RU2481585C1 - Differential diagnostic technique for leiomyosarcoma uteri and proliferating (cell) leiomyomata - Google Patents

Abstract

FIELD: medicine.

SUBSTANCE: what is presented is a differential diagnosis technique for leiomyosarcomas uteri and proliferating (cell) leiomyomatas involving the analysis of molecular genetic variations of microsatellite DNA samples of tumour tissue. The genetic variations of the D17S1161, D10S1213, D10S1146, D10S218, D10S24, D9S942, D9S251 and D3S1295 microsatellites are to be found in the sample of tumour and surrounding normal tissue or peripheral venous blood by polymerase chain reaction (PCR) using specific primers. If observing some variations represented by heterozygosis loss and/or microsatellite instability in at least one of these microsatellites in the sample of tumour tissue, the malignancy is diagnosed.

EFFECT: effective technique for differential diagnosis between leiomyosarcomas uteri and proliferating leiomyomatas by the presence of heterozygosis and microsatellite instability in the mentioned microsatellites in the sample of tumour tissue.

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Description

The invention relates to the study of biological materials, in particular to methods for the molecular diagnosis of malignant neoplasms, and can be used to study a tissue sample for the differential diagnosis of uterine leiomyosarcomas (LMS) and uterine proliferating (cellular) leiomyomas (PLM).

The problems that arise when it is necessary to differential diagnosis of highly differentiated LMS and PLM are due to the fact that the currently used diagnostic methods (morphological, immunohistochemical and other instrumental studies) are not sufficiently informative. This circumstance requires the development of additional criteria for the diagnosis of uterine LMS.

A number of studies are known on the analysis of genetic changes characteristic of uterine LMS (Frequent loss of heterozygosity for chromosome 10 in uterine leiomyosarcoma in contrast to leiomyoma. // American Journal of Pathology. - 1999. - vol. 154, No. 3. - pp. 945-950; Specific loss of heterozygosity of chromosome 3p loci in soft tissue leiomyosarcoma. // Zhonghua Bing Li Xue Za Zhi. - 2003 .-- 32, pp. 124-127; Leiomyosarcoma and malignant fibrous histiocytoma share similar allelic imbalance pattern at 9p // Virchows Archiv. - 2005. - 446, pp. 251-258; Array comparative genomic hybridization analysis of uterine leiomyosarcoma. // Gynecol Oncol. - 2005. - 99 (3). - pp.545-51). The authors of well-known studies found that allelic deletions (loss of heterozygosity (GH) and microsatellite instability (MN)) of loci 3p, 9p, 10p, 10q, 17q are characteristic of this type of sarcoma. However, in each of the above studies, the authors evaluated the genetic changes in the uterine LMS only at individual loci and did not try to consider the totality of these changes and create a diagnostic system for the differential diagnosis of LMS and PLM on their basis.

The invention is aimed at solving the problem of creating a reliable diagnostic test system for malignant smooth muscle neoplasms of the uterus (LMS).

Using in clinical practice the proposed method allows to achieve several technical (therapeutic) results:

- the ability to determine the nature of the tumor disease by identifying signs of genetic instability and biological aggressiveness of the tumor,

- the possibility of a precise diagnosis of uterine leiomyosarcoma.

These technical (therapeutic) results in the implementation of the invention are achieved due to the fact that, as in the known method, the molecular genetic changes of microsatellite DNA are analyzed in samples of tumor tissue.

A feature of the proposed method is that they determine the presence of genetic changes in the totality of the following microsatellites:

- D17S1161 located at locus 17q23,

- D10S1213 located at locus 10q26.13,

- D10S1146 and D10S218 located at locus 10q22.1,

- D10S24 located at locus 10p13,

- D9S942 located at locus 9p21.3,

- D9S251 located at locus 9p21,

- D3S1295 located at the locus 3p14.3

in a tumor sample and a sample of adjacent normal tissue or peripheral venous blood by polymerase chain reaction (PCR) using specific primers. If there is a change in the form of PG and / or MN in at least one of these microsatellites in a sample of tumor tissue, the malignant nature of the tumor is diagnosed.

The invention consists in the following.

It is known that one of the main causes of malignant neoplasms is genetic factors.

Carcinogenesis markers include intragenomic rearrangements of microsatellite DNA that occur in the process of disruption of repair processes. Microsatellite DNAs are tandemly organized, highly repetitive sequences located in the untranslated region of the genome, ranging in length from 1 to 4 pairs of nucleotides. The tumor genome is characterized by the genetic instability of microsatellites. Signs of instability of the repeats are PG - a change in the total length of microsatellites and MN - the emergence of new microsatellites. PG and MN are sensitive markers of tumor cells.

As a result of the studies, the authors of the proposed method revealed a high frequency of GHG and / or MN of the following microsatellite markers:

- D17S1161 located in the chromosomal region of DNA 17q23,

- D10S1213 located in the chromosomal region of DNA 10q26.13,

- D10S1146 and D10S218 located in the chromosomal region of DNA 10q22.1,

- D10S24 located in the chromosomal region of DNA 10p13,

- D9S942 located in the chromosomal region of DNA 9p21.3,

- D9S251 located in the chromosomal region of DNA 9p21,

- D3S1295 located in the chromosomal region of DNA 3p14.3,

which is typical for patients with a histological diagnosis of uterine LMS. Frequency averaged 40%. In the group of patients diagnosed with uterine PLM, no genetic changes were detected. As a result of a comparative analysis of genetic changes between groups of patients with a diagnosis of uterine LMS and uterine PLM, it was found that it is necessary and sufficient for the differential diagnosis of LMS and uterine PLM to determine the presence of genetic changes in the totality of the following microsatellites:

- D17S1161 located in the chromosomal region of DNA 17q23,

- D10S1213 located in the chromosomal region of DNA 10q26.13,

- D10S1146 and D10S218 located in the chromosomal region of DNA 10q22.1,

- D10S24 located in the chromosomal region of DNA 10p13,

- D9S942 located in the chromosomal region of DNA 9p21.3,

- D9S251 located in the chromosomal region of DNA 9p21,

- D3S1295 located in the chromosomal region of DNA 3p14.3,

in a tumor sample when compared with a sample of adjacent normal tissue.

The specificity and sensitivity of this system is 96% and 95%, respectively.

The result of research by the authors of the claimed invention is a single diagnostic test system that allows for differential diagnosis of LMS of the uterus and uterine PLM.

The method is as follows.

1. Clinical material submitted:

- a sample of tumor tissue. A histological specimen should show> 70% of tumor cells,

- a sample of adjacent normal tissue (without pathomorphological changes), and / or if there is none on histological preparations, a sample of the patient's peripheral venous blood.

As a preservative for peripheral venous blood, a 0.5 M EDTA solution is used. When collecting blood into a test tube, 1.8 ml of blood is added to a 20 μl preservative and mixed thoroughly.

2. Isolation of DNA.

DNA is isolated from surgical material fixed in formalin followed by paraffinization using the commercial DNA Sorb (B / AM) kit (InterLabService, RF) and / or, for extremely small amounts of material, the QIAamp DNA FFPE Tissue kit ”(QIAGEN, Germany), in accordance with the attached instructions. The dewaxing of the material is carried out according to the standard scheme.

3. Microsatellite analysis.

The identification of GHGs and / or MNs is carried out by microsatellite analysis, which is PCR using specific primers, which is carried out according to the following scheme:

- to 0.1 μg of genomic DNA add 0.05 μM of each oligoprimer, 200 μM of each deoxynucleotide triphosphate, 1-2 units. Taq polymerase, 5 μl of a single PCR buffer of the following composition: 50 mM KCl, 10 mM Tris-HCl (pH 8.4), 5 mM MgCl2;

- add 30 μl of liquid paraffin, warm the mixture at 94 ° C for 10 minutes and carry out 35 cycles according to the following program: denaturation at 94 ° C for 40 s, annealing at 56-60 ° C for 1 minute 30 s, elongation at 72 ° C - 30 s;

- As a control for PCR, the peripheral blood leukocyte DNA of “healthy” patients (without oncology) is used.

4. The amplification products are separated in a 6% denaturing polyacrylamide gel (PAGE) and stained with silver nitrate.

GHG is assessed as a weakening or absence of a band of one of the alleles on the electrophoregram relative to normal tissue or peripheral venous blood of the patient. MN is assessed as the appearance of an additional band on the electrophoregram relative to normal patient tissue or peripheral venous blood.

Examples of execution:

1. Patient B. Age: 28 years. Clinical data: In mid-January 2011, the myomatous node was removed. The myomatous node was first discovered in July 2010. She was consulted at the Scientific Center for Surgery, where she was diagnosed with PLM. Recommended review glasses histological preparations in Moscow P.A. Herzen. Normal DNA was obtained from peripheral venous blood.

Pathological conclusion: The formal morphological picture is extremely suspicious for LMS, but the low number of mitoses and the non-specific IHC reaction does not allow to confirm the diagnosis categorically.

Conclusion IHC: Ki67: positive in 5% of tumor cells, GMA (smooth muscle actin) positive in tumor cells. The neoplasm has a smooth muscle nature.

Conclusion of the molecular genetic study: A molecular genetic study was carried out by the method of microsatellite analysis of the areas D17S1161, D10S218, D10S1146, D10S24, D10S1213, D9S942, D9S251, D3S1295. GHGs were detected in the D10S218 microsatellite and MHs in the D3S1295 microsatellite.

Conclusion: taking into account the morphological picture and the data of molecular genetic research - LMS G1.

2. Patient K. Age: 49 years. Clinical data: Advisory material from Central Clinical Hospital No. 2 named after N.A.Semashko. In 1999, with the introduction of IUDs, small uterine fibroids were first detected. Over the past 5-6 years, prolonged menstruation was noted (up to 10 days). In 2005, revealed cystadenoma of the right ovary, polyp. In 2010, Hs + WFD was produced. In March 2011, the operation. Normal DNA was obtained from adjacent normal tissue.

Pathologic conclusion: Histology of the Central Clinical Hospital No. 2 named after N.A.Semashko: Cellular mitotically active leiomyoma.

Pathological Department of Moscow P.A. Herzen: Endometrial proliferation stage, cell uterine PLM with rare figures of mitoses. For the categorical exclusion of LMS, a molecular genetic study is desirable.

Conclusion of the molecular genetic study: A molecular genetic study was carried out by microsatellite analysis of GHGs and MHs characteristic of uterine LMS in microsatellites D17S1161, D10S218, D10S1146, D10S24, D10S1213, D9S942, D9S251, D3S1295.

Conclusion: taking into account the morphological picture and the data of molecular genetic research, cell uterine PLM.

3. Patient R. Age: 47 years. Clinical data: In 2004, uterine leiomyoma was diagnosed. In 2009, a neoplasm was discovered in the lung of the cell-fibrous structure. Advisory material from GKB 67, it is impossible to obtain more complete clinical data. Normal DNA is obtained from adjacent normal tissue.

Pathological conclusion: No. 1 in the preparations, the uterine wall with foci of adenomyosis, the endometrium with secreting glands, sections of the cell atypical (bizarre) leiomyoma. No. 2 in lung tissue is the growth of a tumor of a cell-fibrous structure with small glandular-like structures. To clarify the histogenesis and potential of neoplasm in the lung, immunophenotyping is necessary. To exclude leiomyosarcoma, a molecular genetic study is necessary.

Conclusion IHC: Ki67 positive in 20% of tumor cells, desmin, GMA (smooth muscle actin) positive in tumor cells, S100 positive in tumor cells, CD34 positive in vascular endotheliocytes, CD117, NSE, calretinin, HMBE1 negative.

Conclusion of the molecular genetic study: A molecular genetic study was carried out in the material of the uterine tumor and the tumor material of the cell-fibrous structure of the lung by microsatellite analysis of microsatellites D17S1161, D10S218, D10S1146, D10S24, D10S1213, D9S942, D9S251, D3S1295. GHG was detected in microsatellites D3S1295, D9S942, D10S24, characteristic of uterine LMS. These changes were detected both in a sample of a uterine tumor and in a sample of a lung tumor.

Conclusion: taking into account the data of immunohistochemical and genetic studies, the histological picture, a uterine tumor should be regarded as highly differentiated LMS with low mitotic activity, and the node as metastasis of LMS.

4. Patient S. Age: 32 years. Clinical data: Advisory material from Izhevsk, it is impossible to obtain more complete clinical data. Normal DNA is obtained from peripheral venous blood.

Pathological conclusion: Finished glass is not of satisfactory quality. In preparations with paraffin blocks - leiomyoma with areas of epithelioid cell structure, edema and hyalinosis. Figures of mitosis are not detected. A molecular genetic study is needed.

Conclusion of the molecular genetic study: A molecular genetic study was carried out by the method of microsatellite analysis of microsatellites D17S1161, D10S218, D10S1146, D10S24, D10S1213, D9S942, D9S251, D3S1295. MN was detected in microsatellites D9S251, D3S1295, characteristic of uterine LMS.

Conclusion: the results of molecular genetic studies support the sarcomatous nature of the process.

Figure 1 presents the electrophoregram of the products of microsatellite analysis of microsatellite D10S218 patient B.

T is a pathway with amplification products from tumor DNA. Lack of the second strip - GHG.

N is the path with amplification products from DNA of adjacent normal tissue. The presence of two bands is a heterozygous state.

Figure 2 presents the electrophoregram products of microsatellite analysis of microsatellite D3S1295 patient B.

T is a pathway with amplification products from tumor DNA. The appearance of the third additional band - MN.

N is the path with amplification products from DNA of adjacent normal tissue. The presence of two bands is a heterozygous state.

Claims (1)

A method for differential diagnosis of leiomyosarcoma and proliferating (cellular) uterine leiomyomas, including the analysis of molecular genetic changes in microsatellite DNA in tumor tissue samples, characterized in that the presence of genetic changes in the totality of the following microsatellites is determined:
D17S1161 located at locus 17q23,
D10S1213 located at locus 10q26.13,
D10S1146 and D10S218 located at locus 10q22.1
D10S24 located at locus 10p13,
D9S942 located at locus 9p21.3,
D9S251 located at locus 9p21,
D3S1295 located at locus 3p14.3
in a tumor sample and a sample of adjacent normal tissue or peripheral venous blood by polymerase chain reaction (PCR) using specific primers, if there are changes in the form of loss of heterozygosity and / or microsatellite instability in at least one of these microsatellites in a tumor tissue sample is diagnosed malignant nature of the tumor.

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