Field of the Invention

The present invention relates to a diagnostic kit suitable for use in the diagnosis of the Philadelphia-like acute lymphoblastic leukemia (Ph-like ALL) subgroup, which is in the Ph-negative ALL group and is characterized by a gene expression profile similar to that of Ph-positive ALL, a diagnostic method using this kit, and a diagnostic algorithm.

State of the Art

There is a subgroup defined as Ph-positive with BCR-ABL1 fusion in B-ALL type leukemia cases. In recent years, a new subgroup has been defined in which cases progress with a poor prognosis. This subgroup is Ph-like ALL. Although Ph-like ALL is similar in gene expression profile to Ph-positive ALL cases, it does not have the fusion protein of the Ph chromosome like the Ph-negative ALL cases. This group is defined by gene expression panels and has a genetically heterogeneous structure. Since patients with Ph-like ALL are very difficult to diagnose, different diagnostic methods have been developed with studies in recent years.

Today, different pediatric study groups develop different strategies for the diagnosis and characterization of Ph-like ALL based on the patients' ethnicity, the number of patients to be tested, the availability of the genome/transcriptome sequencing infrastructure in the diagnostic laboratories, and the clinical target. The most important parameters to be considered while applying diagnostic methods are the consideration of molecular characterization and identification of molecular lesions potentially suitable for targeted therapy. Gene expression analysis is considered the "gold standard" in the diagnosis of Ph-like ALL. Two different research groups developed low-density gene expression panels (LDA) to identify Ph-like ALL cases in 2009. Although LDAs used in the diagnosis of Ph-like cases have high specificity, In the study of Wells J. et aL, they have determined the overlapping patient profile of the two panels as 18% (Wells et aL, Philadelphia Chromosome-Like Acute Lymphoblastic Leukemia: Progress in a New Cancer, 2017). These two research groups, which were pioneers in identifying the Ph-like subgroup, we cannot distinguish the same cases with 100% confidence. This ratio indicates how specific the diagnosis is for Ph-like ALL cases. When the gene expression profiles evaluated in both studies are examined, it is seen that only seven gene clusters (CCND2, SH3BP5, ABL1 , SOCS2, DUSP6, LST1 , EGFL7) overlap. The COG-TARGET-St Jude research group first identified a subgroup of high- risk classified B-ALL cases with a gene expression profile similar to Ph-positive cases by Gene Set Enrichment Analysis. This group evaluated microarray data of the Ph- positive patient group and high-risk B-ALL patient group by microarray predictive analysis (PAM) with “257 probe Affymetrix gene expression analysis method” to identify the Ph-like group in it (Wells et aL). The other research group performs “Hierarchical cluster analysis with 110 probe sets” for classification of the main pediatric ALL subtypes (T-cell, ETV6-RUNX1 , high hyperdiploid, TCF3-PBX1 , MLL) (Boer et aL, BCR-ABL1 -like acute lymphoblastic leukemia: From bench to bedside, 2017).

Since patients with Ph-like ALL are very difficult to diagnose, recent studies have focused on developing various differential diagnosis methods. In 2016, the World Health Organization (WHO) revised its 2008 publication and drew attention to the diagnostic difficulty of this patient group. Today, different pediatric study groups and centers develop different strategies for the diagnosis and characterization of Ph-like ALL based on the geographical structure of the studies, the ethnicity of the patients, the number of patients to be tested, the availability of the genome/transcriptome sequencing infrastructure in the diagnostic laboratories, and the clinical target. The most important parameters to be considered while applying diagnostic methods are the consideration of molecular characterization and identification of molecular lesions potentially suitable for targeted therapy (Pui et aL, Philadelphia Chromosome-like Acute Lymphoblastic Leukemia, 2017). After identifying the Ph-like group, it will be ensured that the cases receive appropriate intensity chemotherapy. It will be possible to avoid unnecessary and toxic side effects for low-risk ALL.

Today, there are many clinical diagnosis methods for Ph-like ALL, and each method has its advantages and disadvantages. For example, it is impossible to distinguish many gene expression changes seen in the Ph-like subgroup by cytogenetic analysis, so this method has severe limitations.

In the patent documents numbered CN106282357A and CN1 10218790A in state of the art, specific FISH panels are used to diagnose Ph-like patients. However, additional fusion probes or additional tests such as reverse transcription-polymerase chain reaction (RT-PCR) are needed to identify the translocations detected due to the positive finding of the analysis. In the patent document numbered US2017298449A1 , another document in state of the art, It has been explained that it is possible to evaluate the expression of many genes simultaneously with microarray technology. However, there are two different methods involved in array generation. The disadvantage of in-situ synthesis technology in the photolithography method is that it is limited to 25 nucleotides in length. Evaporation and contamination problems are seen in the mechanical fixation process due to the direct contact with the surface and very small amounts of liquid.

Next-Generation Sequencing (NGS), another method used to diagnose Ph-like ALL patients, has many advantages and disadvantages in the art. While the remarkable advantages of the system are formed with the ability to identify newly formed fusions, analyze secondary mutations, and obtain more sensitive and sensitive results, the inability to identify new fusion partners, high cost, long diagnosis times, and the necessity of confirming with basic methods which are considered as the golden rule, is seen as the disadvantages of this method in some NGS-based methods.

When the current applications are examined, it is seen that the methods used in the diagnosis of Ph-like ALL disease are incomplete and have many disadvantages. Accordingly, it is thought that there is still a need for a diagnostic kit that enables the analysis of gene expression differences and similarities related to this particular disease, preferably customized to ethnicity.

Brief Description of the Invention

The present invention is related to a diagnostic kit suitable for diagnosing Ph-like acute lymphoblastic leukemia, which fulfills the abovementioned requirements, eliminates all disadvantages, and brings additional advantages.

The main aim of the invention is to develop a diagnostic kit that enables Ph-like acute lymphoblastic leukemia cases to be differentiated and classified from B-ALL type leukemia cases.

Another aim of the invention is to develop a diagnostic kit that enables the detection of genes suitable for use as biomarkers in identifying Ph-like ALL cases. Another aim of the invention is to develop a diagnostic kit that paves the way for patients with Ph-like ALL, who are separated from those diagnosed with B-ALL, to receive effective treatment.

Another aim of the invention is to develop a diagnostic kit that enables the creation of a specific gene expression panel for patients of a particular ethnicity.

Another aim of the invention is to develop a diagnostic kit that enables the creation of the gene expression panel of Turkish patients.

Another aim of the invention is to develop a diagnostic method and algorithm using said diagnostic kit.

The present invention is a diagnostic kit suitable for use in the diagnosis of Ph-like acute lymphoblastic leukemia to fulfill the above-described aims, comprising the following genes; CD99, NOTCH1, EZH2, MPL, MLLT4, BCL2, IKZF2, FBXW7, CHD1, IRF8, IDH1, CD274, ETV6, IGJ, FLT3, CREBBP, EBF1, MLL, EPOR, JAK2, KDM6A, IRF4, IKZF3, JAK3, KRAS, CRLF2, HOXA9, IL7R, ABL1, LMO1, FGFR1, NF1, IFITM1, CSF1R, CA6, IDH2, CTLA4, MUC4, IKZF1, JAK1, BCL6, AICDA, KLF2, MYC, BLNK, HOXA 10, BCL11B, P2RY8, SEMA6A, SOCS1, PDCD1LG2, RUNX1, PTK2B, NUDT4, PAX5, SOCS2, PIC ALM, NTRK3, STIL, SH2B3, TLX3, TCF3, NRAS, WT1, TP53INP1, TAL 1, PDGFRA, STAT3, PBX1, SETD2, NUP214, PDCD1, PAG1, PTPN1, PDGFRB, STAT5B, ABL2, DNM2, DNTT, LYL 1, NT5C2, NUP98, PTPN11, GAPDH, RAG1, RAG2, SOX11, GPR110, TLX1, G6PD, TYK2, ZCCHC7, BCR, IFITM2, BRAF, ACTB.

The present invention is also a diagnostic kit for use in the diagnosis of Ph-like acute lymphoblastic leukemia, comprising the following process steps; i. Total RNA isolation from bone marrow ii. cDNA synthesis from total RNA iii. Performing gene expression analyzes by a reverse transcription-polymerase chain reaction. iv. Performing statistical analyzes and determining similarity rates with a diagnostic algorithm.

The structural and characteristic features of the present invention will be understood clearly by the following detailed description. Therefore the evaluation shall be made by taking this detailed description into consideration. Detailed Description of the Invention

In this detailed description, the preferred embodiments of the inventive diagnostic kit suitable for use in the diagnosis of Ph-like acute lymphoblastic leukemia are described only for clarifying the subject matter in a manner such that no limiting effect is created.

The inventive diagnostic kit aims to analyze and detect gene expression differences to distinguish the Ph-like ALL patient group from pre-B-ALL cases.

Gene expressions differ in different societies depending on ethnicity, and Ph-like ALL disease shows heterogeneous characteristics. In other words, there may be situations where some genes that are predicted to be important are not expressed at all in inpatient groups in different cohorts. This situation confirms that the panels approved for diagnosis may differ according to the ethnic origin of the patient profile. In this direction, although the diagnostic kit developed within the scope of the present invention is suitable for use on people of all ethnic origin, especially in T urkish people and societies with similar ethnic origins (Cases of Turkish origin living in East Turkestan, Kazakhstan, Kyrgyzstan, Uzbekistan, Turkmenistan, Turkey, Azerbaijan, and Iran) are aimed to be developed to be used in the diagnosis of developing Ph-like ALL disease.

The present invention is a diagnostic kit suitable for use in the diagnosis of Ph-like acute lymphoblastic leukaemia, comprises the following genes; CD99, NOTCH1, EZH2, MPL, MLLT4, BCL2, IKZF2, FBXW7, CHD1, IRF8, IDH1, CD274, ETV6, IGJ, FLT3, CREBBP, EBF1, MLL, EPOR, JAK2, KDM6A, IRF4, IKZF3, JAK3, KRAS, CRLF2, HOXA9, IL7R, ABL 1, LMO1, FGFR1, NF1, IFITM1, CSF1R, CA6, IDH2, CTLA4, MUC4, IKZF1, JAK1, BCL6, AICDA, KLF2, MYC, BLNK, HOXA10, BCL11B, P2RY8, SEMA6A, SOCS1, PDCD1LG2, RUNX1, PTK2B, NUDT4, PAX5, SOCS2, PICALM, NTRK3, STIL, SH2B3, TLX3, TCF3, NRAS, WT1, TP53INP1, TAL 1, PDGFRA, STAT3, PBX1, SETD2, NUP214, PDCD1, PAG1, PTPN1, PDGFRB, STAT5B, ABL2, DNM2, DNTT, LYL 1, NT5C2, NUP98, PTPN11, GAPDH, RAG1, RAG2, SOX11, GPR110, TLX1, G6PD, TYK2, ZCCHC7, BCR, IFITM2, BRAF, ACTB.

According to the preferred embodiment of the invention, the inventive diagnostic kit is in the form of a test panel with at least 96 wells in which the planned primer for the targeted gene is implanted. According to this embodiment of the invention, the volume per well is 20 pl. According to an embodiment of the invention, each well contains a gene in the amount of 2 pl, and the genes contained in the wells are different from each other. As a result of the analyzes made within the scope of the invention, it has been determined that the genes included in the diagnostic kit described in this embodiment of the invention are suitable for use in the diagnosis of Ph-like ALL cases in Turkish patients.

The present invention is also a diagnostic kit for use in the diagnosis of Ph-like ALL, comprising the following process steps; i. Total RNA isolation from bone marrow ii. cDNA synthesis from total RNA iii. Performing gene expression analyzes by a reverse transcription-polymerase chain reaction. iv. Performing statistical analyzes and determining similarity rates with a diagnostic algorithm.

In the method subjected to the invention, the bone marrow is centrifuged with different solutions in multiple steps to isolate the total RNA mentioned in the process step (i). According to one embodiment, peripheral blood material can also be used instead of bone marrow material. In said process step, preferably, bone marrow material is centrifuged with 1000 pl triazole and 200 pl chloroform at 12000 rpm for 15 minutes. Following that, the separated material is incubated for 10 minutes and then centrifuged with 500 pl of isopropyl alcohol at 10000 rpm for 10 minutes. Then, the separated material is centrifuged with 1000 pl of 75% ethanol at 7500 rpm for 5 minutes. Finally, the separated material is dissolved in 100 pl of distilled water for 5 minutes.

The quantity and quality of the isolated RNAs are measured using the UV-Vis Spectrophotometer device. Samples are preferably stored at -800 by considering that the recommended A260/A280 ratio in the panel protocol used is in the range of 1 .8-2.0.

The expression process mentioned in process step (ii) is carried out with a cDNA reverse transcription kit in the method subjected to the invention. In a preferred embodiment of the invention, cDNA is synthesized from the total quality RNA obtained in the process step (i) by PCR device, following the manufacturer's protocol by the "cDNA Reverse Transcription" kit. Said kit contains 0.15 pl of 100mM dNTP, 1 pl of reverse transcriptase enzyme, 1.5 pl of transcription buffer, 0.2 pl of ribonuclease inhibitor (20 U/pl), 1 pl of reverse primer, 6.15 pl of ribonuclease-free H ₂ O. 5 pl of the RNA sample obtained in the (i) processing step is also used. The binding temperature of the random primer to singlestranded RNA is 10 min at 250, 120 min at 370, wh ere reverse transcriptase enzyme is activated, the cDNAs obtained after 5 min incubation cycles at 850 to inactivate the enzyme was stored at -200 until the next process s tep.

In the method subjected to the invention, it is implanted to a plate with 96 or 384 wells in the process step (iii). Ready-to-use test panels are used for gene targets specific to human, mouse, and rat species. The mRNAs of the B-cell Ph-like ALL group, which are considered important in the diagnosis process in state of the art, were evaluated within the scope of the invention. In this direction, mRNAs are determined by literature research, and RT-PCR analysis is performed from the total RNA material. As a result of RNA molecules performing complementary DNA (cDNA) synthesis with the help of reverse transcriptase enzyme isolated from retroviruses, RT-PCR is a fast, sensitive, and multiplex method used to identify the presence of fusion partners, enabling gene expression analyses to be performed, making it possible to determine the amplification of cDNA and its products in a single tube. A wide range of translocation testing is performed using 30 or 50 gene probes per reaction with this methodology.

RT-PCR analysis according to one embodiment comprises the following processes; 15 minutes of reverse transcription at 60°C, 10 minutes of pre-incubation at 95°C, 15 seconds of amplification at 95°C, 30 seconds of amplification at 58°C, and 30 seconds of cooling at 40°C. According to this application, the total number of amplification cycles is 45.

In one embodiment of the invention, the genes whose significance was determined as a result of the analysis are as follows; CD99, NOTCH1, EZH2, MPL, MLLT4, BCL2, IKZF2, FBXW7, CHD1, IRF8, IDH1, CD274, ETV6, IGJ, FLT3, CREBBP, EBF1, MLL, EPOR, JAK2, KDM6A, IRF4, IKZF3, JAK3, KRAS, CRLF2, HOXA9, IL7R, ABL1, LMO1, FGFR1, NF1, IFITM1, CSF1R, CA6, IDH2, CTLA4, MUC4, IKZF1, JAK1, BCL6, AICDA, KLF2, MYC, BLNK, HOXA10, BCL11B, P2RY8, SEMA6A, SOCS1, PDCD1LG2, RUNX1, PTK2B, NUDT4, PAX5, SOCS2, PICALM, NTRK3, STIL, SH2B3, TLX3, TCF3, NRAS, WT1, TP53INP1, TAL 1, PDGFRA, STAT3, PBX1, SETD2, NUP214, PDCD1, PAG1, PTPN1, PDGFRB, STAT5B, ABL2, DNM2, DNTT, LYL 1, NT5C2, NUP98, PTPN11, GAPDH, RAG1, RAG2, SOX11, GPR110, TLX1, G6PD, TYK2, ZCCHC7, BCR, IFITM2, BRAF, ACTB.

The diagnostic kit subjected to the invention is in the form of a 96-well panel in which the oligonucleotides of these genes are implanted. GAPDH, ACTB, and G6PD genes among these genes are on the panel as housekeeping genes. In step (iv) of the method subjected to the invention, the similarity ratio between the control sample and the patient group is determined. To identify the Ph-like group, the Ph-like group is expected to show a similar expression profile as the Ph-positive group when the mRNA expression profiles of Ph-negative and Ph-positive cases are compared. Therefore, the Ho hypothesis, which is meaningful with the definition of difference in statistical analysis, is not meaningful in our study group. The statistical significance of the present study carried out within the scope of the invention confirms the "no difference" hypothesis.

The mRNA expression levels of the patients analyzed within the scope of the invention were evaluated, and it was determined that 96 genes had similar expression levels with the Ph-positive control sample. For similarity classification that can be used to identify Ph-like ALL cases, a diagnostic algorithm is used. The p values between the patient and control groups are analyzed with standard distribution tests. A value of p=0.05 is accepted as the limit for similarity classification according to this algorithm. The "p" value here indicates the amount of possible error when the decision "there is a statistically significant difference" will be made in the comparison. The maximum acceptable level of error was proposed as 0.05 and was accepted. If the P-value found as a result of a test analysis is less than 0.05, there is a significant difference as a result of the comparison. If the P-value is above 0.05, there is no significant difference. As a result of the analysis, cases with a statistical difference (p<0.05) compared to the control sample are defined as having low similarity to our Ph-positive control sample. Cases with no statistical difference (p>0.05) compared to the control sample are highly similar to the Ph-positive control sample.

After the evaluated gene expression profile analysis, it is stated that it is required to add tyrosine kinase inhibitors to treatment protocols if the cases showing the highest similarity to the control samples with the said diagnostic algorithm are in the Ph-like ALL classification.

Ph-like ALL cases can be distinguished from B-ALL cases with the diagnostic kit and method subjected to the invention. Thus it paves the way for patients with Ph-like ALL to receive effective treatment.

Ninety-six genes with potential importance in the identification of Ph-like ALL cases in Turkish patients were identified with the said invention. In the light of the findings obtained within the study; the identified genes are data for the identification of the Ph- like ALL group, a panel containing a total of 96 genes specific to Turkish Ph-like ALL subgroup patients, which can be used in the diagnosis, and an algorithm that enables the bioinformatics evaluation of the panel were created with the analyzes performed. Thus, following the identification of the Ph-like group, classical ALL treatment protocol is envisaged to provide individual benefit and increase survival rates by revising and structuring effective treatment protocols in which new targeted drugs are added to the patients.