Method of simultaneous verification of uveal melanoma course forecast

FIELD: medicine; ophthalmology.

SUBSTANCE: invention is intended for simultaneous verification of uveal melanoma and forecast of metastasis development. Immunohistochemical analysis of protein S-100 and melanin-A expression within tumour cell is accompanied with simultaneous estimation of S-100-positive cells number within visual field. Uveal melanoma is verified if reaction of S-100 and melanin-A is positive with forecasted high possibility of tumour dissemination in case number of S-100-positive cells number is less than 50.

EFFECT: simplified examination associated with high specificity and sensibility of simultaneously verified uveal melanoma and forecasted metastasis.

1 tbl, 1 dwg, 2 ex

 

The invention relates to ophthalmology, namely oftalmologii, and is intended for simultaneous diagnosis and prognosis of metastasis of uveal melanoma (UM).

According to the modern requirements to the diagnosis of human cancers each tumor must be confirmed not only by light or histological level, but also on immunohistochemical tissues. Nowadays, the histologists have a wide range timedifference markers by which conduct verification of tumors. The term "verification" means the determination of its tissue of origin or histogenesis.

For verification of MIND, as well as melanoma of the skin, use a few standard melanomamelanoma markers. These include: S-100, tyrosinase, melanin-And NIV-45, MITF [Guide immunohistochemical diagnosis of tumors of the human - edited Svitava and Nutricline. - Kazan. - 2000 - Str]. The frequency of detection of these markers in MIND, as well as the number of positive tumor cells for these markers in the tumor, according to the literature, varies considerably. This means that the sensitivity of different melanomamelanoma markers for verification of melanomas in different locations varies significantly. In other words, according to some authors, there may be situations when this is the markers do not recognize melanoma, the reaction of this marker will be a false-negative [Beckenkamp G., Schafer H., von Domarus D. Immunocytochemical parameters in ocular malignant melanoma.// Eur j Cancer Clin Oncol, 1988, vol.24, p.41-45; Bumier M.N. Jr., I.W. McLean, Gamel J.W. Immunohistochemical evaluation of uveal melanocytic tumors.// Cancer, 1991, vol.68, p.809-14; Femando S.S., Johnson, S., Bate J. Immunohistochemical analysis of cutaneous malignant melanoma: comparison of S-100 protein, NIV-45 monoclonal antibody and NKI-C3 monoclonal antibody. // Pathology, 1994, vol.26, p.16-19. Heegard, S., Jensen O.A., Prause J.U. Immunohistochemical diagnosis of malignant melanoma of the conjunctiva and uvea: comparison of the novel antibody against melan-A with S-100 protein and HMB-45.// Melanoma Res, 2000, vol.10, p.350-354.]

Therefore, to clarify the situation and to improve the quality of tissues diagnosis, we tried to find the most reliable and accurate markers for verification of these tumors among other intraocular neoplasm. For this we have taken from the archive of our Institute paraffin blocks of tissue MIND, obtained from 82 patients operated for more than 5 years ago, and conducted its own tissues-study. The purpose of these studies was to evaluate the reliability of the use of any marker in the diagnosis or, in other words, the evaluation of their sensitivity. This could be done by the number of false negative cases. To assess the specificity of the markers we also took other intraocular tumors - leiomyosarcoma and lymphoma. The specificity of tissues markers, generally analysed to identify false positive reactions melanocytic markers opuholyah kamelancien series. Along the way we conducted a retrospective analysis of the results of the tissues studies in correlation with clinical data regarding the survival of patients.

It turned out that almost all markers in the majority of cases (94,9-100%) give a positive reaction with S-100, tyrosinase, melanin-A, HMB-45 and MITF. However, the sensitivity of different melanomamelanoma markers really is different. In fairness, we note that this difference was much lower than what was described by these authors (see table 1). The results of this research showed that using a single S-100 cannot with absolute conviction to assert that S-100-positive tumor is melanoma. This marker gave a positive response and tumor myogenic series - leiomyosarcoma. That is talking about the fact that this marker is not highly specific, such as the melanocyte lineage markers tyrosinase, melanin-A, HMB-45, MITF. It is the latter are highly specific markers and not give false positive results. Therefore, their presence is necessary for verification of melanomas. However, they give false negative results. So, from 82 melanoma in 1 case, the reaction with tyrosinase was negative. In practice, this means that using only one of tyrosinase in tissues diagnosis would lead to diagnostic error. Similar to the context would have been and when using only one of MITF. This marker also gave a false-negative reaction with melanoma. Ideal melanomamelanoma markers were NIV-45 and melanin-A (see table).

As shown by our own research, the use of two markers (S-100 and one of melanomacrophage markers: melanin-a or NIV-45) increases the reliability tissues diagnostics. The advantage of melanin-And before NIV-45 is the fact that melanin-And stain premelanosomes, while NIV-45 - Mature melanosomes, and does not stain tumors of other origins. That is, these two markers are responsible for 2 different stages of maturation of melanosomes. And because different populations of tumor cells, as a rule, are at different stages of maturity and, therefore, at different stages of melanogenesis, the MIND will find melanin-A-positive tumor cells. That, in fact, we have obtained empirically.

Our retrospective analysis also showed that there is a direct close relationship between the expression of S-100 and survival of patients. This fact for some reason was not reflected in the literature, or simply not been noticed by other authors. It turned out that the smaller the tumor is detected S-100-positive cells, the worse the prognosis. Statistical retrospective analysis showed that the critical number of S-100-positive TC is the current forecast 50. That is, in other words, the detection in tumors less than 50 S-100-positive cells is the probability of developing metastasis was high and reached 82.2 per cent. As proof here is a graph of the survival of patients with two diametrically opposite indicators in the tumor. Thus, according to this schedule (see drawing), among 28 patients with less than 50 S-100-positive cells in the tumor (stratum A), metastases developed in 9 cases (32% of cases), and the frequency of their 5-year survival was 68%, respectively. Among 29 patients with ≥50 S-100-positive cells in the tumor (stratum) metastasis occurred in 5 (17.8 per cent of cases), and the survival rate reached 82.2 per cent. The difference in survival rates reliable. This is confirmed by 3 different tests quality control of the survey.

Therefore, in our subsequent work we are using these two markers for verification purposes, uveal melanoma, and quantitative analysis of S-100-positive cells in tumors has served us as a marker for prediction of metastasis.

The task of the invention is to provide such a method that would allow at the same time not only to verify uveal melanoma, but also to predict the likelihood of its spread.

The technical result is to solve simultaneously the time of the two tasks (verification of the tumor and the prediction of the probability of metastasis) using only two markers (S-100 and melanin-a) and one-step immunohistochemical studies, that provides simplified way with a positive economic effect.

The technical result is achieved by using two timedifference markers S-100 and melanin, responsible for the affiliation of the tumor to neoplasma melanocyte lineage.

Our method of prediction is as follows.

1. After enucleation of eyes with uveal melanoma prepare paraffin sections with a thickness of 3-5 μm on a standard methodology, deparaffinized in xylene and rehydration in the battery spirits.

2. Immunohistochemical study performed by standard methods. To open antigenic determinants spend processing sections in citrate buffer [pH 6.0] for 30 minutes at 95°With water bath.

3. Incubation with primary antibodies spend over night at 4°C. To visualize the reaction of the antigen-antibody used streptavidin test system LSAB+kit [DAKO Corp] according to the instructions. As a Chromogen use DAB+[DAKO Corp]. As the primary antibodies used rabbit polyclonal antibodies to S-100 (dilution 1:500, DAKO Corp) and melanin-a (dilution 1:80, DAKO Corp).

4. Then the slices Domracheva with hematoxylin Mayer and conclude in canadian balsam.

5. The reaction is evaluated using a light microscope (magnification ×400) according to the following criteria. otricatelniy considered reaction in the absence of specific staining of tumor cells in the entire area of the tumor tissue. A positive reaction was assessed by staining of tumor cells. When a positive response of tumor cells with two markers, the tumor will verify as uveal melanoma.

6. Quantitative assessment of expression of the marker S-100 by slice is defined as the number immunopositive cells in the field of view of the microscope at magnification × 400. In the case where the number of S-100-positive cells was less than 50, define a high risk of developing metastases. If the number of S-100 positive cells in the tumor is greater or equal to 50, it is considered that the risk of metastases to the patient is low.

Example 1. The patient 54 years old. Diagnosis: the MIND in stage T3NoMo on his right eye. Conducted conservative treatment in the amount of iridocyclectomy. Immunohistochemistry paraffin slice of the tumor at the specified method showed S-100-positive and melanin-A-positive tumor, which was verified as melanoma. Estimate the number of S-100-positive cells on the cut MIND when zooming in × 400: they were 17. The patient is classified as at high risk of developing metastases. Assigned shorter intervals between visits to the doctor. After 8 months of follow-up after the operation the patient revealed metastases in the liver. Conducted immunotherapy to slow the rate of metastasis.

Example 2. The patient was 72 years. Moved the nucleation of the left eye about the MIND in the stage T4NoMo. Immunohistochemically showed S-100-positive and melanin-A-positive tumor, which was verified as uveal melanoma. Estimate the number of S-100-positive cells. On the cut MIND when zooming in × 400 of them turned out to be 87. The patient is classified as low risk of developing metastases. Continuous observation of the patient during the 3 years showed favorable course of the disease and the absence of metastases.

Thus, the proposed method with a high degree of probability allows to simultaneously perform both verification uveal melanoma and to evaluate the prognosis of its flow, which allows the selection of patients at risk of developing metastases and justifies the tactics of management of patients with this pathology. The use of only two markers allows to reduce the duration of the study and the economic costs of their implementation.

The method of simultaneous verification of uveal melanoma and to predict metastasis, including immunohistochemical analysis of the expression of protein S-100 and melanin-And simultaneously counting the number of S-100-positive tumor cells in field of view, and verification of uveal melanoma with positive reaction for S-100 and melanin-And predicting a high probability of metastasis of a tumor in the presence of men who e 50 S-100-positive cells.



 

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