 50

Immune/Additional PRAME CFH SPP1 3.01E-11 75

Immune/Additional CXCLIO PTPRC KRT15 3.03E-11 50

Immune/Additional FN1 IRF1 KRT15 3.06E-11 52

Immune/Additional CCL5 SPP1 KRT15 3.07E-11 51

Immune/Additional RGS1 PTPRC KRT15 3.15E-11 52

Immune/Additional CXCLIO PTPN22 KRT15 3.16E-11 50

Immune/Additional CXCLIO ITGB2 KRT15 3.16E-11 50

Immune/Additional CD38 SELL KRT15 3.22E-11 49

Immune/Additional HEY1 CFH KRT15 3.24E-11 79

Immune/Additional IRF1 SPP1 KRT15 3.29E-11 51

Immune/Additional PECAMl SELL PHACTRl 3.33E-11 52

Immune/Additional RGS1 CD38 KRT15 3.33E-11 49

Immune/Additional CXCLIO CCL3 KRT15 3.36E-11 50

Immune/Additional CXCLIO PTN KRT15 3.43E-11 50

Immune/Additional CXCLIO CCL5 KRT15 3.43E-11 50

Immune/Additional HEY1 RGS1 LCP2 3.44E-11 52

Immune/Additional CXCLIO LCP2 KRT15 3.46E-11 50

Immune/Additional CCL3 S100A9 KRT15 3.49E-11 52

Immune/Additional IFI6 PHACTRl CXCL9 3.53E-11 48

Immune/Additional CXCLIO KRT15 HCLS1 3.55E-11 50

Immune/Additional SELL CXCL13 KRT15 3.61E-11 48

Immune/Additional S100A9 SPP1 KRT15 3.66E-11 51

Immune/Additional CCL3 FN1 PRAME 3.67E-11 76

Immune/Additional PRAME ITGB2 KRT15 3.72E-11 52

Immune/Additional PRAME IFI6 CXCL9 3.77E-11 48

Immune/Additional HEY1 RGS1 S100A9 3.82E-11 52

Immune/Additional HEY1 SELL PHACTRl 3.85E-11 52

Immune/Additional HEY1 PHACTRl KRT15 3.85E-11 78

Immune/Additional CXCLIO CFH KRT15 3.96E-11 50

Immune/Additional CCL5 PTPRC CXCL13 4.05E-11 48

Immune/Additional PTPN22 RGS1 KRT15 4.09E-11 52

Immune/Additional CXCL13 LCP2 KRT15 4.09E-11 48

Immune/Additional CXCLIO FN1 KRT15 4.10E-11 50

Immune/Additional CXCLIO SPP1 KRT15 4.23E-11 49

Immune/Additional FN1 BCL2A1 SPP1 4.23E-11 79 Immune/Additional PECAM1 CD38 KRT15 4.24E-11 49

Immune/Additional CCL3 PHACTRl SPP1 4.27E-11 77

Immune/Additional CXCLIO PEC AMI KRT15 4.33E-11 50

Immune/Additional RGSl SELL PHACTRl 4.37E-11 52

Immune/Additional IGJ CCL5 PHACTRl 4.43E-11 50

Immune/Additional CXCLIO CD38 KRT15 4.49E-11 48

Immune/Additional HEY1 RGSl CD38 4.51E-11 49

Immune/Additional PRAME HLA KRT15 4.57E-11 52

Immune/Additional CCL3 PRAME IFI6 4.61E-11 71

Immune/Additional CCL5 SELL PHACTRl 4.81E-11 52

Immune/Additional IGJ SELL KRT15 4.84E-11 50

Immune/Additional FN1 HLA CXCL9 4.87E-11 52

Immune/Additional CXCLIO RGSl PHACTRl 4.95E-11 50

Immune/Additional SELL PHACTRl CXCL9 4.95E-11 52

Immune/Additional CD38 ITGB2 KRT15 4.98E-11 49

Immune/Additional CCL5 CXCL13 KRT15 5.03E-11 48

Immune/Additional CXCL13 KRT15 CXCL9 5.06E-11 48

Immune/Additional PRAME SELL PHACTRl 5.08E-11 52

Immune/Additional RGSl ITGB2 KRT15 5.14E-11 52

Immune/Additional CXCLIO RGSl IRF1 5.15E-11 50

Immune/Additional SELL PHACTRl LCP2 5.22E-11 52

Immune/Additional CCL3 FN1 SPP1 5.30E-11 77

Immune/Additional IFI6 SPP1 KRT15 5.31E-11 72

Immune/Additional RGSl KRT15 HCLS1 5.32E-11 52

Immune/Additional HEY1 IGJ CCL5 5.33E-11 50

Immune/Additional SELL S100A9 PHACTRl 5.34E-11 52

Immune/Additional ITGB2 CXCL13 KRT15 5.37E-11 48

Immune/Additional CXCLIO HLA RGSl 5.43E-11 50

Immune/Additional HEY1 IFI6 SELL 5.47E-11 48

Immune/Additional CD38 LCP2 KRT15 5.53E-11 49

Immune/Additional FN1 CD38 KRT15 5.56E-11 49

Immune/Additional IFI6 CCL5 PTPRC 5.67E-11 48

Immune/Additional IGJ IRF1 KRT15 5.68E-11 50

Immune/Additional FABP7 CD38 KRT15 5.75E-11 49

Immune/Additional HLA CD38 KRT15 6.00E-11 49

Immune/Additional SELL PTPRC PHACTRl 6.02E-11 52

Immune/Additional CD38 PTPRC KRT15 6.03E-11 49

Immune/Additional HEY1 IFI6 CCL5 6.08E-11 48

Immune/Additional CD38 PHACTRl CXCL9 6.09E-11 49

Immune/Additional HEY1 FN1 KRT15 6.28E-11 79

Immune/Additional IFI6 CCL5 KRT15 6.32E-11 48

Immune/Additional HEY1 RGSl CCL5 6.36E-11 52

Immune/Additional FABP7 S100A9 KRT15 6.45E-11 52

Immune/Additional FN1 PHACTRl KRT15 6.46E-11 78

Immune/Additional CCL3 CD38 KRT15 6.61E-11 49

Immune/Additional PECAM1 RGSl KRT15 6.70E-11 52

Immune/Additional PTN S100A9 KRT15 6.72E-11 52

Immune/Additional HEY1 CXCLIO RGSl 6.74E-11 50

Immune/Additional CCL3 CFH SPP1 6.74E-11 77

Immune/Additional CCL3 SELL PHACTRl 6.86E-11 52

Immune/Additional CD38 HLA KRT15 6.89E-11 49

Immune/Additional IRF1 CXCL13 KRT15 6.91E-11 48

Immune/Additional CFH PHACTRl KRT15 6.93E-11 78 Immune/Additional CXCL10 PTPRC CXCL9 6.96E-11 50

Immune/Additional IRF1 SELL PHACTRl 6.99E-11 52

Immune/Additional HLA RGS1 KRT15 7.02E-11 52

Immune/Additional PTPRC CXCL13 KRT15 7.04E-11 48

Immune/Additional CCL3 IFI6 CXCL9 7.06E-11 48

Immune/Additional PTPN22 CD38 KRT15 7.08E-11 49

Immune/Additional PTN CD38 KRT15 7.13E-11 49

Immune/Additional FABP7 CFH KRT15 7.14E-11 79

Immune/Additional HEY1 PTN KRT15 7.16E-11 79

Immune/Additional IGJ LCP2 KRT15 7.17E-11 50

Immune/Additional FN1 PHACTRl CXCL9 7.19E-11 52

Immune/Additional FN1 IFI6 CXCL9 7.20E-11 48

Immune/Additional PTPRC PHACTRl KRT15 7.23E-11 52

Immune/Additional CFH RGS1 SELL 7.26E-11 52

Immune/Additional CD38 KRT15 HCLS1 7.26E-11 49

Immune/Additional CXCL13 KRT15 HCLS1 7.32E-11 48

Immune/Additional S100A9 PHACTRl KRT15 7.48E-11 52

Immune/Additional FN1 S100A9 SPP1 7.49E-11 51

Immune/Additional FN1 HLA CCL5 7.51E-11 52

Immune/Additional FN1 CFH SELL 7.72E-11 52

Immune/Additional HLA LCP2 KRT15 7.75E-11 52

Immune/Additional CXCL10 SELL PHACTRl 7.84E-11 50

Immune/Additional FN1 CFH CXCL9 7.87E-11 52

Immune/Additional RGS1 S100A9 PHACTRl 7.92E-11 52

Immune/Additional PECAMl CXCL13 KRT15 8.25E-11 48

Immune/Additional IFI6 CFH SELL 8.32E-11 48

Immune/Additional FABP7 IGJ CXCL9 8.34E-11 50

Immune/Additional FN1 S100A9 KRT15 8.35E-11 52

Immune/Additional CXCL10 PTPN22 RGS1 8.42E-11 50

Immune/Additional IGJ PTPRC KRT15 8.51E-11 50

Immune/Additional IGJ RGS1 CXCL9 8.59E-11 50

Immune/Additional PRAME PTPRC CXCL9 8.59E-11 52

Immune/Additional PTN PHACTRl CXCL9 8.62E-11 52

Immune/Additional RGS1 HLA KRT15 8.76E-11 52

Immune/Additional IFI6 SELL KRT15 8.77E-11 48

Immune/Additional FABP7 FN1 KRT15 8.88E-11 79

Immune/Additional FN1 BCL2A1 KRT15 8.91E-11 78

Immune/Additional FN1 SELL PHACTRl 8.91E-11 52

Immune/Additional BCL2A1 SELL PHACTRl 8.91E-11 52

Immune/Additional PTPRC PHACTRl CXCL9 8.92E-11 52

Immune/Additional IFI6 IRF1 KRT15 9.11E-11 48

Immune/Additional FABP7 PHACTRl KRT15 9.12E-11 78

Immune/Additional FABP7 BCL2A1 KRT15 9.23E-11 78

Immune/Additional FN1 CFH KRT15 9.28E-11 79

Immune/Additional RGS1 PHACTRl CXCL9 9.60E-11 52

Immune/Additional PHACTRl LCP2 KRT15 9.62E-11 52

Immune/Additional CFH CD38 KRT15 9.70E-11 49

Immune/Additional PTPN22 PHACTRl KRT15 9.76E-11 52

Immune/Additional IFI6 CD38 KRT15 9.78E-11 47

Immune/Additional CCL3 BCL2A1 SPP1 9.79E-11 77

Immune/Additional HLA SELL PHACTRl 9.92E-11 52

Immune/Additional HEY1 BCL2A1 KRT15 l.OlE-10 78

Immune/Additional PTN PHACTRl KRT15 l.OlE-10 78 Immune/Additional FN1 PTPN22 CCL5 1.02E-10 52

Immune/Additional HEY1 RGS1 CXCL9 1.03E-10 52

Immune/Additional FN1 CXCL13 KRT15 1.03E-10 48

Immune/Additional CXCLIO RGS1 HLA 1.04E-10 50

Immune/Additional HLA PTPRC KRT15 1.04E-10 52

Immune/Additional CXCLIO FN1 RGS1 1.05E-10 50

Immune/Additional BCL2A1 S100A9 KRT15 1.05E-10 52

Immune/Additional SELL HLA PHACTRl 1.05E-10 52

Immune/Additional CD38 PHACTRl KRT15 1.06E-10 49

Immune/Additional PTPN22 HLA KRT15 1.07E-10 52

Immune/Additional PTPN22 CXCL13 KRT15 1.07E-10 48

Immune/Additional PTPRC LCP2 KRT15 1.07E-10 52

Immune/Additional HEY1 CFH SELL 1.08E-10 52

Immune/Additional HLA CXCL13 KRT15 1.08E-10 48

Immune/Additional HEY1 CCL3 SPP1 1.09E-10 77

Immune/Additional CXCLIO IGJ RGS1 l.lOE-10 48

Immune/Additional PRAME HLA KRT15 l.lOE-10 52

Immune/Additional CXCLIO IGJ KRT15 1.1 lE-10 48

Immune/Additional IGJ RGS1 KRT15 1.1 lE-10 50

Immune/Additional PTPN22 SELL PHACTRl 1.12E-10 52

Immune/Additional HEY1 CCL5 CD38 1.13E-10 49

Immune/Additional BCL2A1 IFI6 SPP1 1.13E-10 74

Immune/Additional SELL PHACTRl SPP1 1.13E-10 51

Immune/Additional PRAME CD38 KRT15 1.14E-10 49

Immune/Additional S100A9 PHACTRl CXCL9 1.14E-10 52

Immune/Additional RGS1 PTPRC CXCL9 1.15E-10 52

Immune/Additional PTPN22 LCP2 KRT15 1.16E-10 52

Immune/Additional FN1 IGJ CXCL9 1.17E-10 50

Immune/Additional FN1 PTPRC CXCL9 1.19E-10 52

Immune/Additional PTN SELL PHACTRl 1.19E-10 52

Immune/Additional IFI6 CCL5 HLA 1.19E-10 48

Immune/Additional CD38 SPP1 KRT15 1.21E-10 48

Immune/Additional IFI6 CCL5 PHACTRl 1.22E-10 48

Immune/Additional FN1 S100A9 CXCL9 1.24E-10 52

Immune/Additional HEY1 RGS1 IRF1 1.25E-10 52

Immune/Additional CCL3 FN1 CFH 1.26E-10 78

Immune/Additional CXCLIO RGS1 CCL5 1.28E-10 50

Immune/Additional S100A9 PTPRC CXCL9 1.28E-10 52

Immune/Additional HEY1 RGS1 PTPRC 1.30E-10 52

Immune/Additional CXCLIO CXCL13 KRT15 1.30E-10 46

Immune/Additional FN1 PRAME CXCL9 1.30E-10 52

Immune/Additional CD38 PTPRC CXCL9 1.3 lE-10 49

Immune/Additional FABP7 CXCL13 KRT15 1.32E-10 48

Immune/Additional IGJ CFH CXCL9 1.33E-10 50

Immune/Additional CCL3 FABP7 SPP1 1.34E-10 77

Immune/Additional RGS1 CXCL13 KRT15 1.36E-10 48

Immune/Additional CD38 S100A9 KRT15 1.37E-10 49

Immune/Additional HEY1 RGS1 ITGB2 1.40E-10 52

Immune/Additional HEY1 CD38 CXCL9 1.40E-10 49

Immune/Additional FABP7 SELL PHACTRl 1.40E-10 52

Immune/Additional CXCLIO IFI6 KRT15 1.41E-10 47

Immune/Additional HEY1 CD38 SELL 1.43E-10 49

Immune/Additional BCL2A1 PHACTRl KRT15 1.43E-10 78 Immune/Additional IFI6 CCL5 CXCL9 1.44E-10 48

Immune/Additional IFI6 S100A9 CXCL9 1.45E-10 48

Immune/Additional FABP7 FN1 PRAME 1.48E-10 76

Immune/Additional BCL2A1 CFH KRT15 1.48E-10 78

Immune/Additional CXCLIO RGS1 HCLS1 1.54E-10 50

Immune/Additional FABP7 PTN KRT15 1.54E-10 79

Immune/Additional HLA CXCL13 KRT15 1.54E-10 48

Immune/Additional ITGB2 PTPRC KRT15 1.54E-10 52

Immune/Additional CXCLIO CFH RGS1 1.55E-10 50

Immune/Additional IFI6 SELL PHACTRl 1.55E-10 48

Immune/Additional PTN CFH KRT15 1.57E-10 79

Immune/Additional BCL2A1 PTPRC KRT15 1.57E-10 52

Immune/Additional CFH RGS1 CXCL9 1.58E-10 52

Immune/Additional PTPN22 SPPl KRT15 1.58E-10 51

Immune/Additional HEY1 RGS1 HLA 1.60E-10 52

Immune/Additional CFH LCP2 KRT15 1.60E-10 52

Immune/Additional PTPN22 ITGB2 KRT15 1.60E-10 52

Immune/Additional CFH CCL5 SELL 1.61E-10 52

Immune/Additional S100A9 SPPl CXCL9 1.61E-10 51

Immune/Additional HEY1 PECAM1 RGS1 1.62E-10 52

Immune/Additional CCL3 PTPRC KRT15 1.62E-10 52

Immune/Additional IGJ CCL5 CXCL9 1.63E-10 50

Immune/Additional BCL2A1 CD38 KRT15 1.63E-10 49

Immune/Additional PTN PTPRC KRT15 1.64E-10 52

Immune/Additional IFI6 PECAM1 CXCL9 1.64E-10 48

Immune/Additional PTPN22 PTPRC KRT15 1.64E-10 52

Immune/Additional HEY1 PTPN22 RGS1 1.66E-10 52

Immune/Additional CXCLIO FN1 HLA 1.68E-10 50

Immune/Additional CFH RGS1 CCL5 1.68E-10 52

Immune/Additional CCL3 FN1 BCL2A1 1.69E-10 78

Immune/Additional PHACTRl SPPl CXCL9 1.69E-10 51

Immune/Additional CXCLIO SELL PTPRC 1.70E-10 50

Immune/Additional CCL3 PTN SPPl 1.70E-10 77

Immune/Additional CCL3 PTPN22 KRT15 1.70E-10 52

Immune/Additional CXCLIO HLA SELL 1.71E-10 50

Immune/Additional BCL2A1 PTPN22 KRT15 1.73E-10 52

Immune/Additional PRAME RGS1 SELL 1.74E-10 52

Immune/Additional PTPN22 KRT15 HCLS1 1.76E-10 52

Immune/Additional HEY1 RGS1 HCLS1 1.77E-10 52

Immune/Additional HEY1 CD38 KRT15 1.77E-10 49

Immune/Additional IGJ PRAME CCL5 1.77E-10 50

Immune/Additional HEY1 CCL3 CD38 1.78E-10 49

Immune/Additional IGJ SELL PHACTRl 1.79E-10 50

Immune/Additional HEY1 IFI6 CXCL9 1.80E-10 48

Immune/Additional PTN PTPN22 KRT15 1.80E-10 52

Immune/Additional PTN LCP2 KRT15 1.81E-10 52

Immune/Additional FN1 PTN KRT15 1.82E-10 79

Immune/Additional CXCLIO FABP7 RGS1 1.83E-10 50

Immune/Additional PTPRC KRT15 HCLS1 1.83E-10 52

Immune/Additional HEY1 FN1 CCL5 1.84E-10 52

Immune/Additional HEY1 IGJ RGS1 1.84E-10 50

Immune/Additional CXCLIO PHACTRl CXCL9 1.86E-10 50

Immune/Additional FN1 CFH CCL5 1.88E-10 52 Table ZZ

Immune/Additional IFI6 RGSl SPPl KRT15 5.95E-14 72

Immune/Additional FABP7 IFI6 RGSl KRT15 6.01E-14 73

Immune/Additional HEY1 IFI6 RGSl KRT15 6.44E-14 73

Immune/Additional PTN IFI6 RGSl KRT15 6.54E-14 73

Immune/Additional PTN RGSl PHACTRl SPPl 6.54E-14 77

Immune/Additional FN1 IFI6 RGSl KRT15 6.58E-14 73

Immune/Additional IFI6 RGSl PHACTRl KRT15 6.61E-14 73

Immune/Additional IFI6 CFH RGSl KRT15 6.90E-14 73

Immune/Additional FN1 RGSl PHACTRl SPPl 7.42E-14 77

Immune/Additional BCL2A1 IFI6 RGSl SPPl 7.76E-14 74

Immune/Additional BCL2A1 IFI6 RGSl KRT15 8.11E-14 73

Immune/Additional FN1 BCL2A1 IFI6 RGSl 8.15E-14 75

Immune/Additional FABP7 PTN RGSl PHACTRl 8.77E-14 78

Immune/Additional PTN CFH RGSl SPPl 8.97E-14 78

Immune/Additional PTN PRAME RGSl PHACTRl 9.33E-14 76

Immune/Additional CCL3 PTN PRAME KRT15 9.68E-14 76

Immune/Additional FN1 IFI6 RGSl SPPl 9.75E-14 74

Immune/Additional FN1 PTN BCL2A1 RGSl 9.82E-14 78

Immune/Additional FN1 BCL2A1 RGSl PHACTRl 1.01E-13 78

Immune/Additional HEY1 PTN RGSl SPPl 1.10E-13 78

Immune/Additional FABP7 FN1 RGSl PHACTRl 1.14E-13 78

Immune/Additional CCL3 PRAME BCL2A1 KRT15 1.15E-13 76

Immune/Additional FABP7 RGSl PHACTRl SPPl 1.15E-13 77

Immune/Additional HEY1 RGSl PHACTRl SPPl 1.17E-13 77

Immune/Additional CCL3 RGSl PHACTRl SPPl 1.17E-13 77

Immune/Additional FN1 PRAME RGSl PHACTRl 1.26E-13 76

Immune/Additional FN1 PTN RGSl PHACTRl 1.29E-13 78

Immune/Additional CCL3 PRAME SPPl KRT15 1.30E-13 75

Immune/Additional BCL2A1 RGSl PHACTRl SPPl 1.32E-13 77

Immune/Additional CFH RGSl PHACTRl SPPl 1.35E-13 77

Immune/Additional FABP7 FN1 BCL2A1 RGSl 1.38E-13 78

Immune/Additional HEY1 PTN PRAME RGSl 1.43E-13 76

Immune/Additional HEY1 FN1 BCL2A1 RGSl 1.47E-13 78

Immune/Additional HEY1 FABP7 RGSl PHACTRl 1.49E-13 78

Immune/Additional PTN PRAME RGSl SPPl 1.50E-13 75

Immune/Additional HEY1 FABP7 RGSl SPPl 1.53E-13 78

Immune/Additional HEY1 BCL2A1 RGSl SPPl 1.55E-13 77

Immune/Additional HEY1 CCL3 PRAME KRT15 1.56E-13 76

Immune/Additional PRAME RGSl PHACTRl SPPl 1.56E-13 75

Immune/Additional HEY1 FN1 RGSl PHACTRl 1.62E-13 78

Immune/Additional FN1 CFH RGSl PHACTRl 1.67E-13 78

Immune/Additional HEY1 CCL3 RGSl SPPl 1.69E-13 77

Immune/Additional FABP7 PTN RGSl SPPl 1.71E-13 78

Immune/Additional FN1 PRAME RGSl SPPl 1.71E-13 75

Immune/Additional CCL3 PRAME PHACTRl KRT15 1.72E-13 76

Immune/Additional PTN BCL2A1 RGSl SPPl 1.75E-13 77

Immune/Additional PTN CFH RGSl PHACTRl 1.76E-13 78

Immune/Additional HEY1 FN1 RGSl SPPl 1.77E-13 78

Immune/Additional FABP7 PTN PRAME RGSl 1.77E-13 76

Immune/Additional CCL3 FN1 BCL2A1 RGSl 1.78E-13 78

Immune/Additional PTN BCL2A1 RGSl PHACTRl 1.78E-13 78

Immune/Additional HEY1 PTN RGSl PHACTRl 1.81E-13 78

Immune/Additional HEY1 PTN BCL2A1 RGSl 1.82E-13 78 Immune/Additional CCL3 FN1 RGSl PHACTRl 1.82E-13 78

Immune/Additional FN1 BCL2A1 CFH RGSl 1.87E-13 78

Immune/Additional CCL3 FABP7 PRAME KRT15 1.90E-13 76

Immune/Additional CCL3 FN1 PRAME KRT15 1.95E-13 76

Immune/Additional FN1 CFH RGSl SPP1 1.97E-13 78

Immune/Additional HEYl PRAME RGSl SPP1 2.00E-13 75

Immune/Additional HEYl CCL3 FABP7 RGSl 2.02E-13 78

Immune/Additional HEYl FABP7 BCL2A1 RGSl 2.05E-13 78

Immune/Additional CCL3 FN1 RGSl SPP1 2.11E-13 77

Immune/Additional HEYl FN1 PRAME RGSl 2.17E-13 76

Immune/Additional CCL3 PRAME RGSl PHACTRl 2.17E-13 76

Immune/Additional HEYl PRAME RGSl PHACTRl 2.18E-13 76

Immune/Additional HEYl CFH RGSl SPP1 2.26E-13 78

Immune/Additional FABP7 PTN BCL2A1 RGSl 2.26E-13 78

Immune/Additional FABP7 PRAME RGSl PHACTRl 2.27E-13 76

Immune/Additional FN1 PTN RGSl SPP1 2.28E-13 78

Immune/Additional HEYl BCL2A1 RGSl PHACTRl 2.31E-13 78

Immune/Additional FN1 PTN PRAME RGSl 2.31E-13 76

Immune/Additional CCL3 PRAME CFH KRT15 2.38E-13 76

Immune/Additional FN1 PRAME BCL2A1 RGSl 2.40E-13 76

Immune/Additional HEYl FABP7 PRAME RGSl 2.46E-13 76

Immune/Additional FABP7 BCL2A1 RGSl PHACTRl 2.54E-13 78

Immune/Additional HEYl FABP7 FN1 RGSl 2.55E-13 79

Immune/Additional FABP7 FN1 RGSl SPP1 2.63E-13 78

Immune/Additional PRAME BCL2A1 RGSl PHACTRl 2.68E-13 76

Immune/Additional PRAME PHACTRl SPP1 KRT15 2.74E-13 75

Immune/Additional BCL2A1 CFH RGSl SPP1 2.86E-13 77

Immune/Additional HEYl CCL3 PTN RGSl 2.89E-13 78

Immune/Additional CCL3 FABP7 RGSl PHACTRl 2.90E-13 78

Immune/Additional HEYl CCL3 FN1 RGSl 2.95E-13 78

Immune/Additional CCL3 BCL2A1 RGSl PHACTRl 2.99E-13 78

Immune/Additional FABP7 BCL2A1 RGSl SPP1 3.04E-13 77

Immune/Additional CCL3 BCL2A1 RGSl SPP1 3.07E-13 77

Immune/Additional FABP7 CFH RGSl PHACTRl 3.09E-13 78

Immune/Additional FABP7 CFH RGSl SPP1 3.11E-13 78

Immune/Additional FABP7 FN1 PRAME RGSl 3.12E-13 76

Immune/Additional PRAME CFH RGSl PHACTRl 3.13E-13 76

Immune/Additional HEYl CCL3 RGSl PHACTRl 3.17E-13 78

Immune/Additional BCL2A1 CFH RGSl PHACTRl 3.21E-13 78

Immune/Additional CCL3 PTN RGSl SPP1 3.23E-13 77

Immune/Additional FABP7 PTN CFH RGSl 3.26E-13 79

Immune/Additional FABP7 PRAME RGSl SPP1 3.27E-13 75

Immune/Additional CCL3 PRAME RGSl SPP1 3.37E-13 75

Immune/Additional FABP7 PRAME SPP1 KRT15 3.45E-13 75

Immune/Additional PTN BCL2A1 CFH RGSl 3.48E-13 78

Immune/Additional PRAME BCL2A1 RGSl SPP1 3.48E-13 75

Immune/Additional HEYl CFH RGSl PHACTRl 3.49E-13 78

Immune/Additional PRAME CFH SPP1 KRT15 3.56E-13 75

Immune/Additional HEYl CCL3 BCL2A1 RGSl 3.57E-13 78

Immune/Additional CCL3 PTN RGSl PHACTRl 3.58E-13 78

Immune/Additional PRAME BCL2A1 SPP1 KRT15 3.62E-13 75

Immune/Additional HEYl PRAME BCL2A1 RGSl 3.64E-13 76

Immune/Additional HEYl FABP7 CFH RGSl 3.75E-13 79 Immune/Additional CCL3 CFH RGSl SPP1 3.77E-13 77

Immune/Additional PTN PRAME BCL2A1 RGSl 3.78E-13 76

Immune/Additional PRAME IFI6 RGSl SPP1 3.81E-13 70

Immune/Additional IFI6 RGSl PHACTRl SPP1 3.89E-13 72

Immune/Additional HEY1 CCL3 PRAME RGSl 4.17E-13 76

Immune/Additional PRAME CFH RGSl SPP1 4.32E-13 75

Immune/Additional CCL3 FN1 PRAME RGSl 4.39E-13 76

Immune/Additional PTN PRAME CFH RGSl 4.39E-13 76

Immune/Additional HEY1 PRAME SPP1 KRT15 4.41E-13 75

Immune/Additional FN1 PRAME CFH RGSl 4.42E-13 76

Immune/Additional HEY1 IFI6 RGSl SPP1 4.44E-13 72

Immune/Additional HEY1 BCL2A1 CFH RGSl 4.53E-13 78

Immune/Additional HEY1 PRAME CFH RGSl 4.67E-13 76

Immune/Additional CCL3 CFH RGSl PHACTRl 4.76E-13 78

Immune/Additional PTN PRAME SPP1 KRT15 4.92E-13 75

Immune/Additional CCL3 FABP7 RGSl SPP1 5.06E-13 77

Immune/Additional IFI6 CFH RGSl SPP1 5.56E-13 72

Immune/Additional PRAME IFI6 RGSl PHACTRl 5.61E-13 71

Immune/Additional FN1 PRAME SPP1 KRT15 5.68E-13 75

Immune/Additional CCL3 PRAME IFI6 KRT15 5.91E-13 71

Immune/Additional HEY1 FABP7 IFI6 RGSl 5.92E-13 73

Immune/Additional PTN IFI6 RGSl SPP1 6.17E-13 72

Immune/Additional CCL3 IFI6 RGSl SPP1 6.22E-13 72

Immune/Additional CCL3 PTN BCL2A1 RGSl 6.42E-13 78

Immune/Additional FABP7 IFI6 RGSl SPP1 6.57E-13 72

Immune/Additional FABP7 PRAME BCL2A1 RGSl 6.62E-13 76

Immune/Additional CCL3 FABP7 BCL2A1 RGSl 6.78E-13 78

Immune/Additional CCL3 FN1 CFH RGSl 6.93E-13 78

Immune/Additional HEY1 CCL3 CFH RGSl 6.98E-13 78

Immune/Additional HEY1 PRAME IFI6 RGSl 7.06E-13 71

Immune/Additional FABP7 BCL2A1 CFH RGSl 7.06E-13 78

Immune/Additional PRAME BCL2A1 IFI6 RGSl 7.69E-13 71

Immune/Additional FN1 PRAME IFI6 RGSl 7.95E-13 71

Immune/Additional CCL3 PRAME BCL2A1 RGSl 8.06E-13 76

Immune/Additional BCL2A1 IFI6 RGSl PHACTRl 8.07E-13 73

Immune/Additional FABP7 IFI6 RGSl PHACTRl 8.22E-13 73

Immune/Additional CCL3 FABP7 FN1 RGSl 8.25E-13 78

Immune/Additional FABP7 FN1 CFH RGSl 8.35E-13 79

Immune/Additional FN1 IFI6 RGSl PHACTRl 8.44E-13 73

Immune/Additional HEY1 BCL2A1 IFI6 RGSl 8.46E-13 73

Immune/Additional FABP7 BCL2A1 IFI6 RGSl 8.64E-13 73

Immune/Additional CCL3 PTN PRAME RGSl 8.71E-13 76

Immune/Additional PTN PRAME IFI6 RGSl 8.71E-13 71

Immune/Additional FABP7 PRAME IFI6 RGSl 8.83E-13 71

Immune/Additional HEY1 PTN CFH RGSl 9.30E-13 79

Immune/Additional CCL3 BCL2A1 CFH RGSl 9.50E-13 78

Immune/Additional CCL3 FABP7 PRAME RGSl 9.60E-13 76

Immune/Additional HEY1 FN1 PTN RGSl 9.82E-13 79

Immune/Additional PRAME BCL2A1 CFH RGSl 9.87E-13 76

Immune/Additional CCL3 PRAME IFI6 RGSl 1.00E-12 71

Immune/Additional HEY1 IFI6 RGSl PHACTRl 1.02E-12 73

Immune/Additional HEY1 CCL3 IFI6 RGSl 1.03E-12 73

Immune/Additional CCL3 BCL2A1 IFI6 RGSl 1.09E-12 73 Immune/Additional HEYl FN1 IFI6 RGS1 1.15E-12 73

Immune/Additional HEYl FABP7 PRAME KRT15 1.16E-12 76

Immune/Additional FABP7 FN1 PTN RGS1 1.20E-12 79

Immune/Additional PRAME IFI6 CFH RGS1 1.22E-12 71

Immune/Additional HEYl FN1 CFH RGS1 1.25E-12 79

Immune/Additional FABP7 PRAME CFH RGS1 1.27E-12 76

Immune/Additional PTN BCL2A1 IFI6 RGS1 1.30E-12 73

Immune/Additional CCL3 IFI6 RGS1 PHACTRl 1.31E-12 73

Immune/Additional PRAME IFI6 SPP1 KRT15 1.32E-12 70

Immune/Additional FN1 PTN CFH RGS1 1.39E-12 79

Immune/Additional BCL2A1 IFI6 CFH RGS1 1.42E-12 73

Immune/Additional IFI6 CFH RGS1 PHACTRl 1.42E-12 73

Immune/Additional HEYl PRAME CFH KRT15 1.52E-12 76

Immune/Additional FABP7 PRAME PHACTRl KRT15 1.55E-12 76

Immune/Additional PRAME BCL2A1 PHACTRl KRT15 1.55E-12 76

Immune/Additional HEYl PRAME BCL2A1 KRT15 1.57E-12 76

Immune/Additional PTN IFI6 RGS1 PHACTRl 1.57E-12 73

Immune/Additional PRAME CFH PHACTRl KRT15 1.57E-12 76

Immune/Additional FN1 PRAME PHACTRl KRT15 1.61E-12 76

Immune/Additional HEYl PTN IFI6 RGS1 1.69E-12 73

Immune/Additional CCL3 PRAME CFH RGS1 1.76E-12 76

Immune/Additional CCL3 PTN CFH RGS1 1.87E-12 78

Immune/Additional HEYl PRAME PHACTRl KRT15 1.90E-12 76

Immune/Additional FABP7 FN1 PRAME KRT15 1.94E-12 76

Immune/Additional CCL3 FN1 PTN RGS1 2.00E-12 78

Immune/Additional FABP7 PRAME BCL2A1 KRT15 2.01E-12 76

Immune/Additional FABP7 FN1 IFI6 RGS1 2.07E-12 73

Immune/Additional HEYl IFI6 CFH RGS1 2.08E-12 73

Immune/Additional CCL3 CFH SPP1 KRT15 2.20E-12 77

Immune/Additional PTN PRAME PHACTRl KRT15 2.29E-12 76

Immune/Additional FABP7 PRAME CFH KRT15 2.32E-12 76

Immune/Additional CCL3 PTN CFH KRT15 2.36E-12 78

Immune/Additional CCL3 PTN SPP1 KRT15 2.37E-12 77

Immune/Additional FN1 IFI6 CFH RGS1 2.53E-12 73

Immune/Additional FN1 PRAME CFH KRT15 2.58E-12 76

Immune/Additional PRAME BCL2A1 CFH KRT15 2.62E-12 76

Immune/Additional FABP7 PTN PRAME KRT15 2.65E-12 76

Immune/Additional CCL3 FABP7 CFH RGS1 2.67E-12 78

Immune/Additional FABP7 IFI6 CFH RGS1 2.74E-12 73

Immune/Additional CCL3 PHACTRl SPP1 KRT15 2.88E-12 77

Immune/Additional HEYl CCL3 PTN KRT15 2.90E-12 78

Immune/Additional HEYl FN1 PRAME KRT15 2.99E-12 76

Immune/Additional HEYl CCL3 SPP1 KRT15 3.02E-12 77

Immune/Additional CCL3 FN1 SPP1 KRT15 3.09E-12 77

Immune/Additional CCL3 FABP7 PTN RGS1 3.11E-12 78

Immune/Additional CCL3 FN1 PTN KRT15 3.15E-12 78

Immune/Additional HEYl PTN PRAME KRT15 3.27E-12 76

Immune/Additional CCL3 FABP7 SPP1 KRT15 3.35E-12 77

Immune/Additional CCL3 BCL2A1 SPP1 KRT15 3.35E-12 77

Immune/Additional PTN PRAME CFH KRT15 3.39E-12 76

Immune/Additional CCL3 IFI6 CFH RGS1 3.71E-12 73

Immune/Additional CCL3 FABP7 IFI6 RGS1 3.77E-12 73

Immune/Additional HEYl CCL3 PHACTRl KRT15 3.86E-12 78 Immune/Additional CCL3 FN1 IFI6 RGS1 3.87E-12 73

Immune/Additional PTN IFI6 CFH RGS1 4.18E-12 73

Immune/Additional HEY1 CCL3 CFH KRT15 4.23E-12 78

Immune/Additional CCL3 FN1 CFH KRT15 4.32E-12 78

Immune/Additional FABP7 PTN IFI6 RGS1 4.32E-12 73

Immune/Additional FN1 PTN PRAME KRT15 4.34E-12 76

Immune/Additional HEY1 CCL3 BCL2A1 KRT15 4.37E-12 78

Immune/Additional HEY1 CCL3 FN1 KRT15 4.41E-12 78

Immune/Additional CCL3 FN1 PHACTRl KRT15 4.43E-12 78

Immune/Additional FN1 PRAME BCL2A1 KRT15 4.45E-12 76

Immune/Additional HEY1 CCL3 FABP7 KRT15 4.85E-12 78

Immune/Additional CCL3 FABP7 FN1 KRT15 4.90E-12 78

Immune/Additional CCL3 FN1 BCL2A1 KRT15 5.05E-12 78

Immune/Additional CCL3 PTN PHACTRl KRT15 5.16E-12 78

Immune/Additional CCL3 CFH PHACTRl KRT15 5.46E-12 78

Immune/Additional PTN PRAME BCL2A1 KRT15 5.86E-12 76

Immune/Additional CCL3 FABP7 CFH KRT15 5.95E-12 78

Immune/Additional CCL3 PTN BCL2A1 KRT15 6.02E-12 78

Immune/Additional CCL3 FABP7 PTN KRT15 6.09E-12 78

Immune/Additional CCL3 BCL2A1 CFH KRT15 6.33E-12 78

Immune/Additional CCL3 PRAME IFI6 SPPl 6.43E-12 70

Immune/Additional CCL3 FABP7 PHACTRl KRT15 7.08E-12 78

Immune/Additional HEY1 CFH SPPl KRT15 7.22E-12 78

Immune/Additional FN1 PTN IFI6 RGS1 7.22E-12 73

Immune/Additional CCL3 BCL2A1 PHACTRl KRT15 7.24E-12 78

Immune/Additional CCL3 FABP7 BCL2A1 KRT15 8.01E-12 78

Immune/Additional PTN CFH SPPl KRT15 8.62E-12 78

Immune/Additional CCL3 PTN IFI6 RGS1 9.08E-12 73

Immune/Additional CCL3 IFI6 SPPl KRT15 9.33E-12 72

Immune/Additional HEY1 CCL3 IFI6 KRT15 1.02E-11 73

Immune/Additional CFH PHACTRl SPPl KRT15 1.02E-11 77

Immune/Additional FABP7 CFH SPPl KRT15 1.06E-11 78

Immune/Additional HEY1 FABP7 SPPl KRT15 1.23E-11 78

Immune/Additional FN1 CFH SPPl KRT15 1.24E-11 78

Immune/Additional CCL3 FN1 PRAME SPPl 1.29E-11 75

Immune/Additional CCL3 FN1 IFI6 KRT15 1.30E-11 73

Immune/Additional HEY1 PRAME IFI6 KRT15 1.35E-11 71

Immune/Additional CCL3 PTN PRAME SPPl 1.37E-11 75

Immune/Additional PRAME IFI6 PHACTRl KRT15 1.41E-11 71

Immune/Additional HEY1 PHACTRl SPPl KRT15 1.42E-11 77

Immune/Additional FABP7 PRAME IFI6 KRT15 1.43E-11 71

Immune/Additional BCL2A1 CFH SPPl KRT15 1.44E-11 77

Immune/Additional CCL3 IFI6 PHACTRl KRT15 1.51E-11 73

Immune/Additional CCL3 PRAME PHACTRl SPPl 1.54E-11 75

Immune/Additional HEY1 PTN SPPl KRT15 1.59E-11 78

Immune/Additional HEY1 FN1 SPPl KRT15 1.61E-11 78

Immune/Additional PRAME BCL2A1 IFI6 KRT15 1.62E-11 71

Immune/Additional PRAME IFI6 CFH KRT15 1.63E-11 71

Immune/Additional CCL3 PTN IFI6 KRT15 1.67E-11 73

Immune/Additional CCL3 FABP7 IFI6 KRT15 1.73E-11 73

Immune/Additional CCL3 BCL2A1 IFI6 KRT15 1.82E-11 73

Immune/Additional PTN PRAME IFI6 KRT15 1.84E-11 71

Immune/Additional HEY1 BCL2A1 SPPl KRT15 1.91E-11 77 Immune/Additional FN1 PRAME IFI6 KRT15 1.91E-11 71

Immune/Additional CCL3 IFI6 CFH KRT15 2.09E-11 73

Immune/Additional CCL3 FABP7 PRAME SPPl 2.24E-11 75

Immune/Additional HEY1 CCL3 PRAME SPPl 2.63E-11 75

Immune/Additional FABP7 PRAME PHACTRl SPPl 2.75E-11 75

Immune/Additional FABP7 PTN PRAME SPPl 2.77E-11 75

Immune/Additional FN1 PRAME PHACTRl SPPl 2.80E-11 75

Immune/Additional BCL2A1 PHACTRl SPPl KRT15 2.80E-11 77

Immune/Additional FABP7 FN1 PRAME SPPl 2.83E-11 75

Immune/Additional FN1 PHACTRl SPPl KRT15 2.84E-11 77

Immune/Additional PTN PHACTRl SPPl KRT15 2.85E-11 77

Immune/Additional CCL3 PRAME BCL2A1 SPPl 2.86E-11 75

Immune/Additional FABP7 PHACTRl SPPl KRT15 3.01E-11 77

Immune/Additional FABP7 PRAME IFI6 SPPl 3.29E-11 70

Immune/Additional SELL HLA ITGB2 KRT15 3.45E-11 52

Immune/Additional FABP7 BCL2A1 SPPl KRT15 3.50E-11 77

Immune/Additional RGS1 SELL ITGB2 KRT15 3.57E-11 52

Immune/Additional BCL2A1 CCL5 S100A9 KRT15 3.63E-11 52

Immune/Additional IRF1 SELL ITGB2 KRT15 3.66E-11 52

Immune/Additional CCL3 PRAME CFH SPPl 3.69E-11 75

Immune/Additional PRAME BCL2A1 IFI6 SPPl 3.69E-11 70

Immune/Additional RGS1 SELL KRT15 HCLS1 3.69E-11 52

Immune/Additional BCL2A1 RGS1 CCL5 PTPRC 3.73E-11 52

Immune/Additional RGS1 CCL5 PTPRC PHACTRl 3.73E-11 52

Immune/Additional FN1 PRAME IFI6 SPPl 3.74E-11 70

Immune/Additional PRAME RGS1 CCL5 PTPRC 3.75E-11 52

Immune/Additional BCL2A1 CCL5 PTPRC PHACTRl 3.81E-11 52

Immune/Additional BCL2A1 CCL5 PTPRC KRT15 3.81E-11 52

Immune/Additional FABP7 FN1 SPPl KRT15 3.88E-11 78

Immune/Additional CCL5 SELL PTPRC PHACTRl 3.88E-11 52

Immune/Additional FN1 BCL2A1 SPPl KRT15 3.91E-11 77

Immune/Additional CCL3 RGS1 CCL5 PTPRC 3.94E-11 52

Immune/Additional HLA CCL5 SELL KRT15 3.98E-11 52

Immune/Additional PRAME IFI6 PHACTRl SPPl 4.02E-11 70

Immune/Additional FN1 PTN SPPl KRT15 4.06E-11 78

Immune/Additional FABP7 CCL5 S100A9 PTPRC 4.07E-11 52

Immune/Additional FABP7 PTN SPPl KRT15 4.13E-11 78

Immune/Additional PTPN22 CCL5 PTPRC PHACTRl 4.16E-11 52

Immune/Additional CCL5 SELL PTPRC KRT15 4.18E-11 52

Immune/Additional PTN PRAME IFI6 SPPl 4.25E-11 70

Immune/Additional PTN PRAME PHACTRl SPPl 4.31E-11 75

Immune/Additional RGS1 CCL5 PTPRC KRT15 4.31E-11 52

Immune/Additional FABP7 BCL2A1 CCL5 PTPRC 4.34E-11 52

Immune/Additional PTN BCL2A1 SPPl KRT15 4.35E-11 77

Immune/Additional RGS1 CCL5 PTPRC LCP2 4.36E-11 52

Immune/Additional HEY1 RGS1 CCL5 PTPRC 4.37E-11 52

Immune/Additional HLA RGS1 CCL5 PTPRC 4.37E-11 52

Immune/Additional IRF1 CCL5 SELL PTPRC 4.39E-11 52

Immune/Additional FABP7 CCL5 PTPRC PHACTRl 4.45E-11 52

Immune/Additional CCL5 PTPRC LCP2 HCLS1 4.50E-11 52

Immune/Additional HEY1 CCL5 S100A9 KRT15 4.52E-11 52

Immune/Additional CCL5 S100A9 LCP2 KRT15 4.52E-11 52

Immune/Additional FABP7 CCL5 S100A9 KRT15 4.55E-11 52 Immune/Additional RGS1 CCL5 HLA PTPRC 4.56E-11 52

Immune/Additional SELL S100A9 PHACTRl KRT15 4.56E-11 52

Immune/Additional FABP7 RGS1 CCL5 PTPRC 4.57E-11 52

Immune/Additional HEY1 FABP7 PRAME SPPl 4.61E-11 75

Immune/Additional PRAME RGS1 SELL KRT15 4.61E-11 52

Immune/Additional CCL3 BCL2A1 CCL5 PTPRC 4.63E-11 52

Immune/Additional CCL5 SELL S100A9 KRT15 4.65E-11 52

Immune/Additional PRAME PTPN22 CCL5 PTPRC 4.67E-11 52

Immune/Additional BCL2A1 CCL5 S100A9 PTPRC 4.67E-11 52

Immune/Additional PTPN22 CCL5 PTPRC LCP2 4.68E-11 52

Immune/Additional PRAME CCL5 PTPRC PHACTRl 4.70E-11 52

Immune/Additional CCL5 SELL ITGB2 KRT15 4.71E-11 52

Immune/Additional HEY1 PRAME IFI6 SPPl 4.72E-11 70

Immune/Additional BCL2A1 CCL5 HLA PTPRC 4.73E-11 52

Immune/Additional IRF1 CCL5 S100A9 KRT15 4.74E-11 52

Immune/Additional HEY1 PTPN22 CCL5 PTPRC 4.75E-11 52

Immune/Additional PRAME CCL5 S100A9 KRT15 4.75E-11 52

Immune/Additional PTPN22 CCL5 PTPRC KRT15 4.75E-11 52

Immune/Additional RGS1 SELL S100A9 KRT15 4.77E-11 52

Immune/Additional PTN RGS1 CCL5 PTPRC 4.81E-11 52

Immune/Additional CCL5 SELL PTPRC LCP2 4.82E-11 52

Immune/Additional HEY1 BCL2A1 CCL5 PTPRC 4.84E-11 52

Immune/Additional RGS1 CCL5 S100A9 PTPRC 4.84E-11 52

Immune/Additional CCL3 CCL5 S100A9 KRT15 4.85E-11 52

Immune/Additional CCL5 HLA S100A9 KRT15 4.87E-11 52

Immune/Additional BCL2A1 PTPN22 CCL5 PTPRC 4.89E-11 52

Immune/Additional PTPN22 RGS1 CCL5 PTPRC 4.89E-11 52

Immune/Additional CCL5 PTPRC LCP2 CXCL9 4.91E-11 52

Immune/Additional HEY1 CCL5 PTPRC KRT15 4.93E-11 52

Immune/Additional PRAME CCL5 PTPRC KRT15 4.93E-11 52

Immune/Additional RGS1 CCL5 SELL PTPRC 4.94E-11 52

Immune/Additional PTPN22 CCL5 HLA PTPRC 4.95E-11 52

Immune/Additional PRAME BCL2A1 SELL KRT15 4.96E-11 52

Immune/Additional BCL2A1 HLA CCL5 PTPRC 4.98E-11 52

Immune/Additional FABP7 CCL5 PTPRC LCP2 5.02E-11 52

Immune/Additional CCL5 SELL KRT15 HCLS1 5.06E-11 52

Immune/Additional CCL3 CCL5 SELL KRT15 5.07E-11 52

Immune/Additional PRAME BCL2A1 PHACTRl SPPl 5.07E-11 75

Immune/Additional RGS1 IRF1 CCL5 PTPRC 5.07E-11 52

Immune/Additional CCL3 PTPN22 CCL5 PTPRC 5.09E-11 52

Immune/Additional PRAME IFI6 CFH SPPl 5.11E-11 70

Immune/Additional RGS1 SELL LCP2 KRT15 5.11E-11 52

Immune/Additional CCL5 S100A9 PTPRC LCP2 5.12E-11 52

Immune/Additional BCL2A1 CCL5 PTPRC LCP2 5.13E-11 52

Immune/Additional CCL5 SELL LCP2 KRT15 5.14E-11 52

Immune/Additional CCL3 CCL5 PTPRC PHACTRl 5.18E-11 52

Immune/Additional CCL3 FABP7 CCL5 PTPRC 5.20E-11 52

Immune/Additional CCL5 PTPRC PHACTRl LCP2 5.20E-11 52

Immune/Additional CCL5 PTPRC PHACTRl KRT15 5.21E-11 52

Immune/Additional PRAME BCL2A1 CCL5 PTPRC 5.24E-11 52

Immune/Additional CCL5 S100A9 PHACTRl KRT15 5.25E-11 52

Immune/Additional HLA CCL5 S100A9 KRT15 5.26E-11 52

Immune/Additional FN1 PRAME BCL2A1 SPPl 5.29E-11 75 Immune/Additional PRAME CCL5 HLA PTPRC 5.29E-11 52

Immune/Additional FABP7 PRAME CCL5 PTPRC 5.31E-11 52

Immune/Additional PRAME IRF1 SELL KRT15 5.31E-11 52

Immune/Additional CCL5 S100A9 KRT15 CXCL9 5.31E-11 52

Immune/Additional CCL5 S100A9 PTPRC PHACTRl 5.34E-11 52

Immune/Additional PTN PRAME KRT15 CXCL9 5.37E-11 52

Immune/Additional FABP7 PTN CCL5 PTPRC 5.38E-11 52

Immune/Additional IRF1 SELL S100A9 KRT15 5.38E-11 52

Immune/Additional FABP7 PTPN22 CCL5 PTPRC 5.40E-11 52

Immune/Additional CCL5 HLA PTPRC PHACTRl 5.40E-11 52

Immune/Additional CFH CCL5 S100A9 KRT15 5.42E-11 52

Immune/Additional FABP7 CCL5 SELL PTPRC 5.44E-11 52

Immune/Additional PECAM1 RGS1 CCL5 PTPRC 5.44E-11 52

Immune/Additional CCL5 S100A9 PTPRC KRT15 5.45E-11 52

Immune/Additional FABP7 PRAME SELL KRT15 5.47E-11 52

Immune/Additional HLA SELL KRT15 CXCL9 5.48E-11 52

Immune/Additional PTPN22 IRF1 CCL5 PTPRC 5.50E-11 52

Immune/Additional CCL5 HLA S100A9 PTPRC 5.50E-11 52

Immune/Additional PTPN22 CCL5 S100A9 PTPRC 5.51E-11 52

Immune/Additional IRF1 CCL5 PTPRC LCP2 5.53E-11 52

Immune/Additional PRAME CCL5 S100A9 PTPRC 5.55E-11 52

Immune/Additional PRAME SELL HLA KRT15 5.56E-11 52

Immune/Additional CCL5 PTPRC LCP2 KRT15 5.56E-11 52

Immune/Additional CCL3 PRAME CCL5 PTPRC 5.61E-11 52

Immune/Additional RGS1 SELL KRT15 CXCL9 5.61E-11 52

Immune/Additional PRAME SELL PTPRC KRT15 5.62E-11 52

Immune/Additional BCL2A1 IRF1 CCL5 PTPRC 5.62E-11 52

Immune/Additional RGS1 CCL5 PTPRC CXCL9 5.63E-11 52

Immune/Additional S100A9 PTPRC LCP2 KRT15 5.63E-11 52

Immune/Additional PTN RGS1 KRT15 CXCL9 5.65E-11 52

Immune/Additional SELL HLA S100A9 KRT15 5.65E-11 52

Immune/Additional SELL ITGB2 PHACTRl KRT15 5.65E-11 52

Immune/Additional CCL5 S100A9 KRT15 HCLS1 5.66E-11 52

Immune/Additional PTN PRAME SELL KRT15 5.68E-11 52

Immune/Additional FABP7 S100A9 PTPRC KRT15 5.70E-11 52

Immune/Additional HEY1 CCL5 PTPRC PHACTRl 5.71E-11 52

Immune/Additional CCL3 RGS1 SELL KRT15 5.71E-11 52

Immune/Additional FN1 RGS1 CCL5 PTPRC 5.71E-11 52

Immune/Additional CFH RGS1 CCL5 PTPRC 5.74E-11 52

Immune/Additional CCL3 CCL5 S100A9 PTPRC 5.75E-11 52

Immune/Additional HEY1 FABP7 CCL5 PTPRC 5.76E-11 52

Immune/Additional HEY1 PRAME PHACTRl SPP1 5.77E-11 75

Immune/Additional HLA PHACTRl KRT15 CXCL9 5.77E-11 52

Immune/Additional HLA PTPN22 CCL5 PTPRC 5.79E-11 52

Immune/Additional SELL S100A9 PTPRC KRT15 5.80E-11 52

Immune/Additional CFH PTPN22 CCL5 PTPRC 5.82E-11 52

Immune/Additional CCL5 HLA PTPRC KRT15 5.82E-11 52

Immune/Additional FABP7 SELL S100A9 KRT15 5.84E-11 52

Immune/Additional PRAME CCL5 SELL PTPRC 5.84E-11 52

Immune/Additional IRF1 CCL5 SELL KRT15 5.84E-11 52

Immune/Additional PRAME SELL PHACTRl KRT15 5.85E-11 52

Immune/Additional PRAME CCL5 PTPRC LCP2 5.86E-11 52

Immune/Additional RGS1 CCL5 PTPRC HCLS1 5.87E-11 52 Immune/Additional CCL3 PRAME SELL KRT15 5.88E-11 52

Immune/Additional PTN BCL2A1 CCL5 PTPRC 5.88E-11 52

Immune/Additional FABP7 CCL5 HLA PTPRC 5.90E-11 52

Immune/Additional PTN PTPN22 CCL5 PTPRC 5.90E-11 52

Immune/Additional PRAME SELL KRT15 CXCL9 5.90E-11 52

Immune/Additional PECAM1 SELL S100A9 KRT15 5.90E-11 52

Immune/Additional HEY1 CCL5 PTPRC LCP2 5.92E-11 52

Immune/Additional CCL3 S100A9 PTPRC KRT15 5.94E-11 52

Immune/Additional HEY1 PRAME CCL5 PTPRC 5.96E-11 52

Immune/Additional RGS1 CCL5 S100A9 KRT15 5.96E-11 52

Immune/Additional BCL2A1 PECAM1 CCL5 PTPRC 6.00E-11 52

Immune/Additional HEY1 PRAME SELL KRT15 6.02E-11 52

Immune/Additional PTN CCL5 S100A9 KRT15 6.02E-11 52

Immune/Additional PTPN22 CCL5 SELL PTPRC 6.02E-11 52

Immune/Additional S100A9 PTPRC PHACTR1 KRT15 6.05E-11 52

Immune/Additional BCL2A1 CCL5 SELL KRT15 6.06E-11 52

Immune/Additional CCL5 S100A9 ITGB2 KRT15 6.06E-11 52

Immune/Additional CCL5 HLA PTPRC LCP2 6.07E-11 52

Immune/Additional HLA RGS1 SELL KRT15 6.08E-11 52

Immune/Additional PRAME HLA SELL KRT15 6.09E-11 52

Immune/Additional PRAME LCP2 KRT15 CXCL9 6.1 lE-11 52

Immune/Additional HEY1 CCL3 CCL5 PTPRC 6.14E-11 52

Immune/Additional IRF1 CCL5 PTPRC PHACTR1 6.15E-11 52

Immune/Additional HEY1 CCL5 S100A9 PTPRC 6.16E-11 52

Immune/Additional S100A9 PTPRC KRT15 CXCL9 6.16E-11 52

Immune/Additional PRAME CCL5 SELL KRT15 6.18E-11 52

Immune/Additional RGS1 CCL5 SELL KRT15 6.19E-11 52

Example 5

[00170] In this example we determined a model for differentiating melanoma samples from nevus samples based on a computed score.

Methods

[00171] Approximately 600 skin lesions were acquired from two separate sites (coded "Munich" and "Provitro"), with relatively equal numbers from both locations. Each site provided both malignant and benign samples, with all major histological subtypes represented. The diagnosis for each case was confirmed, using a second dermatopathologist who was blinded to the diagnosis of the first

dermatopathologist. If there was discordance, a third dermatopathologist adjudicated the diagnosis. [00172] An H&E stained slide from each case was then reviewed by a pathologist, and the lesion of interest identified for each case. The corresponding tissue was macrodissected from 5 unstained slides (4 mm thickness) and pooled into a single tube. The RNA was then extracted from the tissue, the RNA was DNAsed using DNAse I and cDNA synthesized. We then pre-amplified all genes of interest including 7 housekeeper normalization genes) in one multiplex reaction. Finally, quantitative PCR was used to measure the expression of each gene. The expression values were calculated by determining the CT (Crossing Threshold) of each gene. Each sample was run in triplicate by splitting each sample into 3 aliquots after the cDNA synthesis.

[00173] The three measurements of each gene were then averaged and normalized by the averaged expression of all seven housekeeper genes. Each gene was studied to determine if its expression could differentiate between malignant melanoma and benign nevi samples; if genes were effective at this, they were further analyzed to see which genes had correlating data (an indication that these genes measure the same biological pathway). Genes with correlating data were grouped together in sets, with the average expression of the set used to differentiate melanoma and nevi.

Results

[00174] We acquired -600 samples from two German sites (labeled Munich and Provitro [Berlin]). Each site contributed roughly even numbers of malignant and benign samples, with all major histologic subtypes represented in the samples from each site. We first identified the lesion of interest in each sample and then extracted the RNA from each sample and measured the RNA expression level of the potential signature genes and 7 housekeeper normalization genes (Table 1 1 ).

Table 11- List of potential signature genes tested and housekeeper genes used for normalization.

HLA-DRA Potential Signature CXCL 13 Potential Signature

HLA-DMA Potential Signature CXCL9 Potential Signature

BCL2A1 Potential Signature CENPF Potential Signature

FABP7 Potential Signature PBK Potential Signature

PTPRC Potential Signature CXCL 10 Potential Signature

IFI6 Potential Signature FOXM1 Potential Signature

S 100A9 Potential Signature PLK1 Potential Signature

FN 1 Potential Signature CEP55 Potential Signature

ITGB2 Potential Signature DTL Potential Signature

PTPN22 Potential Signature SKA1 Potential Signature

PHACTR1 Potential Signature CCL5 Potential Signature

CCL3 Potential Signature CLTC Housekeeper

PRAME Potential Signature MRFAP 1 Housekeeper

KRT15 Potential Signature PPP2CA Housekeeper

SPP 1 Potential Signature PSMA1 Housekeeper

LCP2 Potential Signature RPL 13A Housekeeper

IRF 1 Potential Signature RPL8 Housekeeper

SELL Potential Signature TXNL 1 Housekeeper

[00175] The ability of each gene was then analyzed to determine if its expression was effective in differentiating the malignant melanoma and benign nevi samples. We determined that PRAME was a very effective biomarker (Figure 5).

Furthermore, we also found that a large number of immune genes were also able to strongly differentiate melanoma and nevi. We further investigated 8 of these immune genes and found that their data was highly correlated, as the expression of each individual immune gene had a linear relationship with the average of all 8 immune genes (Figure 6). This may indicate that they are measuring the same biological process, and they were thus grouped together into an "immune" set. The averaged expression of all 8 immune genes was calculated and then used when analyzing the dataset. Finally, we also noted that the cell cycle gene S 100A9 also was able to differentiate many melanoma and nevi samples.

[00176] We next created a combined diagnostic model, using these three sets of biomarkers (PRAME, the immune genes, and S 100A9). When the expression of each biomarker was graphed against the other, we did not see a high correlation, indicating that each biomarker is likely measuring a different biological process and has independent value (see Figure 7). We ran the data through a logistic regression model, to best determine how each biomarker could be weighted in the model, to maximize the model's ability to differentiate melanoma and nevi. The most effective model was found with the following weightings for the expression of each biomarker set: (0.525 x

PRAME) + (0.677 x Immune) + (0.357 x S 100A9). The model then takes the data from each patient and generates a score, which can be used to differentiate melanoma and nevi (see Figure 8). This current dataset was then used to generate a ROC curve (see Figure 9), which had an AUC of -0.96.

Example 6

[00177] In this example we determined a diagnostic model for

differentiating between malignant melanoma and non-malignant nevi and then further refined the diagnostic model.

Methods

[00178] Patient samples were acquired and prepared as described above in Example 5. Likewise, RNA extraction, preparation, and quantification of gene expression were also carried out in the same fashion as described above in Example 5. The same list of potential signature genes and the same housekeeper genes were assayed as in Example 5 (Table 1 1 ). Like Example 5, the measured expression of each gene was averaged and normalized by the averaged expression of all seven housekeeper genes. Each gene was analyzed to determine if its expression could differentiate between malignant melanoma samples and benign nevi samples. Genes that were effective at differentiating between malignant melanoma samples and benign nevi samples were further analyzed to determine which genes had correlating data. Correlating data indicated that the genes measured the same biological pathway. Genes with correlating data were grouped together in sets, with the average expression of the set used to differentiate melanoma and nevi.

Results

Building the diagnostic model

[00179] As described in Example 5, each gene was analyzed to determine if its expression was effective in differentiating the malignant melanoma samples from the benign nevi samples. In this analysis, we used forward selection to choose predictors for inclusion in a diagnostic model that would differentiate melanoma and nevi samples. As in Example 5, we selected PRAME, S 100A9, and an immune component score as the predictors to differentiate the malignant melanoma samples from the benign nevi samples. The immune component score was made up of eight immune genes (CXCL9, CCL5, CXCL 10, IRF 1 , PTPN22, PTPRC, LCP2, AND CD38) that all had highly correlated data (Figure 6). These eight immune genes were grouped and their values averaged to create an immune component score. As in Example 5, we also determined that these three predictors (PRAME, S 100A9, or the immune component) had

expression patterns only moderately correlated with each other (Figure 7), indicating that each predictor largely provides independent information when distinguishing melanoma and nevi samples.

[00180] We next created a combined diagnostic model using PRAME, S 100A9, and the immune component score. The combined diagnostic model that we created was a linear model based on the gene expression data from the three predictors (PRAME, S 100A9 and the immune component). In generating the linear model, we used generalized logistic regression to calculate the best weightings for each of the three predictors that would most effectively differentiate the melanoma and nevi samples in this dataset. The calculated best weightings were as follows: 1.149 for PRAME, 0.922 for S 100A9, and 0.698 for the immune component score. Using these best weightings, the gene expression data for the three predictors was then used to generate a score that could be used to help differentiate between benign nevi samples and melanoma samples. The gene expression data for each predictor was multiplied by the respective best weighting and then the three weighted values were combined. This combined value, as shown here, represented a score that could be used to help differentiate between benign nevi samples and melanoma samples:

Score = (1.149 x PRAME) + (0.922 x S 100A9) + (0.698 x Immune component)

[00181] We then generated and plotted scores for all 544 samples (Figure 10). In the plotted scores we observed a bi-modal distribution. Nearly all samples with a score greater than zero were melanoma (dark gray bars), while nearly all nevi samples (light gray bars) had a score less than zero. Using this data, a ROC curve was generated that had an AUC of ~ 0.95 and an associated p-value of 2.0 x 10-63 (Figure 1 1). From this ROC curve we selected a cutoff point to differentiate between melanoma samples and nevi samples. In selecting the cutoff point we sought to maximize the sensitivity of the model, while maintaining the highest possible specificity. We selected a cutoff point with a sensitivity of 0.89 and a specificity of 0.93. We then adjusted the calculation of the score so that the selected cutoff point would be at a value of zero. Thus the adjusted score calculation was:

Adjusted ₌ (1.149 x (0.922 x (0.698 x Immune

score PRAME) S 100A9) component) ^"

Refining the diagnostic model

[00182] To improve the robustness and precision of the assay, we wanted to determine additional measurements for the PRAME and S 100A9 predictors. We tested additional amplicons corresponding to PRAME and S 100A9. We sought to determine if any other amplicons produced a reliable and correlated signal for these genes. We tested four additional PRAME amplicons.

[00183] We also tested amplicons that corresponded to six other genes that we determined would have expression that was highly correlated to S 100A9 : S 100A7, S 100A8, S 100A10, S 100A12, S 100A14 and PI3.

[00184] We determined that one PRAME amplicon had low failure rates and had values highly correlated with the PRAME amplicon previously used in the model. We averaged its measurement with the other PRAME measurement to provide a PRAME component.

[00185] We chose four other potential genes which produced data with low failure rates and were highly correlated with the S 100A9 amplicon selected in the training set. These four genes, S 100A7, S 100A8, S 100A12, and PI3 were averaged with S 100A9 to yield a single S l OO-related component, or S I 00 score.

[00186] Thus, we were able to create a refined signature for the three predictors, PRAME, S l OO-related component, and the immune component. The refined signature included additional measurements for the PRAME and S l OO-related predictors determined above. The refined signature included a total of 15 amplicons that measured 14 signature genes comprising two measurements of the PRAME gene, one measurement each of S l OO related genes S 100A9, S 100A7, S 100A8, S 100A12, and PI3, and measurements of eight highly correlated immune genes (CXCL9, CCL5, CXCLI O, IRF l , PTPN22, PTPRC, LCP2, AND CD38)(Table 12). Along with these 15 signature amplicons, we also included amplicons corresponding to nine different housekeeper genes for normalization, for a total of 24 amplicons in our refined signature (Table 12).

Table 12- Signature and housekeeper genes comprising the refined signature.

[00187] Lastly, we performed a concordance study to verify that the changes to the multiplex PCR reaction would not alter the data generated from the 10 signature amplicons retained from the initial signature. The qPCR assay relies on the fact that all the measured genes are pre-amplified in a single multiplex PCR reaction. Since the refined signature differed from the initial signature, it was important to ascertain that the different amplicon set of the refined signature did not alter the multiplex PCR reaction and consequently alter the data generated from the 10 amplicons retained from the initial signature set. Therefore, we retested RNA expression in 74 RNA samples from the initial training set using the refined signature. The scores of the retested RNA samples demonstrated an extremely high correlation (correlation coefficient of 0.99) when compared to the scores generated from the training set. Thus, the refined signature did not alter the data generated from any of the amplicons of the refined signature.

[00188] Accordingly, the refined signature produced a refined, adjusted score calculation of:

Refined, (1 .149 x O Q?? S 1 00 (0.698 x

adjusted = PRAME + " + Immune - 0.334 x. component) _x .

score component) ¹ ' component)

Example 7

[00189] In this example we clinically validated a diagnostic model for differentiating between malignant melanoma and non-malignant nevi.

Methods

[00190] A validation cohort was generated by acquiring more than 400 skin lesions from four separate sites: Cleveland Clinic, Moffit Cancer Center, Northwestern University and the University of Utah (Table 13).

Table 13 - Sources of samples for the clinical validation cohort.

Only samples that produced analyzable results were included.

[00191] Each site provided both malignant and benign samples, with all major histological subtypes represented. The diagnosis for each case was confirmed, using a second dermatopathologist who was blinded to the diagnosis of the first

dermatopathologist. If there was discordance in the diagnoses, a third

dermatopathologist adjudicated the diagnosis. The melanoma subtypes included superficial spreading melanoma, nodular melanoma, lentigo maligna melanoma, desmoplastic melanoma, and subtypes classified as a not otherwise specified (Table 14).

Table 14 - Clinical information for melanoma samples used in the clinical

validation.

SSM = Superficial Spreading Melanoma, Nod = Nodular melanoma, LMM = Lentigo Maligna Melanoma, Desm = Desmoplastic, NOS = Not Otherwise Specified subtype

Only samples that produced analyzable results were included.

[00192] The nevi subtypes included blue nevi, compound nevi, junctional nevi, dermal nevi, deep penetrating nevi, dysplastic nevi, and subtypes classified as a not otherwise specified (Table 15).

Table 15 - Clinical information for nevi samples used in the clinical validation.

Comp = Compound, June = Junctional, Derm = Dermal, Deep Pen = Deep Penetrating, NOS = Not Otherwise Specified subtype, Dyspl = Dysplastic.

*Dysplastic nevi were scored as an attribute of a subtype, not as a specific subtype.

[00193] The patient samples were prepared as described above in Example 5. RNA extraction, RNA preparation, and quantification of gene expression were also carried out as described above in Example 5. The same list of potential signature genes and the same housekeeper genes were assayed as in Example 5 (Table 1 1). Likewise, as in Example 5, the measured expression of each gene was averaged and normalized by the averaged expression of all seven housekeeper genes. Then, a refined, adjusted score was generated for each patient sample using the refined, adjusted score calculation of:

Refined, (1 .149 x (0.698 x

(0.922 x S 100

adjusted PRAME + + Immune - 0.334 component)

score component) component)

Results [00194] The refined, adjusted scores were plotted for each patient sample (Figure 12). The plotted refined, adjusted scores resulted in a bimodal distribution similar to that seen in Figure 10 of Example 6. Nearly all melanoma samples had scores greater than zero (Figure 12, upper panel) and nearly all nevi samples had scores less than zero (Figure 12, lower panel). This data was then used to generate a validation ROC curve that had an AUC of ~ 0.96 with a sensitivity of 0.9 and a specificity of 0.91 (Figure 13). The p-value associated with the validation ROC curve was 3.7 x 10 ^"63 . The validation ROC curve was then compared to the ROC curve generated in Example 6 (Figure 1 1) to determine if the diagnostic model had been validated. The validation ROC curve had an AUC of ~ 0.96, a sensitivity of 0.9, and a specificity of 0.91 compared to the ROC curve of Example 6 which had an AUC of ~ 0.95, a sensitivity of 0.89, and a specificity of 0.93. The close agreement of the validation ROC curve and the ROC curve of Example 6 indicated that the validation cohort validated the diagnostic model of Example 6.

[00195] Next, the performance of the diagnostic model was analyzed within individual histological subtypes for those subtypes with 30 or more samples (Table 16).

Table 16 - Assay performance within individual subtypes in the clinical validation.

Only subtypes groups with > 30 samples were reported.

[00196] There were two benign subtypes, compound and dermal, with more than 30 samples. The scoring of compound and dermal subtypes resulted in specificities of 94% and 98%, respectively, with an overall specificity of 91 % for all nevi. There were three malignant subtypes, superficial spreading melanoma, nodular melanoma, and lentigo maligna melanoma with more than 30 samples. The scoring of superficial spreading melanoma, nodular melanoma, and lentigo maligna melanoma resulted in sensitivities of 86%, 97%, and 90%, respectively, with an overall sensitivity of 90%> for all melanomas.

[00197] All publications and patent applications mentioned in the specification are indicative of the level of those skilled in the art to which this invention pertains. All publications and patent applications are herein incorporated by reference to the same extent as if each individual publication or patent application was specifically and individually indicated to be incorporated by reference. The mere mentioning of the publications and patent applications does not necessarily constitute an admission that they are prior art to the instant application.

[00198] Although the foregoing invention has been described in some detail by way of illustration and example for purposes of clarity of understanding, it will be obvious that certain changes and modifications may be practiced within the scope of the appended claims.