POLYMORPHISM

BACKGROUND OF THE INVENTION

Field of the Invention

[0001 } The invention is directed to a risk stratification method for a patient and particularly to a patient having a polymorphism and a tumor, such as a hemangioma and for determination of placenta health, Including prediction of diseases such as an intrauterine growth restriction, pre-eclampsia, eclampsia and placental insufficiency in a patient.

Background

[0002] Infantile hemangioma (IH) is the most common tumor of the pediatric age group, affecting up to 4% of newborns ranging from inconsequential blemishes, to highly aggressive tumors. Following well defined growth phases (proliferative, plateau involutional) IH usually regress into a fibro-fatty residuum. Despite the high prevalence of IH, little is known regarding the pathogenesis of disease.

[0003] Infantile hemangioma (IH) is the most common tumor of the pediatric age group, affecting up to 4% of newborns with nearly 60% localized to the head and neck. These vascular lesions range from inconsequential blemishes to highly aggressive tumors that can cause eye obstructions or blindness, blockage of airways, facial deformations and ulcerations. During the first year, hemangiomas demonstrate both histology and behavior that are also commonly noted in malignancy: immature vascular channels, high mitotic indices, and strong positivity for proliferative markers such as Ki-67,

[0004] Despite these ominous beginnings, IH remain benign. The growth velocity slowly reverses leading to a "quiescent or piateau" phase of non-growth {1-2 years) and then transitions into a regressive or Involuting" phase by replacing the once proliferative endothelium with a fibrofatty residuum (2-10 years). However, these growth phases are a matter of clinical judgment alone and the exact timing of each varies considerably among studies. Despite its prevalence, !itt!e is known regarding the pathogenesis of disease. Insulin Like Growth Factor 2 (IGF2) has been

I implicated as an important p!ayer in driving the growth of these lesions. SGF2 decreases over six fold from proliferative to involuting IH samples. Furthermore, Beckwith-Wiedmann Syndrome (BWS), where hemangiomas are considered a supportive finding of the diagnosis, is associated with duplications or a !oss of imprinting of the IGF2/H19 iocus that leads to SGF2 overproduction, fvloreover, exp!ani hemangioma cultures grow strongly in response to exogenous IGF2.

[0005] IGF2 is an imprinted gene that is usually only expressed from the paternal copy. Commonly, DNA methylation of cytosines preceding guanines (CpG's) reinforce DNA imprinting. These so called epigeneiic marks in part determine and are determined by the array of DNA binding proteins capable of interacting with specific chromatin structures. The end result of this process is diploid DNA thai is potentially identical in sequence but chemically, transcriptionally and architecturally distinct in a parent of origin specific manner. This leads to activation of one given parental allele and reciprocal silencing of another. The IGF2/H19 region of chromosome 11 15.5 serves as a model for the production of muitipie imprinted transcripts.

[0006 ]: There is evidence to support the existence of a common genetic program between placental and hemangioma endothelium. The idea of a placental origin of hemangioma is evaluated through molecular profiling and genome-based analysis as described in "Evidence by Molecular Profiling For A Placental Origin of Infantile Hemangioma", Proceedings of the National Academy of Sciences of the United States, December 19, 2005, Carmen . Barnes et al.; the entirety

incorporated by reference herein. The transcriptomes of human placenta and infantile hemangioma are shown in these studies to be sufficiently similar and therefore suggest a placenta! origin for this tumor. The unique hemangioma cycle is marked by rapid endothelial ceil proliferation, endothelial eel! apoptosis, and tumor involution which may mirror the lifetime of a placental endothelial ceils.

SUMMARY OF THE INVENTION

[000?]: The invention is directed to a risk stratification method for a patient and particularly to a patient having a polymorphism and a tumor, such as a hemangioma and for determination of placenta health, including prediction of diseases such as an intrauterine growth restriction, pre-eclampsia, eciampsia and placenta! insufficiency in a patient and prediction of development of postnatal infantile hemangioma, identification of the expression of CTCF with parent of origin specific determination from the mother and/or from the maternal placenta and/or from the fetus or fetal placenta may be used to predict placenta! health and stratify patients for diseases such as an intrauterine growth restriction, pre-eclampsia, eclampsia and placental insufficiency.

1000 j The placenta functions as a fetomaternal organ with two components, the fetal placenta, Chorion frondosum, which develops from the same blastocyst that forms the fetus, and the maternal placenta, Decidua basalis, which develops from the maternal uterine tissue. A blood sample from the mother may be taken for DNA analysis of both the mother and the fetus. In some cases, a simpiy check swap may be used to provide a sample for DNA analysis of the mother. Both of these techniques are non-invasive. In some cases, a blood sample may be taken from the placenta or from the fetus for DNA analysis as described herein.

[0009] Brother of the regulator of imprinted sites or BORIS, also known as transcriptional regulator transcriptional repressor CTCFL, is a protein that in humans is encoded by the CTCFL gene.

[OO!Oj With respect to infantiie hemangiomas, a reported six fold decrease in IGF2 expression (correlating with transformation of proliferative to involuted lesions) prompted a study of the IGF-2 axis further. As described herein, it has been discovered that IGF2 expression in IH is strongly related to the expression of a cancer testes and suspected oncogene BORIS (paralog of CTCF), placing !H in the unique category of being the first known benign BOR!S positive tumor. SGF2 expression was strongly and positively related to BORIS transcript expression.

Furthermore, a stronger association was made when comparing BORIS levels against the expression of CTCF via either a percentage or difference between the two, or a rate of increase of %CTCF. A patient with a reducing rate of %CTCF may be a higher risk patient, as this may indicate a more aggressive tumor, as described herein, A common C T polymorphism at CTCF BS6 appeared to modify the correlation between CTCF/BORIS and IGF2 expression in a parent of origin specific manner. Moreover, these effects may have phenotypic consequences as tumor growth also correlates with the genotype at CTCF BS6. This may provide a framework for explaining the clinical variability seen in IH and suggests new insights regarding CTCF and BORIS related functionality in both normal and malignant states.

[001 ) I Investigations into the differential regulation and potential role of IGF2 as it pertains to SH, as well as the expression of CTCF, a known chromatin insulator element for IGF2 and its antagonist, BORIS (also known as CTCFL~for CTCF like) at both the transcript and protein levels are described herein. The nearby imprinted and maternally expressed H19 gene, which shares enhancers wit IGF2, was also quantified. These results were then correlated with methylation analysis of key regulatory regions in the IGF2 and H19 locus. This analysis suggests that a common polymorphism within CTCF Binding Site Six, the critical imprinting control region of H19/IGF2, may have both cellular and phenoiypic consequences in a parent of origin specific manner. These findings may serve as a predictor of clinical behavior of !H and may enable risk stratification for patients having a tumor, and specifically SH.

[0012] In an exemplary embodiment, a risk stratification method for a patient in a disease state is accomplished by first determining whether said patient is a homozygote patient or a heterozygote patient with respect to CTCF biding site 8. When the patient is a homozygote patient it is then determined if the patient has a Thymine/Thymine, (TT) allelic expression or a Cytosine/Cytosine (CC) allelic expression for said CTCF biding site 6. When the patient is a heterozygote, it is the determined whether the cytosine is methylated; whereby when the cytosine is not methylated the patient has a Thymine/Cytosine, (TC) ai!elic expression for said CTCF biding site 6; and whereby when the cytosine is methylated the patient has a Cytosine/Thymine (CT) allelic expression for said CTCF biding site 6. A patient with a TC allelic expression has a maternai cytosine and a patient with a CT allelic expression has a paternal cytosine. Risk stratifying can then be determined based on the patient's allelic expression for CTCF binding site 8, wherein the patient with a TT allelic expression is classified as a highest risk patient, the patient with a TC allelic expression i classified as a second highest risk patient, the patient with a CT allelic expression is classified as a third highest risk patient and the patient with a CC allelic expression is classified as a lowest risk patient. Furthermore, unique to the disease of vascular tumors in general and infantile hemangioma specifically is the risk of ulceration. A painful condition where the epithelium of the lesion degrades, creating an open wound prone to bleeding. The TT allelic expression is classified at the highest risk for u ice ration.

[0033] A highest risk patient having an IH may be prone to a rapidly growing tumor, ulceration of the tumor, facial deformations, eye obstructions, blockage of airways or any combination of these conditions. A second highest risk patient having SH may be prone to moderately to rapidly growing tumors, facial deformations, eye obstructions, blockage or airways or any combination of these conditions. A third highest risk patient having IH may have a sto to moderately growing tumor, facial blemishes. A lowest risk patient having IH may have a slow growing tumor, facial blemishes,

[0014] Any suitable method may be used to for determining for a heterozygote patient whether the cytosine is methylated including, bisulfite conversion and quantitative methyiation sensitive pyrosequencing or directly sequencing parental DNA, for example,

[0015] A risk stratification method, as described herein, may be initiated for a patient in a diseased state, wherein the patient is In a diseased state by identification of a patient tumor. A patient may be considered to be in a diseased state when the identif ied tumor is an infantile hemangioma, or any other tumor such as a cancerous tumor including, but not limited to, breast cancer, ovarian cancer, testicular cancer, liver cancer, lung cancer, brain cancer, skin cancer, esophageal cancer, throat cancer, bladder cancer, cervical cancer, colorectal cancer, kidney cancer, ieuikemia, melanoma, non-Hodgkin lymphoma, ovarian cancer, pancreatic cancer, prostate cancer, skin cancer, thyroid cancer, uterine cancer and any other cancer.

[0016]: The risk stratification as described herein, may further comprise the step of identification of an abnormal expression or mutation/function of a gene regulated by CTCF biding site 6. The abnormal expression or mutation functio of a gene product regulated by CTCF biding site 6 may be IGF2, H19, H19 antisense, SGF2 antisense, a micro ma within the gene locus, or the gene product may be an isoform.

001 ? ] The risk stratification as described herein, may further comprise the step of identification of an abnormal expression or mutation/function of a binding gene product that binds to the CTCF biding site 8. The abnormal expression or mutation/function of a binding gene product that binds to the CTCF biding site 6 may be CTCF, BORiS or a biding isoform.

[0038] The risk stratification as described herein, may further comprise the step of identification of an abnormal expression or mutation/function of a binding partner of CTCF or BORIS.

[0019] In an exemplary embodiment, a risk stratification method further comprising the steps of determining an expression ieve! of BORIS and an expression level of CTCF with respect to CTCF biding site 8, and then determining a percentage of CTCF transcript, such as the percentage of CTCF with respect to the total of CTCF and BORIS. In an alternative embodiment, a difference in the amount of CTCF to the amount of BORIS may be used. A risk stratification method may take into account the afleiic expression, as described herein, and may further take into account the %CTCF, wherein, when the % CTCF is less than 20%, the patient is classified as a highest %CTCF risk patient, wherein when the %CTCF is less than 50% but greater than 20%, the patient is classified as a second highest %CTCF risk patient, wherein when the %CTCF is less than 80% but greater than 50% the patient is classified as a third highest %CTCF risk patient and wherein when the %CTCF ratio is less than 100% or less and greater than 80% the patient is classified as a lowest %CTCF risk patient. Furthermore, the risk stratification method may include the rate of reduction of %CTCF, wherein a rapid drop in %CTCF may indicated a higher risk of an aggressive tumor, or a tumor that grows quickly.

[0020] In an exemplary embodiment, a risk stratification method further comprising the steps of determining an expression leve! of BORIS and an expression level of CTCF with respect to CTCF biding site 6, and then determining a CTCF- BORIS difference which is a difference in the expression ievel of CTCF to the expression ievel of BORIS. The patient may then be risk stratified according to said CTCF-BOR!S difference wherein, when the CTCF-BORIS difference is less than zero, the patient is classified as a highest CTCF-BORIS risk patient; and when the CTCF-BORIS difference is greater than zero, the patient is classified as a lowest CTCF-BORIS risk patient.

[0021]: In an exemplary embodiment, the risk stratification method as described herein, may be used to predict a response of medication to the tumor. For example, a patient with a TT allelic expression and classified as a highest risk patient may have a predicied medication response that is most effective, a patient with a TC allelic expression and classified as a second highest risk patient may have a predicted medication response that is second most effective, a patient with a CT allelic expression and classified as a third highest risk patient may have a predicted medication response that is third most effective and a patient with a CC allelic expression and classified as a iowest risk patient may have a predicted medication response that is least effective. Patients classified as the highest risk patients may have the most effective response to medication whereas patients ciassified as the iowest risk patients may have the least effective response to medication. Medication may be beta blockers, cortical steroids, alpha interferon, and/or 1GF2 receptor blockers. Preferred medications are beta blockers and cortical steroid. A most effective response or highly effective response to medication includes stowing of the growth of the tumor, reduction in size of the tumor without rebound or relapse, closure of ulceration or healing or ulceration, A second most effective response to medication includes reduction in size of the tumor without rebound or relapse, T third most effective response to medication includes reduction in size of the tumor without rebound or relapse. A least effective response to medication includes reduction in size of the tumor, or no reduction in size of the tumor,

S 0221 The medication response prediction method may further comprise the step of determining the disease state of said patient by identification of an abnormal expression or mutation/function of a gene product produced by CTCF biding site 6. The abnormal expression or mutation/fu notion of a gene product regulated by CTCF biding site 6 may be SGF2, H19, H19 aniisense, IGF2 antisense, a micro rna within the gene locus, or the gene product may be an isoform.

[0023] The method of determining this polymorphism and allelic expression of the gene product regulated by CTCF binding site 6 for the fetus or for an infant, may also be used to predict placental health and risk stratify mothers for placenta! diseases including an intrauterine growth restriction, pre eclampsia, eclampsia, placental insufficiency. DNA analysis of the mother and/or the fetus may be used in this predictive model. As with the risk stratification model described herein, a predicted model may be based on the allelic expression for CTCF binding site 6 of the mother, the fetus or some combination thereof . A mother with a TT allelic expression may be classified as a highest stratified risk patient, a mother with a TC allelic expression may be classified as a second highest risk patient, a mother with a CT aiieSic expression may be classified as a third highest risk patient and a mother with a CC allelic expression may be classified as a lowest risk patient. The various combinations of allelic expression of the mother and fetus may further be used in a risk stratification method. When the mother and the fetus both have a TT allelic expression , this may correlate with the highest risk patient for placental health issues, the mother and fetus have a CT or TC allelic expression, this may correlate with a second highest risk patient for placental health issues and when both the mother and fetus have a CC allelic expression, this may correlate with the lowest risk patient for placental health issues. It is to be noted that data to determine

correlations between the allelic expression of CTCF binding site 6 of the mother and/or the fetus may a iter the risk stratification categories as provided herein,

{0024] A G/A polymorphism approximately 130 base pairs downstream of rs10732516 was found to be in strong linkage disequilibrium with the (C/T) polymorphism at CTCF 8S6. This polymorphism is rs2107425. This polymorphism was detected by using differential primer binding, see incorporated reference Tost et al, for details. The A polymorphism at rs2107425 was strongly associated with the T polymorphism at CTCF BS 6 (rs10732516} in the samples. Thus, it is conceivable that nearby polymorphisms within the CTCF BS6 locus would be used as a surrogate for a test at rs10732518. This concern is underscored by the fact that large areas of linkage disequilibrium of over 500 base pairs have been documented, shoemaker et al ¹ . This has included regions in IGF2, Shoemaker et al. Given the hig level of linkage disequilibrium we observed at CTCF binding six between re10732516 and rs2107425 we propose that the sequence between CTCF binding site 5 and CTCF binding site seven (this includes CTCF binding site six and rs10732518} be considered one quantitative trait locus. Therefore, for the purposes of this invention, a polymorphism as described herein, may be determined by analysis of any sequence between CTCF binding site 5 and CTCF binding site 7. The placenta! health predictive indicators as well as the risk stratification methods can be based on analysis of an sequence between CTCF binding site 5 and CTCF binding site 7. [0025] ASieSe-specific methy!ation is prevalent and is contributed by CpG- SMPs in the human genome. The risk stratification method, as described herein, may be characterized from CTCF binding site 6, or a range upstream and downstream, because of high linking disequilibrium, as described in Robert shoemaker, jie Deng, Wei Wang and un Zhang Genome Research 2010 Jul; 20(7): 883-889, the entirety of which is hereby incorporated by reference. The risk stratification method of the present invention may be preformed on base pairs up to 500 base pairs from CTCF binding site 6, or up to about 350 base pairs from CTCF binding site 6, or from about 150 base pairs from CTCF binding site 6, or from CTCF binding site 5 to CTCF binding site 7.

[0026] The summary of the invention is provided as a general introduction to some of the embodiments of the invention, and is not intended to be limiting.

Additional example embodiments including variations and alternative configurations of the invention are provided herein.

BRIEF DESCRIPTION OF THE DRAWINGS

[0027] The accompanying drawings are included to provide a further understanding of the invention and are incorporated in and constitute a part of this specification, illustrate embodiments of the invention, and together with the description serve to explain the principles of the Invention.

[0028] Figure 1 shows a drawing of an infantile hemangioma.

[0029] Figure 2A shows a diagram representing the genotyping via direct sequencing of biood samples.

[0030]: Figure 26 shows a chart of bisulfite conversion and quantitative methylation sensitive pyrosequencing.

[003 i ] Figure 3A and 3B show a chart of IGF2 transcription by clinical stage wherein FIG, 3A shows IGF2 Transcript and FIG. 38 shows %CTCF,

[0032] Figure 4A shows a graph of the percent CTCF with respect to CTCF and BORIS versus the age of the lesion.

[0033] Figure 4B shows the CSUW of %CTCF versus the age of the lesion.

[0034 ] Figure 5 shows a graph of IGF2 Transcript versus the %CTCF. [0035] Figure 6 shows Western Analysis of 24 IH Samples Via 5 Independent Western Blots.

[0036] Figure 7 shows a schematic integrating CTCF and BORIS expression via both transcript and protein with clinical stage.

[ O37j Figures 8A and 8B show a graph of the parent of origin, paternal and maternal respectively, specific effects of CTCF BS8 on IGF2 and H19 transcription,

[0038] Figure 9A shows a graphs of percent methyiation versus CpG number,

[0039] Figure 9B shows a graph of percent methylation of the H19 promoter by pyrosequencing versus CTCF transcript.

[0040]: Figure 9C shows methylation of the H19 promoter.

[0041] Figure 9D shows a sequence showing the H19 promoter, CpG#4 of the bisulfite sequencing test corresponds to the CCCGGG Pst1 digestion site of the Southern analysis.

[0042] Figure 10A shows a graph of the diameter of an infantile hemangioma versus the age of the lesion in days for TT and non-TT patients.

[0043] Figure 108 shows a graph of the diameter of an infantile hemangioma versus the age of the lesion in days for TC, C/T and C/C patients.

[0044] Figures 11 A to 1 1 D show portions of Table 1 , as the print for ease of viewing.

DETAILED DESCRIPTIO OF THE ILLUSTRATED EMBODIMENTS

[0045] Corresponding reference characters indicate corresponding parts throughout the several views of the figures. The figures represent an illustration of some of the embodiments of the present invention and are not to be construed as limiting the scope of the invention in any manner. Further, the figures are not necessarily to scale, some features may be exaggerated to show details of particular components. Therefore, specific structural and functional details disclosed herein are not to be interpreted as limiting, but merely as a representative basis for teaching one skilled in the art to variously employ the present invention.

[0046]: As used herein, the terms "comprises," "comprising," "includes," "including," "has," "having" or any other variation thereof, are intended to cover a non-exclusive inclusion. For example, a process, method, artide, or apparatus that to comprises a iisl of e!emenis is not necessariiy iimited to only those elements but may include other elements not expressly listed or inherent to such process, method, article, or apparatus. Also, use of "a" or "an" are employed to describe elements and components described herein. This is done merely for convenience and to give a general sense of the scope of the invention. This description should be read to include one or at least one and the singular also includes the piural unless it is obvious that it is meant otherwise.

[0047] Certain exemplary embodiments of the present invention are described herein and are iliustrated in the accompanying figures. The embodiments described are only for purposes of illustrating the present invention and should not be interpreted as limiting the scope of the invention. Other embodiments of the invention, and certain modifications, combinations and improvements of the described embodiments, will occur to those skilled in the art and all such alternate embodiments, combinations, modifications and improvements are within the scope of the present invention.

[0048] This application incorporates by reference in its entirety, the following publication:

Brent Schultz, Xiaopan Yao, Yanhong Deng, Milton aner, Christopher

Spock, Laura Tom, John Persing, Deepak Narayan, {2015} A Common Polymorphism Within The IGF2 Imprinting Control Region Is Associated with Parent of Origin Specific Effects in Infantile Hemangiomas, PLOS ONE | DOI:10.1371/journai. pone.01 13188

Materials and Methods

[0049]: This article describes a study that involved analyzing post excisional tissue from surgicai candidates. The decision to operate was in no way influenced by the study. Clinical data was gathered retrospectively from this same group. Ail surgical candidates had clinical measurements available for analysis. All samples and clinical data were collected in accordance with the approved HIC protocol (#0507000430) as reviewed by the Yaie University Medical School IRB. This protocol was approved specifically for this study. Written consent was obtained from each patient's legal guardian prior to surgery, Alt data obtained including clinical measurements were stored in a de-identified format.

Specimen Collection:

Π [0050] Please refer to the Master Data Tab!e for details. Those specimens later confirmed to be hemangioma tissue, via Giut-1 positive histology, were considered for this project. Further, only discrete solitary lesions that were not found in the setting of a syndrome were considered. Those patients where prior surgical resections of the iesion were performed were also deemed ineiigib!e. Of note, those lesions previously treated with laser were not excluded, as the effects of laser treatment are relatively superficial. However, during specimen collection, all areas that appeared grossly to be affected by laser treatment were excised before further processing. Briefly, forty-two samples were collected (See Table 1 for details.) Of these, two samples (#41 and #42), were excluded from all analyses; sample #41 was Giut-1 negative on histology and #42 was subject to prior resections. Nineteen SH samples were seiected at random for methyiation analysis of the H19 promote by southern. These 19 samples were also analyzed for methyiation specific

pyrosequendng of the same region. An additional two samples (numbers 1 and 14) were also subjected to H19 methyiation specific pyrosequencing to bolster the number of samples with both H19 methyiatio and transcriptional data. Regarding transcriptional analysis, nineteen samples were found to have suitably intact RNA for quantitative RT PGR. Specimens for transcriptional analysis were separated into three categories: 1 ) Proliferative, 2) Quiescent, and 3) involuting phases. As a lesion's stage is, by definition, dinicai, an experienced physician staged the IH at the time of surgery. Data regarding clinical stage was gathered prospectively. The determination of the clinical stage was made by one of three highly experienced surgeons regarding vascular anomalies, using interval growth, patient age and the color/turgor of the lesion at the time of resection as criteria. Genera! characteristics of these categories are as foliows: 1 ) proliferative hemangiomas were generally iess than 1.5 years of age with interval growth between the last two clinical visits preceding surgery, no lightening of Iesion color was noted. 2) Quiescent

hemangiomas: no interval growth between the last two clinic visits preceding surgery, lightening of color also played a factor in these determinations. Involuting hemangiomas: interval regression by measurement between the last two clinic visits preceding surgery, further color changes were often but not always noted.

Table 1 :

[005 !]: Table 1. Master Data Table. Ail samples are assigned arbitrary

numbers for ease of reference. Samples are categorized according to which set of experiments were performed, then by paternal/maternal genotype regarding the IGF2 rtPCR experiment. Ail sub categories are then sorted by age at resection. All quantitative data is collated with clinical descriptors. Please see methods section under specimen collection for details regarding the selection of individual samples for each experiment. Table 1 is provided in four expanded portions in FIGS. 1 1 A to 1 1 D.

[0052 | In totai 34 samples were genotyped for a polymorphism within CTCF BS6 and parental contributions were determined for heterozygotes (see Fig 2 and bisulfite sequencing methods for details.) Only iesions of the head and neck were included. Of these samples, 3 were excluded because they were not on the head or neck. Two other samples were excluded because one was not Giut-1 positive on histology and one had a previous resection of the same lesion prior to evaluation, (See Table 1 for details.) Thus 29 individuals were included In this analysis. Charts were retrospectively reviewed from patients treated either at the Yaie University

J3 P!astic Surgery Center (New Haven CT), or the Vascular Birthmarks institute (New York. New York), The age of the iesion was then compared to the size of the Iesion as determined below. These data were plotted and separated by CTCF BS8 genotype and parental contribution in heterozygotes. ANCOVA analyses were then performed on putative growth curves. The age at the time of resection, with corresponding size, was used only for proliferating lesions. For those lesions that were resected at the time of involution, or quiescence, the size of the Iesion at the clinical visit where quiescence was first noted was used, in the case of medical interventions, the size of the iesion before a response was noted was used. This information was used to create a clinical table of results were factors such as ulceration, steroid/chemoiherapeutic, and Saser treatment were also noted (note that no beta blockers were used in the sampie population.) Thus, different ages are associated with most individuals when comparing the clinical data table and the master data table. For instance: Sample #7 has an age of 2304 days assigned in the Master Data Table 1. However, in the Clinical Table the age assigned to sampie #7 is 1050 days. The difference in age assignments represent the age of the patient when the lesion was excised (this age was used for the molecular analyses) versus the age of the patient either before the first response to medical intervention was noted or whe the iesion first entered the quiescent phase. Thus, for sampie number 7: The lesion entered the quiescent phase (as determined retrospectively) at age 1050 days but was then excised at age 2304 days. Regarding the assessment of iesion size, if multiple dimensions were given, the largest was used, in some cases, only one dimension was given so volumetric estimations could not be calculated for every patient. Thus, patients' lesions were standardized to a greatest diameter equivalent. This measurement was correlated with ciinical photographs when available. Ail data utilized varied by iess than 10% between stated

measurement and photographic estimation when available. Lesions were classified into one of three growth phases; proliferative, quiescent and involuting. Oniy sizes of lesions that were in the proliferative or quiescent phase were used in this study. All data was stored in a de-identified format with a unique accession number for each patient.

DDNA Preservation and Extraction

Ϊ4 [0053] immediately following tissue resection, DMA was isolated using the Giagen DNeasy Tissue Mini Kit according to the manufacturer's protocol. Only samples with an A260/A280 measurement of 1.8 or above that ran as a single band on the gel were further analyzed.

[0054] Fig 2. Deducing Parental Contributions From Direct Sequencing and Bisulfite Pyrosequencing. Fig 2A: 29 patients were genotyped via direct sequencing of b!ood samples for a known polymorphism within the core CTCF SS6 sequence (rs10732516.) All homozygous genotypes could be deduced from this information alone. Fig 2B: All samples (heterozygotes and homozygotes) were subjected to bisulfite conversion and quantitative methyiation sensitive pyrosequencing,

ethylation occurs only on the paierna! chromosome for CTCF BS8. In normal tissue, such as patient matched control biood, this assay is capable of isolating the genotype of the paternal chromosome. As thymidine cannot be methylated, those individuals with a paternal T at rs10732516 were not methylated at CpG#5. Paternal C carrying individuals were methylated at CpG#5. Thus, the maternal and paternal contribution to CTCFBS6 can be deduced. This assay sidesteps the need for directly sequencing parents' DNA and eliminates the potential ambiguity ensuing from heterozygous parents. Note: The methyiation values of this assay are subject to primer bias, Tost et a! (25.) This is evident by the 3 distinct groupings of methyiation levels, which are artifactual.

RNA Preservation and Extraction

[0055] Immediately following tissue resection, 100-500mg of tissue was stored in Quiagen RNA Later solution according to the manufacturer's protocol. RNA was extracted via liquid nitrogen powder homogenization using Invitrogen Trizof reagent according to the standard protocol 10 pg of total RNA from each sample was then treated with DNase Qiagen mini-elute columns according to manufacturer's specifications. RNA integrity was then assessed using Agilent bioanaSyzer 2100 (provided as a service of the Keck Center at Yale University.) Those samples with 18s/28s ratio of 1.8 or greater were converted into cDNA using the ABI 4368813 cDNA archive kit. Ail samples were then stored at -80 degrees C.

Quantitative rtPCR for CTCF, BORIS, H19 and SGF2 [0056] Nineteen 1H samples with suitable RNA, as previously specified, were subjected to fluorescent quantitative RT-PCR using ABS Ta man primers that were previousiy validated by the manufacturer and spanned intron exon boundaries. For reasons of sample scarcity, not all samples were subjected to each assay (See Fig 1 for details.) The assays were: SGF2— assay number Hs00171254_m1 ₍ H19— assay number Hs00399294_g1 , CTCF—assay number Hs00198081_m1 , and BORIS— assay number Hs00540744_m1 , Gene quantification was performed using the standard curve method via pooled sample cDNA (equal contributions from each sample) and successive two fold dilutions, beginning from 50 ng and ending with 0,39 ng. All reactions were performed on the ABS 79005 thermocycier using default cycling conditions previously optimized for these assays. Reactions were performed in duplicate and average CT values, if they agreed within 0.4 cycles, were used to calculate absolute quantity. Three runs of RT PGR were performed with overlapping samples in each run to allow normalization of the

data. Not all samples were subjected to every assay depending upon sample quantity. Of Note: Sample #4 does not have an H19 transcription value, as on duplicate plating for rtPCR, the CT values did not agree within .4 cycles.

Furthermore, samples 15-19 were the final rtPCR of the three runs performed and due to sample scarcity and the need to construct standard curves from pooled samples, only CTCF and BORIS rtPCR's were performed.

Western Analysis

[0057] 24 samples were subjected to Western analysis. As this process is tissue intensive, younger samples such as #21 and #23 could only be used for this analysis as insufficient tissue was left for further

processing. Other samples were selected biased toward analyzing those samples with transcriptional data in order to compare transcriptional phenomena to translational. However, as the analysis proceeded, presentation gels were constructed to demonstrate key transition points in CTCF and BORIS translation in samples that had not been treated with steroids. Briefly, 50 mg of each

sample were processed with a rotary homogenteer in 200ml of R!PA lysis buffer. After centrifugation iysates were created using a standard beta-mercapto-ethanol with SDS. PAGE was performed with 36pg of protein per well in NuPage 10% Bis- Tris precast gels infvlOPS buffer at 100 volts. PAGE separated proteins were then

J 6 transferred for two hours to a PVDF membrane (Bio-Rad) in a standard transfer buffer at lOOmAmps. Αηΐί BORIS antibody (Abeam 18337} was used at 1/

5000 dilution in TBST with 5% cows milk overnight. Two concentrations of anti-CTCF were used— 1 :10,000 and 1 :5,000— to better visualize Sate and eariy rises in CTCF protein {see Fig 3 legend for details.) As anti-CTCF and anti-BORiS were both rabbit po!yc!onal antibodies they couSd be visualized simultaneously on the same fiim following incubation with the anti-rabbit secondary conjugated to horseradish peroxidase and ECL treatment, images were then scanned and adjusted

for brightness and contrast in Adobe Photoshop.

Bisulfite Methylation Analysis Using Quantitative Pyrosequencing

[0058] This method was first described by Grunau et al and Dupont et al. Protocols specific for each assay in this study are avaiiabie in S1 Supplementar information or upon request. Incomplete bisulfite conversion was detected by designing amplicons that contained at least 1 unmethyiated cytosine. Primer bias was controlled for by establishing methylation curves of 100% methylated DMA titrated against known amounts of whole genome amplified PGR products thai, by definition, are unmethyiated. These methylation curves allow experimental samples to be calibrated against known standards. Primers for Exon 9, the HI 9 promoter and CTCFBS6 as well as the bisu i ite-con verted sequences they amplify are avaiiabie in S1 Supplementary information. The presence of an A/G polymorphism,

approximately 130 base pairs downstream of CTCF BS6 leads to primer bias and distorts the absolute methylation values of CTCF BS6. Tost et al.

Deducing Parental Contributions of Alleles at CTCF BS6

[0059] All samples were subjected to direct sequencing of CTCF BS8 containing the polymorphism rs 10732516. Primer design and reaction conditions are available in S1 Supplementary Information. The DNA samples were subjected in parallel to methylation sensitive pyrosequencing of the same polymorphism; please see the section titled "Specimen Collection" for further details. Comparing these results allows each parental contribution to be deduced, see Fig 1 for fu!S details..

[0060]: As shown in Fig 3, !GF2 transcription by clinical stage shows an inverse relationship to the %CTCF of identical stage. Fig 3A: IGF2 mRNA levels were approximately 6X lower in the involuting samples compared to their

J 7 proliferating counterparts. Proliferating vs. involuted p = .02, Proliferating vs.

quiescent p ~ .0081 , Quiescent vs involuting p ~ .01. Wilcoxian Rank Sum Test. Erro bars represent standard deviation. Fig 3B; %CTCF changes significantly according to clinical stage. Prolif vs. quiescent p ~ .01 , quiescent vs. invo! p ~ .006, proiif vs. invoi p - .63, Wilcoxian two sample test. Error bars represent standard deviation. Note: All samples in the IGF2 anaiysis were included in the %CTCF analysis, with additional samples.

Genomic Southern Analysis for the H19 Promoter

[006!]: Eighteen were analyzed at a CUA certified molecular diagnostics laboratory where this assay is performed as a clinical test for Beckwith-Wiedemann Syndrome. The assay is originally described by Debaun et al. Norms for this test were previously established with 30 normal controls at 55% methylation with a standard deviation of 5%. All samples were run with a normal and Beckwith- Wiedemann control. The assay exploits a CCCGGG site in the H19 promoter that is cut by the methylation sensitive restriction enzyme Pst1.

Statistical Analysis

[0062] Descriptive statistics were used to present patient characteristics. The difference in expression of IGF2 transcript across the three developmental stages of !H was evaluated using a Kruskai-Wailis test. To evaluate if the relative amount of CTCF compared to BORIS transcript changes predictably over time, change point analysis was performed. Change point anaiysis indicates the likelihood that a change in transcript expression occurred in the sample population by confidence level and a confidence interval regarding when those changes occur. The %CTCF

[CTCF/{CTCF + BORIS) xlQQ] was used to develop a change point model that was then compared against clinical staging and IGF2 expression in the sample population. A fuil explanation of the methods used, as well as a shareware change- point analyzer is presented as an online resource: Taylor, Wayne A, (2000), "Change-Point Anaiysis; A Powerful New Tool For Detecting Changes,"

(http://wwvv.variation.com/cpa/tech/changepointhimS,} To evaluate the association of iGF2 transcript and the relative amounts of CTCF, a linear regression model was fitted, with the %CTCF and age as covahates. The correlation and partial correlation

J 8 were also calculated. Partial correlations indicate what percentage of variance in SGF2 can be explained by CTCF% alone. Analysis of the covarianee model

(A COVA) was fitted to examine if the correlation between the SGF2 transcript and the difference between CTCF and BORIS varied by the paternal genotype at CTCF BS6, once adjusted by age.

Results Master Data Table

10063] in total, 40 samples were analyzed on a molecular basis. A description of basic demographics, with genotypes at CTCF BS6 and transcript expression values for IGF2, H19, CTCF and BORIS with correlative methyiation data was compiled. Please see Fig 1 ; Master Data Table for details, This table can be utilized to confirm any statistical analysis presented in this study.

Expression of IGF2, CTCF and BORIS

10064] FIGS. 4A and 4B Analyzing the Percentage of CTCF Compared to Total CTCF and BORIS Transcript.

[0065]: FIG. 4A Using the two samples with the lowest %CTCF (420 and 418 days as common points {Purple}} two curves with high correlation to age can be appreciated. %CTCF steadily decreases as lesions age until approximately 400 days (red and purple points), then CTCF once again increases compared to BORIS (purple and blue points). This roughly correlates with the transition from proliferative to quiescent lesions. FIG. 4B CSUM of %CTCF demonstrates staiisticaiiy significant variation about the mean of %CTCF according to age. For a full explanation of the CSUM data and commensurate change point analysis,

[0066] IGF2 transcription differed significantly by clinical stages (p<0.0001 , Kruskai-Waliis test). Plateau stage lesions expressed significantly higher levels of SGF2 than proliferating (p - 0.0081 , Wilcoxon rank sum test) and involuted sampies (p - 0.02). Involuted hemangiomas expressed the lowest levels of SGF2,

approximately 6X lower than their proliferating counterparts (p ~ 0.01 ).

[006?] To potentially explain the changes in IGF2 transcription, quantitative RT-PCR was performed fo CTCF and BORIS. CTCF and BORIS are co-expressed in ait samples. However, the percentage of CTCF transcript compared to total CTCF and BORIS in a given sample [CTCF/ (CTCF + BORIS) x 100] varied significantly

J 9 over developmental time (Figs 3B and 4A). This was confirmed by a change point model (Fig 4B): the Y axis is the cumulative sum (CUSUM) of the differences between %CTCF and the average value of %CTCF, A segment of the CUSUM chart with an upward slope indicates a period where the values tend to be above the overaii average. Likewise, a segment wit a downward slope indicates a period of time where the values tend to be below the overaii average. Based on this analysis, two change points, one estimated at 418 days and the other at 1277 days, were detected. Prior to approximately 418 days (90% Cf; 368-547 days), the vaiue of %CTCF tends to maintain a higher level with an average vaiue in this stage equal to 59%, In the second stage (418-1277 days), the level of %CTCF is tow with an average 28%. After the second change point at approximately 1277days (90% CI: 780-1500 days). %CTCF has recovered to a high tevei again with the average 66%. See S2 Supplementary Information for a bar graph analysis of the change point model of %CTCF expression. This result is highly similar to the results obtained by separating samples according to clinical stage (S3 Supplementary Information) %CTCF transcription in IH varies according to clinical stage (Fig 36) Furthermore, the %CTCF varied inversely with IGF2 transcription (compare Fig 3A and 3B) These two graphs clearly demonstrate that a higher %CTCF corresponds with tower levels of 1GF2 expression. Moreover, a strong positive correlation was detected between BORIS and IGF2 transcription (p - 0.0028). Though CTCF alone does not

significantly correlate with 1GF2 transcript levels, taking both CTCF and BORIS into account using %CTCF is the strongest predictor of IGF2 mRNA expression p - .0004(Fsg 5).

[0068]: As described herein, a reduction, especially a rapid reduction, in the %CTCF, as expressed in FIG. 4A, 4B, and 5, may be an indication of an aggressive tumor and may be used to risk strafify the patient, A %CTCF may be determined as a proportion of the sum of the expression level of CTCF and expression level of BORIS, it has been found that a reduction in the %CTCF overtime, or rate of %CTCF, in indicative of a more aggressive tumor, thereby putting the patient in an higher risk classification. For exampie, as shown in FIG. 4A, if the line fit of %CTCF versus age in days has a negative slope, then a patient may be classified in as a highest risk patient and if the slope is positive, then the patient may be classified as a lowest risk patient. [0069] As shown in Fig. 5, IGF2 transcript levels correlate inversely with the percentage of CTCF compared to a total of CTCF + BORIS. This data represents the first demonstration of the potentiaily antagonistic effects of CTCF and BORIS on a target gene's transcription through a continuous curve. P - ,0004 ANCOVA Mode!. Age effect was not significant in the model p - ,241. The %CTCF is a stronger statistical predictor of SGF2 expression than BORiS alone .0004 vs. .0028

respectively. (N = 15}

[0070] Fig 8. shows Western Analysis of 24 IH samples via 5 independent Western Biots. Twenty samples with 4 blots depicted, demonstrates 4 stages of CTCF and BORIS expression. 6-1 : A Sow concentration of anti-CTCF (1 : 10,000) demonstrates the complete spectrum of CTCF expression with increases early (210 to 418} and late (760 to 987) in protein expression, (Note, a testes negative controi was included as well as a venous malformation denoted as "V .") 8-2 through 6-4 were probed with 1 :5000 concentration of anti-CTCF that more clearly demonstrates the early rise in CTCF thai occurs after 367 days. 6-2 suggests an early increase in BORiS with precipitous downregulation after 244 days. 6-3 and 6-4 expand this critical age range demonstrating a period from 244 days to 367 where BOR!S is downreguiated but CTCF is not yet upreguiated. Note, samples marked with an asterisk were also subjected to CTCF and BORIS rtPCR.

[007 ij Western analysis of CTCF and BORIS confirms and expands upon the transcript data (Fig 6) As expected in proliferating lesions, BORIS transcript and protein levels steadily rise in early stage samples (Fig 4A transcript data, Fig 6-2 Western Analysis.} Furthermore, during the transition from quiescent to involuting samples, CTCF mRNA and protein increase compared to BORIS (Figs 3B, 4A and 4B transcript, and Fig 6-1 protein.) Thus, the western and transcriptional data globaiiy confirm one another at the endpoints of IH development. However, the protein data suggests a third change in CTCF and BORIS levels that the transcript change point analysis does not. This third proteomic change appears to take place at the fate proliferating to eariy quiescent phase, it coincides with the so-called late proliferative stage in IH that is suggested by clinicians but not universally accepted. These data provide the first molecular support for what was previously a clinical category: the iate proliferative stage of IH growth. The relative expression of CTCF and BORIS via both transcript and protein levels, is predictive of clinical stage and IGF2 expression. See Fig 7 for details.

[0072] Fig 7. shows a schematic integrating CTCF and BORIS expression via both transcript and protein with clinical stage. The Western analysis suggests 4 stages of CTCF and BORIS (see Fig 3, panels 1 to 4) each stage leading to higher levels of CTCF expression relative to BORIS. These interval changes in protein expression closely correlate with clinical stage. Furthermore, bars above the stages represent the 95% confidence intervals of the two change points identified by quantitative rtPCR. These data show remarkable agreement reinforcing the idea that relative CTCF and BORIS expression levels closely mirror the clinical stage of the lesions tested. Of note, the CTCF and BORIS protein data also suggest a molecular basis for a late proliferative stage.

[0073] The CTCF to BORIS transcript difference (G~B) predicts 1GF2 transcription according to the paternal allele at CTCF BS6, This study utilizes existing technologies: Direct sequencing of the known polymorphism of CTCF BS6 (rs10732516) with a previously described methylation assay for CTCF BS6. Applying these two assays in a novel manner (see Fig 2) allowed us to deduce both maternal and paternal contributions to CTCF BS6— which will be referred to as the maternal and paternal contribution (Fig 8.) This paternal contribution likely has significant effects on IGF2 production as it relates to CTCF and BORIS.

[0074] IGF2 mRNA is demonstrated to be inversely related to CTCF and positively correlated to BORIS transcripts when plotted against %CTCF (Fig 5).

[0075] By using the difference between CTCF and BORIS (C-B) rather than the %CTCF, this relationship can be differentiated by the paternally contributed allele at CTCF BS8 (Fig 8A). The paternal allele of a common C/T polymorphism within CTCF BS6 (rs10732516) corresponds with two strikingly different CTCF-BORIS vs. SGF2 curves. After age adjustment, the effect of CTCF-BORIS on IGF2 transcription was significantly different between patients bearing different paternal alleles {p ~ 0.05, ANCOVA model) and there is a strong correlation between IGF2 expression and CTCF-BORIS (p - 0.0007). The samples bearing a paternal C allele, appear to demonstrate a six fold steeper slope of SGF2 mRNA relative to the CTCF-BORIS difference, compared to their paternal T bearing counterparts, This allele was identified in both tissue and patient matched control blood. Of note, heterozygote analysis revealed no dear relationship regarding these factors according to the maternal allele (p ~ 0.95, ANCOVA model), it remains a possibility that the paternal allele effect may be steroid treatment driven as more samples with the paternal T allele were treated with steroids than the paternal C allele. This potential bias was investigated with an odds ratio calculation sorting steroid treatment according to paternal genotype. The odds ratio suggested that paternal T samples were more likely to be treated with steroids but this result did not reach statistical significance. As the allele specific analysis was done on only proliferative samples the odds ratio calculation was performed twice, once including involuted samples and once to their exclusion. However, it is acknowledged that the odds ratio suggested a potential steroid treatment bias in paternal T samples, which may have become significant in a larger patient cohort. Lastly, IGF2 expression in !H was mono-allelic in all 5 informative heterozygoies tested for a known IGF2 polymorphism in exon 9. SGF2 imprinting status appears to be maintained despite BORIS expression.

[0076] H19 transcript levels correlate positively with CTCF mR A according to the maternally contributed allele at CTCF8S6. After age adjustment, CTCF transcript levels alone correlated positively with H19 transcription but only when separated by maternai genotype ({p - 0.0150, Fig SB) Moreover, this positive correlation is significantly different among patients with different maternai alleles (p = 0.0129, ANCOVA). The correlation between CTCF and H19 transcription is stronger in patients bearing a maternal C allele compared to their maternal T counterparts. There were no identifiable relationships between H19 transcription and either the paternal genotype at CTCF BS6 or BORIS mRNA, p ~ .8 ANCOVA (S4A

Supplementary Information.) There also appeared to be no relationship between H19 expression and clinical stage of the hemangioma (S4C Supplementary information).

[0077] It remains a possibility that the maternal allele effect may be steroid treatment driven as more samples with the maternal T allele were treated with steroids than the maternai C allele. This potential bias was investigated with an odds ratio calculation sorting steroid treatment according to maternal genotype. The odds ratio suggested that maternal T samples were more likely to be treated with steroids but this result did not reach statistical significance (See S4F Supplementary information.) However, it is acknowledged that this odds ratio may have become statistically in a larger sample size; the effect was not great enough to significantly bias the sample size.

[0078] CTCF transcript levels alone correlate with demethylation of the H19 Promoter. Ail !H samples tested demonstrated significant hypomethyiatson at the H19 promoter compared to matched patient blood controls (Fig 9A). This was demonstrated with bisulfite specific pyrosequencing (Fig 9B) and confirmed with methylation sensitive enzyme digest and southern analysis {Fig 9C). As

pyrosequencing is quantitative, these data were correlated with CTCF and BORIS expression in matched sampies. Decreased promoter methylation correlated with higher levels of CTCF (p - 0.015; simple regression. Fig 9B). Yet, this finding may be subject to confounders as it is not statistically significant after age adjustment (p ~ 0,17), As the H19 promoter is paternally methylated and maternally demethyiated, a methylation level below 50% would entail demethylation of the paternal allele in at least a subset of cells within a given sample. However, no bt-allelic expression of H 9 could be detected in 5 heterozygote samples (S5 Supplementary Information.) Furthermore, Hi 9 promoter demethylation did not strongly correlate with H19 expression (S6 Supplementary information.)

[0079] Fig 9, shows CTCF expression and HI 9 promoter methylation. Fig 9A: increased CTCF transcript level correlates with demethylation of the H 9 Promoter, Those samples with the highest CTCF expression were the least methylated ranging from 34% to 14%. However, demethylation of the H19 promoter did not correlate strictly with H19 transcript expression (S8 Supplementary information). Fig 9B and 9C~~The H19 promoter (see Fig 9D) is hypomethylaied, demonstrated by bisultife converted pyrosequencing (9B) and methylation sensitive restriction digest with southern hybridization (9C.) 25 IH samples, and 13 matched blood controls were subjected to bisulfite converted pyrosequencing. 13 IH sampies and 13 matched blood controls were subjected to southern analysis with methylation sensitive Pst1 digestion. Two representative gels show, 5 IN samples, a Beckwith-Weidman positive control and a 50% methylated normal control. Fig 9D: sequence showing the H19 promoter— CpG#4 of the bisulfite sequencing test corresponds to the CCCGGG Pst1 digestion site of the Southern analysis. Other CpG's tested are in bold. This CpG is in close proximity to the transcription start site of H19 (blue arrow) and an overlapping putative CTCF binding site identified by positional weight matrix analysis.

[0080] Multiple imprinted sites within the SGF2/H19 locus are abnormally methylated in IH compared to matched control b!ood.

[0081 j it remains a forma! possibility that the normalization of CTCF to BORIS ratios, as well as decreased IGF2 transcription, in involuting and involuted samples is not due to an intracellular phenomenon but rather the incremental replacement of abnormal IH tissue (vascular stroma) with normal tissue (fat,) Thus, the results presented are the product of tissue heterogeneity. It is acknowledged that IH lesions transform from a vascular tumor into a fibrofatty residuum; therefore, the transitional phases are by definition composed of heterogeneous cell populations. However, no evidence was observed that the fibrofatty residuum of an involuted SH represents "norma!" tissue. To the contrary, many of the methylation abnormalities discovered by this study are either stab!e or progressive from early to late ciinica! stages. For instance, the H19 promoter is significantly demethyiated in all IH samples (see Fig 9A-9C). However, the demethyiation is progressive with age (S7 Supplementary information.) Furthermore, it was also found focal demethyiation at Exon 9 and hypermethy!ation at D R0, deviating from the expected 50% for these known imprinted sites. Both findings remained consistent in ail !H ciinica! types and were age independent (SB and S9 Supplementary information for details. ) !f IH tissues were being replaced by norma! fat it wouid be expect the methylation abnormalities demonstrated in this work to normalize, not remain constant or even progress with age. Given this argument, the simplest explanation for the methylation data is that !H tissue begins as epigeneticai!y abnormal vascular stroma and transforms into epigeneticaily abnormal fat or is at least replaced by the like.

CTCF BS6 Genotypes Correlate With C!inica! Outcomes

[0082] Mechanisms aside, parent of origin specific effects are demonstrated at the molecular level regarding expression patterns of both !GF2 and H1 , However, the question remains whether these molecular phenotypes may translate into clinically significant growth patterns. Table 2 is a complete table of all patients participating in this retrospective clinical study. For details of subject inclusion piease see methods section. Size of the !esion as well as the date of examination was included with relevant clinical information such as sex, medical treatments utiiized and presence of ulceration during clinicai course. Each patient was sorted according to CTCF Binding Site Six Genotype and paternal contribution for heterozygotes. By plotting the size of IH lesions against the CTCF BS6 genotypes, four distinct growth curves emerge (Fig 10A and 10B).

Table 2: Summary of clinicai data. Retrospectively collected results with associated descriptive information.

[0083] As shown in Table 2, many of the patients were treated with a cortical steroid-methyl pregnlsone, either fay injection or systemic oral intake. Table 3 shows that the patients with a non-TT allelic expression had a much better response to this medication, wherein only 25% had a failure of response to the medication, in contrast, the patients with aa IT allelic expression had the least effective response to the Cortical steroid treatment.

Table 3

[0084]: The results of this analysis shows that the type of allelic expression can be used to predict the effectiveness of Cortica! steroid treatment. The odds ratio from this stud is 10.5 that the TT group will fail steroids, with a sensitivity of 58.33% (95% CI 27-84%) and a specificity of 88.33% (95% CI 63-98%). The failure of steroids is defined in this analysis as the patient requiring surgical treatment despite the treatment with the medication, cortical steroids.

[0085] Fig 10. Clinical Correlation of Hemangioma Growth Rates with Parental Contributions to CTCF BS6, Fig 10A; This retrospective analysts of 29 samples, 3 TT, 20 non TT, demonstrates significantly distinct growth curves over a large age range. The ANCOVA model has identified age as a predictor of size p =.0007., The association between tumor size and age is significantly different among the genotypes of TT, C/T, T C and CC p < ,0001 . Of Note the paternal contribution is presented first and the maternal is second. The interaction terms of parentally specific genotypes allowed us to test if the slopes of the curves between tumor size and age are different among the genotypes. This analysis indicted that an increase in 1 day of age is associated with .0 6cm of growth in the TT group. This is significantly higher than the non TT group p - .0019. Fig 10B: Growth analysis focusing on the "non TT" group. Each non TT growth curve varied independently and significantly from the TT samples (CC vs. TT; P<0.0001 , CT vs. TT. PO.0008, TC vs. TT: P - 0.0025). Furthermore, these data suggest parent of origin specific effects as those samples with identical genotypes but opposite parental contributions displayed statistically significant differences in growth curves. The paternal

T/rnatemai C genotype grew at approximately twice the rate as their paternal C/ ^' maternal T carrying counterparts (p = .05). The homozygous C group appeared to have a roughly flat growth rate between the heierozygotes and did not significantly vary with either hetero2ygote group (CC vs. C/T p = ,99,CC vs. T/C p = .74)

[0086] The association between tumor size and age (days) are significantly different among these four genotypes (separating heierozygotes by their respective parental contributions) CC, C/T, T/C, TT (p = 0.0162, ACOVA.) The most impressive growt phenotype was exhibited by homozygous T samples reaching an average of 7.8cm before excision {Fig 1 1A) Comparing the TT group against all non-TT subjects, the difference in !esion size, increased significantly with age (p ~ 0.0001 , ANCOVA), Thus in this study, TT lesions grew more rapidly than non-TT genotypes, in fact, each growth curve— separated by maternal and paternal genotype— varied independently and significantly from the TT samples (CC vs. TT: PO.0001 , CT vs. TT: PO.0008, TC vs. TT: P = 0.0025). Although genotype appears to have no effect at approximately 100 days, after three months, lesions begin to distinguish themselves suggesting distinct growth velocities. Furthermore, these data suggest parent of origin specific effects as those samples with identical genotypes but opposite parentai conitibutions displayed statistical significant differences in growth curves. Namely, those lesions carrying the paternal T /maternal C genotype grew at approximately twice the rate as their paternal C/ maternal T carrying counterparts p - .05 (Fig 10B) Furthermore, each heterozygote growth curve varied by age with high correlation of r squared above ,85. However, it must be emphasized that sample size is relatively low (particularly in the C patema!/T maternai group) and the p-value just reached the threshold of significance.

Table 4

CLINICAL TABLE OF ULCERATION BY TT AND NON-TT GENOTYPE

Retrospectively coJIeetetj results wittt associated descriptive statistics: TT Lesions have a significantly higher associated odds ratio for ulceration. This proposed clinical test may be most useful in ruling out the chance of ulceration early in the disease course as sensitivity and negative predictive value are high. A

larger prospective study is. warranted.

Sensitivity ~ 100%

Specificity - 88-98% Odds Ratio ~ 76.1

Positive Predictive Value ^™ 95% Ci = 3.4-1676

Negative Prediciiive Value P ~ ,006

[0087] TT Lesions Have a Significantly Higher Associated Odds Ratio for Ulceration. This proposed ciinica! test couid be most useful in ruling out the chance of u!ceraiion early in the disease course as sensitivity and negative predictive value are high. A iarger prospective study is warranted.

[0088] Lastly, size is a highly significant clinical outcome when studying IH. However, of similar importance is ulceration. Once an IH ulcerates, it is usually painful for the patient and Is given to bleeding which can be clinically significant. Ulceration is usually a marker of rapid disease progression and heralds an escalation of care. This can entail the institution of laser therapy, pharmacologic intervention or surgical excision. Not surprisingly, ulceration alone can prompt surgical treatment regardless of size or location of the iesion. To study the risk of ulceration an odds ratio calculation was performed comparing TT and non-TT lesions. The TT iesions had an odds ratio of 76.1 for ulceration p = .006 (Table 4). Although this is a small sample cohort preliminary specificity was performed, sensitivity and positive and negative predictive value calculations (Table 4). These early results suggesi the highest clinical usefulness of the proposed test in ruling out potential future ulceration. Although encouraging, these results wiS! need to be corroborated prospectively in a larger cohort,

[0089] It will be apparent to those skilled in the art that various modifications, combinations and variations can be made in the present invention without departing from the spirit or scope of the invention. Specific embodiments, features and elements described herein may be modified, and/or combined in any suitable manner. Thus, it is intended that the present invention cover the modifications, combinations and variations of this invention provided they come within the scope of the appended claims and their equivalents.

[0090] The following references are hereby incorporated by reference in their entirety;

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