Use of WBP2 as a prognostic and predictory biomarker for tamoxifen resistance in breast cancer

Abstract

The invention relates to a method for predicting response or resistance to treatment in patients using tamoxifen in the treatment of breast cancer for use in biotechnology, bioinformatics and healthcare. In particular, the invention relates to a method for predicting the resistance mechanism of tamoxifen used as first-line treatment in ER+ breast cancer subtype and the use of the WBP2 gene as a prognostic and predictive biomarker

Description

[0001] USE OF WBP2 AS A PROGNOSTIC AND PREDICTORY BIOMARKER FOR TAMOXIFEN RESISTANCE IN BREAST CANCER

[0002] Field of the Invention

[0003] The invention relates to a method for predicting response or resistance to treatment in patients using tamoxifen in the treatment of breast cancer for use in biotechnology, bioinformatics and healthcare.

[0004] In particular, the invention relates to a method for predicting the resistance mechanism of tamoxifen used as first-line treatment in ER+ breast cancer subtype and the use of the WBP2 gene as a prognostic and predictive biomarker

[0005] State of the art

[0006] Tamoxifen is used as first-line treatment in the ER+ breast cancer subtype, which accounts for 70% of breast cancer. It is of great importance to provide effective tamoxifen treatment in this patient group. In patients using tamoxifen for breast cancer, toxicity occurs due to active drug use and patients may develop resistance to treatment. De nova and acquired resistance in breast cancer patients complicate treatment strategies. However, there is a need to develop personalized strategies, aiming for patients to get the best benefit from tamoxifen treatment, which is the most commonly used treatment in clinics. The molecular mechanism associated with developing tamoxifen resistance has not yet been fully elucidated. New studies are needed to determine the effective dose of drugs used in clinics and to predict the development of resistance, to identify and reduce side effects in patients, and to increase the patient's response to treatment.

[0007] At this point, it is aimed to identify prognostic biomarkers and provide new perspectives in the treatment protocol with groundbreaking gene therapy methods in recent years. In this context, understanding the molecular mechanisms in the development of tamoxifen resistance is of great importance both for the effective treatment of the patient and for the prediction of poor prognosis in patients, considering that ineffective treatment increases drug costs. Especially in patients using tamoxifen drug in breast cancer, there is a need for a development that will contribute to the health sector economically, both to reduce toxicity due to effective drug use and to cover drug expenses in patients who have developed resistance for a treatment that will not be effective by the Ministry of Health.

[0008] In the literature search, document numbered W02008091326A1 is encountered. The application relates to a method for estimating tamoxifen sensitivity in a subject. The method comprises determining the level of WWOX expression in a biological sample from the subject; and comparing the level of WWOX expression in the biological sample with a control. Another application in the prior art, WO2018013466 A2, relates to a method for determining the likelihood that breast cancer in a subject will respond to an endocrine therapy. However, there is no explanation in the documents regarding the use of WBP2 for the detection of drug resistance, especially tamoxifen resistance.

[0009] The document numbered WO2012030302A1 , found in the research conducted in the literature, is related to methods and kits for determining susceptibility to cancer or diagnosing and / or treating cancer. Here, WBP2 is identified as a target of tyrosine phosphorylation of estrogen signaling via EGFR and it is suggested that phosphorylation of WBP2 a Tyr192 and Tyr231 can be regulated by c-Src and c-Yes kinases using RNAi. In addition, only downregulation of the gene was achieved by phosphorylation, and phospho-WBP2 was shown to be more effective than chemotherapeutic agents. Another application in the art, WO2021 177898A1 , relates to a method for predicting resistance to treatment for homologous recombination deficiency (HRD), comprising detection of copy number alteration (CNA) burden in a breast cancer patient. In document WO2021177898A1 , RAD51, MCM10, TROAP, HJURP, EX01, DEPDC1, MTFR2, AUNIP, UBE2T, GINS1, RAD51AP1, TICRR, DSCC1, DDIAS, PRR11, TOP2A, PARPBP, EME1, RFC4 and XRCC2 genes were identified as biomarkers. However, neither document mentions the use of WBP2 as a predictive and prognostic biomarker for tamoxifen resistance in the ER+ group via the Hippo pathway.

[0010] Application numbered US2018363062A1 generally relates to methods and reagents for the diagnosis and prognosis of estrogen receptor 1 ESR1) positive breast cancer and breast cancer patients responsive to endocrine therapy. In the application, evidence was presented that FOXA1, GATA3, ESR1 can be used as biomarkers. The European patent application numbered EP2669682A1 relates to new biomarkers to predict response or resistance to certain treatments and to predict the prognosis of the tumor in a breast cancer patient. However, the documents do not contain any information about the use of WBP2 as a biomarker for the detection of tamoxifen resistance. As a result, due to the abovementioned disadvantages and the insufficiency of the current solutions regarding the subject matter, a development is required to be made in the relevant technical field.

[0011] Objective of the Invention

[0012] The present invention relates to the biomarker for detection of tamoxifen resistance in breast cancer treatment which fulfils the abovementioned requirements, eliminates all disadvantages and brings some additional advantages.

[0013] The primary objective of the invention is to provide a biomarker for the detection of tamoxifen resistance developing in breast cancer treatment and a method for the use of the biomarker. WBP2 is used as a predictive and prognostic biomarker in detecting tamoxifen resistance in ER+ breast cancer patients.

[0014] Objective of the invention is to understand the molecular mechanisms of tamoxifen resistance, which can develop in the ER+ group, which constitutes 70% of breast cancer, in the hormone replacement therapy of tamoxifen, which is routinely used in every patient, and can be understood by recurrence and distant organ metastases in the following years. The object is to predict poor prognosis in patients, to detect a treatment that will not be effective, and to ensure effective treatment of the patient. In this direction, our invention covers the use of WBP2 as a prognostic and predictive biomarker in the resistance to tamoxifen treatment, which is routinely used as preventive treatment in ER+ breast cancer.

[0015] The other objective of the invention is to reduce toxicity due to the use of effective drugs by preventing ineffective treatment in patients with resistance. In addition, by preventing the use of an ineffective drug, drug costs are reduced, thus contributing to the health sector economically. This will contribute to increasing the competitiveness of the health sector in Turkey and its share in the world market.

[0016] In order to fulfil the aforementioned objectives, the invention is a method for predicting the response or resistance to treatment in patients using the drug tamoxifen for the treatment of breast cancer, comprising the following; i. Isolating total RNA separately by sectioning the tumor and normal parts of paraffinized or fresh tissue, ii. Synthesis of cDNA from the total RNA obtained, iii. Determination of WBP2gene expression level by RT-PCR analysis, iv. Calculation of gene expression analysis results of tumor and normal tissues by Ct method, normalization of average Ct values obtained from raw data with reference housekeeping genes, calculation of fold change and fold regulation according to delta delta Ct values obtained, v. Comparison of WBP2 expression level in tumor tissue sample with normal tissue and control gene expression; wherein, the increase in WBP2 expression in the said biological sample compared to the control indicates tamoxifen resistance developed in patients

[0017] In order to fulfil the above-mentioned aims, the invention is the use of the WBP2 gene as a prognostic and predictive biomarker for predicting the response to treatment or the occurrence of resistance in patients using tamoxifen in the treatment of breast cancer.

[0018] 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.

[0019] Figures Clarifying the Invention

[0020] Figure 1 : WBP2 ROC Curve analysis

[0021] Figure 2: WBP2 tumor normal tissue expression (t-test)

[0022] Detailed Description of the Invention

[0023] In this detailed description, the preferred embodiments of the invention are described only for clarifying the subject matter in a manner such that no limiting effect is created.

[0024] The subject of the invention is a method for predicting the response to treatment or the occurrence of resistance in patients using the drug tamoxifen in the treatment of breast cancer ER+ breast cancer subtype, comprises i. Isolating total RNA separately by sectioning the tumor and normal parts of paraffinized or fresh tissue, ii. Synthesis of cDNA from the total RNA obtained, iii. Determination of WBP2 gene expression level by RT-PCR analysis, iv. Calculation of gene expression analysis results of tumor and normal tissues by Ct method, normalization of average Ct values obtained from raw data with reference housekeeping genes (GAPDH or ACTB), calculation of fold change and fold regulation according to delta delta Ct values obtained, v. Comparison of WBP2 expression level in tumor tissue sample with expression of normal tissue and control gene, wherein, the increase in WBP2 expression in the said biological sample compared to the control indicates tamoxifen resistance developed in patients.

[0025] Here, the statistically significant increase in WBP2 expression compared to the expression of GAPDH, which is the internal control, and normal tissue provides information about the development of tamoxifen resistance.

[0026] The housekeeping genes mentioned in the process step (iv) of the method of the invention are GAPDH (glyceraldehyde 3-phosphate dehydrogenase) or ACTB (beta actin).

[0027] The invention relates to the use of the WBP2 gene as a prognostic and predictive biomarker to predict the response to treatment or the formation of resistance in patients using tamoxifen in breast cancer treatment, especially in the ER+ breast cancer subtype.

[0028] As in many biological systems, it is an evolving technology intended to be used routinely for high-throughput gene expression analyzes in cancer. With this method, it is possible to analyze the expression of many genes simultaneously. The most important advantages of gene expression panel platforms, which are used for prognosis determination or prediction in disease diagnosis, are that that they provide information on many genes at the same time, provide fast results, have a short experimental period, are reliable, and are cheaper than other methods after optimization of the system.

[0029] In an application of the method subject to the invention; Total RNA Isolation from paraffinized Tissue (ii), total RNA isolation is obtained using the Trizol method. The quantity and quality of the isolated RNAs were measured using a UV-Vis Spectrophotometer (Beckman Coulter, USA) and the samples were stored at -800, taking i nto account that the A 260 / A 280 ratio recommended in the panel protocol used was in the range of 1 .8-2.0. The materials used in Total RNA Isolation, the process performed and the execution conditions are given in Table 1 .

[0030] Suitable kit protocols for RNA isolation are the protocols included in commercialized isolation kits that enable RNA isolation from paraffinized tissue with various enzymes and buffers. Within the scope of the invention, manual isolation was carried out with trizol. In the mentioned Trizol method; Trizol is added to the sample / tissue and works by lysing cells and cell components while preserving RNA integrity during tissue homogenization. After centrifugation, chloroform is added to separate the solution into aqueous and organic phases, leaving the RNA only in the aqueous phase. Table 1 Materials and conditions used in Total RNA Isolation

[0031] Synthesis of cDNA from total RNA (iii) is preferably performed from the obtained quality total RNA with the ‘cDNA Reverse Transcription’ kit by following the protocol of the manufacturer. The obtained cDNAs are stored at -20cC until the ne xt processing step. cDNA Synthesis protocol and conditions are given in table 2.

[0032] Table 2 cDNA Synthesis protocol and conditions

[0033] Determination of WBP2 gene expression level by RT-PCR analysis (iv); one-step RT-PCR analysis is preferably performed with the TaqMan™ Gene Expression Master mix protocol, which provides high specificity and high sensitivity for the evaluation of gene expression. The master mix comprises the buffer required for the reaction, Taq DNA Polymerase enzyme, dNTP and Mg(CI)2. For RT-PCR analysis, master mix, distilled water, WBP2 primer and cDNA samples are preferably loaded into one well for a 96-well plate, with a final volume of 10 pl. Here, preferably 5 pl of master mix, 3.5 pl of distilled water, and 1 pl of cDNA and 0.5 pl of WBP2 primer are loaded into the well.

[0034] WBP2 mRNA expression levels are analyzed quantitatively on "The Applied Biosystems® StepOneTM Real-Time PCR" device, preferably using primers with TaqMan probes. In the method of the invention, instead of using Taqman probe primers in RT-PCR analysis, the SYBR Green method can also be used. The WBP2 expression level in the sample provides prediction of tamoxifen resistance when compared with the gene control that we designed and determined significance. The content of the RT-PCR Programme used for mRNA Expression Analysis is given in Table 3.

[0035] Table 3 RT-PCR Program Used for mRNA Expression Analysis

[0036] A study was conducted to evaluate the correlation between YAP, TAZ genes in the tamoxifen- associated hippo pathway and the expression of WWOX and WBP2 associated with tamoxifen resistance using the method of our invention. The clinical data of ER+ breast cancer patients in this study are given in Table 4. In the study, the correlation between YAP and TAZ genes in the tamoxifen-related hippo pathway and WWOX and WBP2 expression associated with tamoxifen resistance was evaluated. It was determined that there was an increase in the expressions of WBP2, WWOX, YAP, TAZ compared to normal tissue, and there was a correlation between the expressions of WBP2, WWOX, YAP, TAZ. (*p<0.001 ). A correlation was observed between tamoxifen resistance in patients and WBP2, YAP, WWOX (p<0.05). Additionally, a correlation was observed between YAP expression level and necrosis (p=0.041 ), perineural-invasion (p=0.048), and tumor-size (p=0.035). A significant difference was detected between TAZ expression and necrosis (p=0.031 ), tumor-grade (p=0.017), venous-vascular-invasion (p=0.014). WPB2 ROC Curve analysis is given in Figure 1. Here the area under the ROC curve value is 0,517105; standard error 0.0623; confidence interval 0,413352-0,619786; z statistic (Standardization) was calculated as (0,275).

[0037] An exemplary study was carried out with the method of our invention. The data obtained in the study and the clinical characteristics of the patients, including tamoxifen drug resistance data, were compared using Independent T test (parametric, non-parametric) and Chi-Square tests. Figure 2 shows the WBP2 tumor-normal tissue expression (t-test) graph. SPSS 25.0 and GraphPad Prism 8.1 programs were used for all statistical analyses. mRNA expression levels and fold changes were analyzed using the ‘Data Analysis Center-QIAGEN’ platform. Ct values obtained from the study were normalized with reference genes. CT value , defined as Control Gene Thresholds value, refers to the first cycle in which the increase in fluorescence (ARn) begins. The fluorescence emission of all reacting components of the loaded sample is defined as Rn(+), the fluorescence emission of the negative control and unreacted samples is defined as Rn(-). ARn is the basic indicator used to calculate the Ct value. The data to be obtained as a result of the RT-PCR reaction were evaluated by the 2-ACT method using the web-based ‘RT2Profiler™ PCR Array Data Analysis’ programm. In all analyses, the confidence interval was 95% and (p<0.05) was considered statistically significant.

[0038] Table 4 Clinical Data of ER+ Patients

[0039] Percentage Characteristic features Number

[0040] (%)

[0041] Age

[0042] < 50 41 54%

[0043] > 50 35 46%

[0044] Degree of Tumor 1 / II 59 78%

[0045] III 17 22%

[0046] Lymph node

[0047] NO: node-negative 41 53.9%

[0048] N1 : Metastasis with 1-3 nodes 35 46.1%

[0049] Tumor size (cm)

[0050] < 3 cm 23 30.3%

[0051] >3 cm 53 69.7%

[0052] Ki-67

[0053] <14% 34 %44.7

[0054] 14%-35% 23 30.2%

[0055] >35% 19 25%

[0056] In situ component

[0057] No-DCIS (0) 4 5.3%

[0058] Low-DCIS (<25%) 29 38.2%

[0059] High-DCIS (>25%) 42 55.5%

Claims

CLAIMS1. A method for predicting the response or resistance to treatment in patients using the drug tamoxifen for the treatment of breast cancer, characterized by comprising the following; i. Isolating total RNA separately by sectioning the tumor and normal parts of paraffinized or fresh tissue, ii. Synthesis of cDNA from the total RNA obtained, iii. Determination of WBP2gene expression level by RT-PCR analysis, iv. Calculation of gene expression analysis results of tumor and normal tissues by Ct method, normalization of average Ct values obtained from raw data with reference housekeeping genes, calculation of fold change and fold regulation according to delta delta Ct values obtained, v. Comparison of WBP2 expression level in tumor tissue sample with normal tissue and control gene expression; wherein, the increase in WBP2 expression in the said biological sample compared to the control indicates tamoxifen resistance developed in patients2. The method according to claim 1 , characterized in that; in process step (i) total RNA isolation from paraffinised sample / tissue is performed using the appropriate kit protocol or Trizol method.

3. The method according to claim 1 , characterized in that; in process step (ii) cDNA synthesis from total RNA obtained is performed with a cDNA Reverse Transcription kit.

4. The method according to claim 1 , characterized in that; the master mix, distilled water, WBP2 primer and cDNA samples for RT-PCR analysis mentioned in process step (iii) are preferably loaded in a final volume of 10 pl per well for a 96-well plate.

5. The method according to claim 1 , characterized in that; 5 pl master mix, 3,5 pl distilled water and 1 pl cDNA, 0,5 pl WBP2 primer are loaded in a 96-well plate for RT-PCR analysis as mentioned in process step (iii).

6. The method according to claim 4 or 5, characterized in that; the said master mix comprises Buffer for reaction, Taq DNA Polymerase enzyme, dNTP and Mg(CI)2.

7. The method according to claim 1 , characterized in that; the reference housekeeping genes mentioned in process step (iv) are GAPDH or ACTB.

8. Use of the WBP2 gene as a prognostic and predictive biomarker to predict response or resistance to treatment in patients using the drug tamoxifen for the treatment of breast cancer.

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