RU2547583C2 - Diagnostic technique for breast cancer - Google Patents

Abstract

FIELD: medicine.

SUBSTANCE: invention refers to medicine, particularly to oncology, and aims at sub-typing breast cancer. RIL (PDLIM4) expression is measured in a patient's tumour tissue sample by the sequencing technology of the following NGS generation or by northern hybridisation methods and real-time PCR. If the RIL (PDLIM4) expression tends to decrease twice as much as its level in the normal tissues, a sub-type of a malignant breast new growth is diagnosed.

EFFECT: invention provides effective diagnostic technique for breast cancer subtype - tumours characterised by incomplete epithelial-mesenchymal transition and relatively minimally invasive.

2 dwg, 1 ex

Description

The invention relates to medicine, in particular to the field of oncology, and relates to a new tumor marker.

Breast cancer ranks first among all malignant diseases in women. According to modern data, the incidence of breast cancer in Russia over the past 15 years has more than doubled. It is known that the incidence of women living in large cities and industrial areas is higher than that of rural women. Breast cancer also occurs in men. Many do not even suspect the possibility of developing such a disease in them, which leads to untimely access to doctors, late diagnosis and the start of treatment, and, as a result, low therapeutic results.

Therapy for the disease is currently based on a combination of early diagnosis and one or more treatments, such as surgery, radiation therapy, chemotherapy and hormone therapy. The course of treatment in a particular case of breast cancer, as a rule, is chosen on the basis of a number of prognostic parameters, including the analysis of specific tumor markers.

There are a lot of diagnostic tumor markers. Most of them are successfully used in diagnostics. The main application of tests to determine tumor markers is monitoring the course of the disease and the effectiveness of surgical treatment and / or radio, chemo and hormone therapy. It was shown that the dynamics of the level of the tumor marker reflects a change in the patient's condition. In addition, sometimes it is possible to differentiate benign and malignant diseases by the rate of increase of the marker level, which is extremely low with benign diseases. A number of tests can also be used as diagnostic in screening examinations.

Thus, the determination of the level of tumor markers seems appropriate in the following cases:

1. Monitoring the course of the disease.

2. Monitoring therapy. The ability to detect relapse and / or metastases in 6 months. and more before the onset of clinical manifestations, which is important for determining the tactics of further treatment.

3. Identification of residual tumors. The absence or slight decrease in the concentration of the tumor marker after surgery indicates incomplete removal of the tumor or the presence of multiple tumors.

4. Prediction of the development of certain tumors.

5. Early detection of cancer in high-risk patients. Oncomarkers can be either produced by the tumor itself or produced by the body in response to the development of the tumor. The biomarkers used to predict and diagnose a tumor may have a different nature and origin. In modern medicine, the following categories are distinguished: genetic, epigenetic, protein biomarkers, glycans (all mono- and polysaccharides) and visual markers.

Currently, the field of application of genetic and protein biomarkers as the most reliable and simple to measure is actively developing.

The most common diagnostic test systems are based on the analysis of biomarkers in physiological fluids, blood and urine, however, biopsy material is also good material for obtaining eloquent research results.

In science and medicine, tumor markers are mainly used for three main purposes:

1. Diagnostic - for the diagnosis of cancer in the early stages.

2. Prognostic - to predict the degree of aggressiveness of the tumor, its characteristics and assess the risks and chances of recovery.

3. Predictive - to predict the behavior of the tumor in response to different types of treatment. It is necessary both when choosing the type of therapy, and when selecting specific medications.

Risk assessment.

Evaluation of tumor markers associated with genetic mutations and epigenetic rearrangements makes sense with an individual approach to determine patients' predisposition to the development of a particular type of cancer. It is known that mutations in certain genes can contribute to the development of cancer, and when they are detected in patients, it is concluded that there is an increased predisposition to the corresponding type of cancer.

So, for example, mutations in the KRAS, p53, EGFR, erB2 genes are characteristic of cancer of the intestine, stomach, liver and pancreas; mutations in the BRCA1 and BRCA2 genes - for breast and ovarian cancer; abnormal methylation in the p16, CDKN2B and pl4ARF cancer suppressor genes for brain cancer, hypermethylation of MYOD1, CDH1 and CDH13 for cervical cancer, and p16, p14 and RB1 for oral cancer.

Diagnostics.

The use of tumor markers is very valuable in the direct diagnosis, in particular, determining whether a tumor belongs to primary or metastases by comparing chromosomal abnormalities found in primary and repeated tumors. If the disorders coincide, then the secondary tumor is the primary metastasis, and if they differ, then the neoplasm that has arisen independently.

Prediction and prediction of therapy. Oncomarkers are also used in predicting the disease after the cancer has been diagnosed. Based on the presence or increased expression of certain biomarkers, for example, one can determine the degree of cancer aggressiveness and its potential response to therapy. An example of such prognostic biomarkers is the metallopeptidase inhibitor 1 (TIMP1), a marker associated with more aggressive types of myeloma; and an increased level of estrogen (ER) or progesterone (PR) receptors is characteristic of patients with better survival in breast cancer; overexpression of the HER2 gene in breast cancer suggests a good potential response to trastuzumab therapy; and, for example, a tumor with a mutation in exon 11 of the c-KIT proto-oncogen is highly likely to be susceptible to imatinib therapy.

Pharmacodynamics and pharmacokinetics.

Oncomarkers are also used to select the most optimal treatment regimen for each individual patient. Due to the difference in the effectiveness of metabolic processes, different people can respond differently to certain drugs, which, accumulating in the body, can pose a great danger to some. All these indicators are necessarily taken into account when calculating the dosage of drugs during therapy. For example, patients with a mutation in the TPMT gene are not able to metabolize large amounts of the drug mercaptopurine used in the treatment of leukemia, therefore, treatment with this drug is prescribed to them taking into account the characteristics of the body.

Monitoring the effectiveness of therapy. Another use of biomarkers has been found in monitoring the effectiveness of anti-cancer therapy over time. For example, SlOO-beta protein with a positive response to therapy is significantly reduced in number in a number of melanomas and its regular quantitative assessment allows us to evaluate the effectiveness of the treatment used.

Relapses.

The indicators of some tumor markers also make it possible to predict the likelihood of relapse. Search for drugs for cancer therapy.

Tumor markers are very useful not only in the diagnosis of diseases, but also for the search for new drugs for chemotherapy, acting as targets in screening libraries of chemical compounds.

The prior art patent RU 2473555, publ. 2013-01-27, which discloses a method for evaluating a clinical prognosis for an individual suffering from breast cancer based on measuring gene expression in a breast cancer sample in the following descriptor groups: (1) ERBB2, STAED3, GRB7, THRAP4, PPARBP; (2) ESR1, GATA3, XBP1, TFF3, ACADSB; (3) KRT5, KRT17, TRIM29, GABRP; (4) KRT18, KRT8, PPP2CA, KRT8L2; (5) PLAU, COL5A2, FAP, THY1; (6) STAT1, UBE2L6, TAP1, LAP3, and (7) SEACAM5, SEACAM6, SEACAM7. Moreover, overexpression of most genes in the descriptor group compared to the normal breast tissue sample indicates that the tissue sample is positive for this group, and the absence of overexpression of most genes in the descriptor group compared to the normal breast tissue sample indicates that a tissue sample is negative for this group.

Known application EA 200971079 A1, concerning certain genetic variants on Chr5pl2 and Chrl0q26 as variants of predisposition to breast cancer. Methods of controlling the course of the disease are described, including diagnosing an increased and / or decreased predisposition to breast cancer, methods for predicting a response to treatment, and methods for determining the prognosis using such options.

US 20110251082 relates to the use of a 2-dimensional differential gel (2D-PHLP) and a mass spectral method for identifying biomarkers of breast cancer in transformed breast cells. Numerous putative markers for various stages of breast cancer have been identified. The expression of various proteins, including PdLIM1, was evaluated. However, a conclusion was drawn about the potential importance as a marker of parvabumin and the opposite effect of PdLIM1. No PdLIM4 information provided.

Application WO 2009124251 relates to a method for diagnosing the metastatic potential of tumors, including breast cancer cells. This approach proposes the measurement in patient cells of levels of five or more markers selected from the group of TGF typing genes [Beta]:

Although the PDLIM4 gene is mentioned among the genes used, its use as the only marker of breast cancer outside of combinations with other genes has not been disclosed.

At the moment, there are no tests for tumor markers with 100% sensitivity and specificity. This circumstance seriously limits their use in the primary diagnosis of cancer, therefore, to achieve a reliable result, it is necessary to combine the indicators of several markers. The gradual accumulation of knowledge regarding the mechanisms of tissue malignancy and disorders that potentially underlie tumor formation leads to the fact that within the existing, known types of tumors, subgroups are identified that have different typical profiles of genetic disorders and for which it is possible to develop highly specific therapeutic approaches. Thus, the development of new tumor biomarkers contributes to a more accurate and detailed classification and, as a result, makes it possible to apply more effective therapeutic approaches.

The objective of the invention is the development of a method for the diagnosis of breast cancer and its use to identify a single subtype of malignant neoplasms.

The problem is solved by a method for the diagnosis of breast cancer using a biomarker based on the epigenetic suppression of the PDLIM4 / RIL gene, which can be used to diagnose and treat breast cancer, assess the aggressiveness of the disease, and select a method for treating the tumor.

A method for diagnosing a breast cancer subtype is proposed, based on determining the level of PDLIM4 / RIL expression in tumor tissue using next-generation sequencing (NGS) technology or real-time Northern hybridization and PCR. The decrease in PDLIM4 / RIL expression in the patient’s sample by more than two times compared with its level in normal tissues indicates a malignant process (breast cancer by RIL-negative type) and an unfavorable prognosis.

The PDLIM4 / RIL gene was first mapped to the long arm region of chromosome 5 (5q31), often deleted in a number of human cancers. In addition to deletions, the PDLIM4 / RIL gene often undergoes epigenetic suppression, as a result of which its expression is suppressed. In such cases, a lesser degree of morphological transformation and a lower frequency of tyrosine kinase activation are noted.

The technical result achieved by using the invention is the rapid diagnosis of a subtype of breast cancer by measuring the level of transcripts of the RIL gene will contribute to an adequate choice of therapy. The proposed approach will make it possible to make a correct diagnosis in the early preclinical and clinical stages of development, in cases of a low-symptom, atypical course of the disease, to clarify the nature of breast tumors, and to determine treatment tactics in a shorter time. Due to the significant diversity of the course of the disease in patients, the medicine of the future will focus more on personalized approaches to therapy. In this sense, the invention is relevant because it is aimed at identifying a common subtype of breast cancer that requires a personalized approach to treatment. Also, the determination of the detailed mechanism of malignancy of tumors of this subtype opens up prospects for the creation of highly selective effective chemotherapeutic drugs aimed at key components of impaired signaling pathways.

We first noticed the correlation of PDLIM4 / RIL gene suppression with the morphology and biochemical properties of tumor cells and established the role of tumor suppressor in the malignant transformation of breast cells. The results of our studies indicate that within the group of mammary adenocarcinomas characterized by a triple negative phenotype, a large subgroup of tumors can be distinguished, in which the epigenetic suppression of PDLIM4 / RIL is the basis of tumor transformation. The suppression of PDLIM4 / RIL leads to an increase in the functional activity of PTP-BL phosphatase, which, in turn, dephosphorylates and converts the receptor-unassociated tyrosine kinase c-Src, which is responsible for the activation of proliferation, suppression of pro-apoptotic signals, an increase in cellular mobility. The possibility of carrying out the invention can be demonstrated by the following examples.

Example 1. Determining the role of PDLIM4 / RIL in malignant transformation.

The analysis of its mRNA expression in samples of malignant neoplasms from different organs compared with surrounding normal tissue was performed by Northern hybridization with Cancer Profiling Array I (BD Clontech) (241 pairs of tumor / norm samples from 8 human tissues). It was found that the expression level of PDLIM4 / RIL was suppressed 2 or more times in 43% (23 of 53) samples of malignant breast tumors (Fig. 1). To independently confirm these data, the level of PDLIM4 / RIL was analyzed in a panel of 8 human breast carcinoma cell lines. In four cases out of 8, the expression of PDLIM4 / RIL was lower than the level of detection; in the remaining 4 cell lines, the level of PDLIM4 / RIL was comparable to the control (Fig. 2a, b).

A comparison of PDLIM4 / RIL status with cell morphology showed that a high level of expression of PDLIM4 / RIL correlates with increased aggressiveness of breast cancer cultures MDA-MB-231, -435S, -436, and BT-474 (Fig. 2c). The cells of these lines are characterized by a star-shaped or spindle-shaped form, a weak degree of adhesion and flattening, a high growth rate and a visible absence of intercellular contacts. Slightly aggressive cell lines MCF-7, BT-20, T-47D and MDA-MB-468 with undetectable PDLIM4 / RIL levels, on the contrary, retain epithelial morphology and intercellular contacts. Analysis of the expression of the epithelial marker E-cadherin confirmed the effects observed at the level of cell morphology. Cells of the MDA-MB-231, -435S, -436, and BT-474 lines do not express E-cadherin (by immunoblotting and RT-PCR) and do not have intercellular contacts, i.e. underwent epithelial-mesenchymal transformation, which is a necessary condition for a high degree of aggressiveness of epithelial tumors. The MCF-7, BT-20, T-47D, and MDA-MB-468 lines, on the contrary, retained high levels of E-cadherin, intercellular contacts, and the epithelial phenotype (Fig. 2).

However, neither the ectopic expression of PDLIM4 / RIL in slightly aggressive breast cancer cell cultures, nor the suppression of its expression in highly aggressive lines using short interfering RNAs led to phenotype reversal or a change in the status of E-cadherin. It follows that the inverse correlation between the expression of these two proteins is a consequence of their coregulation by a single signaling cascade.

It is known that expression of E-cadherin is regulated at the transcription level by three repressor proteins Snail, Slug and Twist, which in turn are components of TGFβ / R-, NFkB-, ERK- or β-catenin-dependent signaling pathways. A correlation was found between the expression of PDLIM4 / RIL and the Slug transcriptional repressor, which inversely correlated with the level of E-cadherin mRNA (Fig. 2a), and these events were independent of NFkB, ERK, and β-catenin cascades. An immunoblot analysis in total protein extracts of mildly aggressive breast cancer cell lines revealed a processed form of TGFβ1, which under normal conditions performs the function of a signal for differentiation. The binding of transforming factor β to its receptor leads to phosphorylation, heterodimerization, and transport of Smad2 protein, the transcription factor responsible for the activation of target genes of the TGFβ signaling cascade, into the nucleus. Identification of the phosphorylated form of Smad2 confirmed the activation of TGFβ in these cells (Fig. 2b). Moreover, the total amounts of Smad2 in all the studied cells were comparable.

The expression of PDLIM4 / RIL is suppressed in malignant neoplasms of the breast and tumor cell lines of the same origin, which is accompanied by activation of the signaling cascade initiated by TGFβ1 and the expression of the epithelial marker E-cadherin is preserved. Overexpression of the closely related Mystique protein is known to increase cell dependence on the substrate. PDLIM4 / RIL shows a similar effect, and its shutdown promotes cell independence from attachment and more efficient metastasis. When removing the problem of adhesion dependence using any other mechanism, cells with increased expression of PDLIM4 / RIL may be selected, which will enhance their migration and proliferation, increasing the aggressiveness of the tumor.

Thus, we have shown that a low level of expression of the PDLIM4 / RIL gene can serve as an effective diagnostic marker for detecting a single subtype of breast malignancies - tumors characterized by incomplete epithelial-mesenchymal transition and relatively minimally invasive.

Fig. 1. Expression of PDLIM4 / RIL mRNA in human tumors of various origins compared with normal tissues. Northern analysis of total RNA samples presented on a Cancer Profiling Array I micropanel (BD Clontech) using a PDLIM4 / RIL-specific probe. All values are normalized by GAPDH mRNA. a is a view of the autoradiogram of a micropanel (O is a sample of total RNA from a tumor; H is from surrounding morphologically normal tissue from the same patient); b - the proportion of “norm: tumor” pairs in which the expression of PDLIM4 / RIL mRNA in the tumor is suppressed by 2 or more times (N / O> 2), not changed (0.5 <N / O <2) or increased by 2 or more times (N / O <0.5). The dotted line marks the region of the autoradiogram of the blot (a) and the bar graph (b), corresponding to the pairs “norm: tumor” from the mammary gland.

Fig. 2. Expression of PDLIM4 / PIL and features of the morphology and signaling cascades of human breast cancer cell lines. a - expression of mRNA PDLIM4 / RIL, E-cadherin and Slug according to RT-PCR; b - Western blot analysis of the expression of proteins PDLIM4 / RIL, E-cadherin, transforming growth factor beta (TGFβ) and activated Smad2; c - cell morphology of the used cultures of breast tumors and primary epithelium. Light microscopy. The black frame denotes cell cultures with high expression of PDLIM4 / RIL, and the gray dotted line indicates the lines where PDLIM4 / RIL expression is undetectable.

Claims (1)

A method for diagnosing a breast cancer subtype, characterized in that the level of RIL expression (PDLIM4) in a patient’s tumor tissue sample is determined using next-generation NGS sequencing technology or real-time Northern hybridization and PCR methods with a decrease in RIL expression (PDLIM4) of more than twice in comparison with its level in normal tissues, a subtype of breast cancers is diagnosed - tumors characterized by incomplete epithelial-mesenchymal transition and relatively few asive.

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