RU2279089C1 - Differential diagnosis method for determining squamous cell skin carcinoma and keratoacanthoma cases - Google Patents

Abstract

FIELD: medicine.

SUBSTANCE: method involves studying nuclear Ki-67 or P-63 marker expression localization in epidermis and derma layers. Marker being basally localized, keratoacanthoma is to be diagnosed. Marker being diffusely localized, squamous cell skin carcinoma is to be diagnosed.

EFFECT: high accuracy of diagnosis.

4 dwg, 2 tbl

Description

The invention relates to medicine, namely to pathomorphology and dermato-oncology.

A known method for diagnosing the transformation of keratoacanthomas into squamous cell carcinoma, including the study of venous blood with the addition of antigen, followed by radiometric determination of the level of leukocyte blasttransformation, the results of which determine the value of the stimulation index characterizing the diagnosis (Patent RF №2050003, IPC G 01 N 33/53, publ. 1995).

The disadvantage of this method is that the initial diagnosis of keratoacanthoma is based on clinical data that require long-term follow-up, which is unacceptable in the case of squamous cell carcinoma.

A known method for the differential diagnosis of squamous cell carcinoma of the skin and keratoacanthoma, including pathomorphological studies of biopsy material (Sanchez-Yus E., Simon P., Requena L., Ambrojo P., de-Eusebio E. Solitary keratoacanthma: a self-healing proliferation that frequently becomes malignant. Am. J. Dermatopathol. 2000, Aug; 22 (4). P.305-310).

The disadvantage of this method is the low accuracy of the diagnosis, since the basis of histological changes in keratoacanthoma (CA) is pseudo-carcinomatous epidermal hyperplasia, in which many signs characteristic of the malignant process can be detected: a large number of pathological mitoses, deep invasion of epidermal complexes.

The closest is a method for the differential diagnosis of keratoacanthoma and squamous cell carcinoma, including pathomorphological studies with the addition of immunohistochemical markers, in particular the nuclear marker Ki-67, determination of the proliferation index (Mayumi Matsuta, Sadakatsu Kimura, Gen Kosegawa, Saiichi Kon and Morimasa Matsuta "Immunohistochemical -67 in Epithelial Skin Tumors in Formalin-fixed Paraffin-embedded Tissue Sections Using a New Monoclonal Antibody (MIB-1). "The Journal of Dermatology. Vol.23: 147-152, 1996).

The disadvantage of this method is the low accuracy, since the determination of the proliferation index, i.e. the study of only quantitative characteristics of the marker in most cases is comparable in both diseases.

The task of the invention is to remedy these disadvantages due to an additional, in addition to quantitative, study of marker redistribution in the epidermis layers.

To this end, in a method for differential diagnosis of squamous cell carcinoma of the skin and keratoacanthoma, including an immunohistochemical study using a nuclear marker Ki-67, it was proposed to study the localization of the expression of a nuclear marker Ki-67 or p-63 in the layers of the epidermis and dermis, and if the marker is localized, it is basal to diagnose keratoacanthoma, and with diffuse marker localization, diagnose squamous skin cancer.

The proposed studies allow us to make an accurate diagnosis since the expression of the gene protein product p-63 and the protein of the nuclei of the proliferating cells Ki-67 is detected in the cells of the outer basal layer of the warty-acanthotic growths of the epidermis in the keratoacanthoma. In squamous cell carcinoma, the expression zone of this protein expands, nuclei specifically stained for Ki-67 and p-63 are detected both on the periphery and in the central departments of epidermal tumor cords and complexes.

Figure 1 shows the expression in the nuclei of epidermocytes in the keratoacanthoma nuclear marker p63; figure 2 - the same marker Ki-67, localized on the periphery of tumor growths in the basal sections; figure 3 shows the expression in the nuclei of epidermocytes in the squamous cell carcinoma of the nuclear marker p63, which are randomly scattered over acanthotic outgrowths and disembodied complexes of the atypical epithelium, i.e. expression is detected diffusely and the expression zone expands beyond the basal layer; figure 4 is the same marker Ki-67.

The method is as follows.

The study was carried out in accordance with the methodology of Taylor C.R., Cote R. and Sternberger L.A.

For immunohistochemical (IHC) studies, the biopsy material was fixed in a 10% formalin solution buffered according to Lilly (pH 7.2), then carried out on a battery of alcohols and xylenes and poured into paraffin according to a standard method. Used special paraffin with a melting point of 54 ° C.

Prepared serial paraffin sections with a thickness of 3-5 μm, which were applied to glass with polylysine coating. Sections were dewaxed according to the standard scheme.

Before incubation with primary antibodies, the sections were processed in the microwave at a power of 750 W, in citrate buffer (pH 6.0) - 2 times for 5 minutes.

Then the slices were cooled at room temperature for at least 15-20 minutes.

Then, the corresponding primary antibodies were applied to the sections, incubated with primary antibodies: 30 minutes at room temperature, then left overnight (14-18 hours) at 4 ° C.

Antibodies Clone Firm Breeding p63 4A4 Santa crus 1: 500 Ki-67 B56 BD Biosciences 1:10

Sections were washed thoroughly in phosphate buffer at pH 7.4-7.6.

Then, EnVision complex (anti-mouse and anti-rabbit, DAKO company) was applied to the sections and incubated for 30-40 minutes at room temperature.

Sections were washed thoroughly in phosphate buffer at pH 7.4-7.6. Next, a staining solution visualizing the reaction was applied to the sections. As a visualization system, a solution of diaminobenzidine (DAV) - DAB + from DAKO was used, then the sections were washed in distilled water and stained with Mayer hematoxylin.

Further conducted microscopic studies. Depending on the nosology, different localization of expression (staining of nuclei in brown color under the influence of a marker) of Ki-67 or p-63 markers is observed in the biopsy sample.

According to our data, the expression in the nuclei of epidermocytes of the CA of the gene protein product p-63 is detected along the periphery of tumor growths in the basal sections. In RCC, nuclei expressing p-63 are randomly scattered over acanthotic outgrowths and atypical complexes of the atypical epithelium, i.e. the expression zone of this marker expands.

The protein of the nuclei of proliferating cells Ki-67 is expressed in the cells of the outer (basal) layer of the warty acanthotic growths of the epidermis in the CA. With RCC, the expression zone of this protein expands; nuclei specifically stained for Ki-67 are detected both on the periphery and in the central departments of epidermal tumor cords and complexes.

When detecting the protein of the nuclei of proliferating cells in a reaction with AT to another protein, PCNA, the same topography of the location of Ki-67-expressing nuclei was revealed, but their number increased, which is explained by PCNA expression in all phases of the cell cycle, unlike Ki-67, which is expressed only in the late G1, S, G2, and M phases.

No difference in the expression of anti-apoptotic protein products of p-53 and CVC between CA and RCC was noted.

Example 1

Patient D, 65 years old, was admitted to the Department of Dermatovenereology and Dermato-Oncology with a presumptive diagnosis of nodular shoulder basal cell carcinoma. From the anamnesis: the formation appeared 6-7 months ago, on apparently unchanged skin, until recently, it did not subjectively bother, the last 2-3 weeks began to notice pain on palpation. On examination: the formation of a hemispherical shape, pink with ulceration in the center by closed horny masses, the size of the formation is 4 cm in diameter, the height of the formation is 3.5 cm. During a pathomorphological examination, the structure resembles keratoacanthoma, but the histological changes are more consistent in the number of pathological mitoses and the depth of invasion not high, but low-grade skin cancer. The patient was referred to the regional oncology clinic for surgical removal of the tumor with the capture of healthy skin on the periphery of the formation of 1.5-2 cm, and a skin graft transplant from the donor area. An immunohistochemical study using the p-63 marker and additional control with the Ki-67 marker revealed a basal distribution of both markers, the patient was prescribed Reaferon therapy at a dose of 1 million IU No. 10 hedgehog. as in the treatment of atypical keratoacanthas, and by the 4th injection of the drug, a tendency to regress appeared. After 3 weeks from the onset of the disease, the tumor resolved. At the site of formation, a small focus of depigmentation.

Example 2

Patient G., 57 years old, was ill for about 2 years, when he first noticed a nodule with a pinhead in the area of the left slope of the nose, which slowly increased, and a year and a half after the onset of the disease reached a diameter of 0.6 cm. Then, over the past three months, the patient noted a rapid growth of education, which at the time of treatment reached a diameter of 1.8 cm. On examination: on the skin of the left slope of the nose, a node 1.8 cm in diameter with an expression in the center. The peripheral rim of the tumor consists of pale pink nodules, and the central part is represented by the tumor shaft with ulceration in the center.

The rapid growth of the formation was regarded as a malignancy of the process, but after the proposed immunohistochemical study with the marker p-63, diffuse marker localization was not detected. He was diagnosed with a keratoacanthoma that developed on basal cell carcinoma and received appropriate treatment. Long-term results confirmed the diagnosis.

Example 3

Patient A., 73 years old, was admitted with a presumptive diagnosis of keratoacanthoma of the lower lip. According to the above method, a study was conducted with a nuclear marker p-63.

The results of an immunohistochemical study with a nuclear marker p-63, which is expressed both on the periphery and in the central departments of epidermal tumor complexes, i.e. showed diffuse marker localization. He was diagnosed with squamous cell cancer and treated accordingly.

According to the proposed method, studies were conducted in 24 patients, squamous cell cancer was diagnosed in 13 patients, and keratoacanthoma in 11 patients. Conducted treatment. Long-term results showed the correctness of the diagnosis in 100% of cases.

The proposed method eliminates the possibility of a multi-stage surgical intervention in case of incorrect diagnosis of squamous cell carcinoma with a keratoacanthoma, which is actually present. In cases of keratoacanthomas, an accurate diagnosis allows timely implementation of adequate therapy.

Claims (1)

A method for differential diagnosis of squamous cell carcinoma of the skin and keratoacanthoma, including an immunohistochemical study using a nuclear marker Ki-67, characterized in that the localization of the expression of a nuclear marker Ki-67 or P-63 in the layers of the epidermis and dermis is examined and keratoactantum is fundamentally diagnosed when the marker is localized, and with diffuse marker localization, squamous cell skin cancer is diagnosed.

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