The diagnostic value of the p16ink marker for verification of tumors of unknown primary site in women with isolated lesion of inguinal lymph nodes.

Carcinomas with an unknown primary site are a heterogeneous group of metastatic tumors, numbering from 3% to 5% of all malignant carcinoma phenotypes. An isolated lesion of the inguinal lymph nodes is relevantly associated with primary localization of tumors in the pelvis, anal canal, lower abdomen, lower limbs, and more towards in the reproductive system (vulva, vagina and cervix for women, penis for men). But in the absence of clinical signs of cancer after careful research, the histological biopsy of the lymph node with additional immunohistochemical staining with organ-specific markers becomes the diagnostic method of choice. A retrospective analysis of the histological, morphometric and immunohistological characteristics of the biopsy material of 59 patients with isolated lesions of the inguinal lymph nodes (35 women and 24 men) aged from 20 to 87 years (mean 59.26±15.86; median 62 years) was conducted. The distribution of variants of an isolated lesion of the inguinal lymph nodes in women showed 14 of 35 (40.00%) metastatic tumors (the other 21 are lymphoproliferative conditions that require phenotyping, but not finding the primary localization). Of 14 metastatic lesions in women, 6 cases demonstrated a p16 ink (+) phenotype. For comparison, of 24 cases in men, only 6 (25.00%) were of metastatic origin, of which p16 ink (+/-) was partially 1 metastasis of melanoma (16.67%). After a thorough immunohistochemical study with a panel of organo-specific markers among isolated metastatic lesions of the inguinal lymph nodes of tumors in women, half of the revealed localized sites (7 out of 14) were in reproductive organs, namely: 4 p16 ink (+) to squamous cervical cancers, 2 p16 ink (+) to serous ovarian adenocarcinoma, 1 p16 ink (-) to leiomyosarcoma of the uterus. Other localizations did not have a clear location, as they related to metastases of carcinoma from Merkel cells and melanomas, as a result of frequent reduction of the primary tumor lesion in the skin.

P16 ink is a specific marker for identifying HPVdependent cervical neoplasia. Inhibitor of p16 ink cyclokinase is a protein that blocks the cell cycle by terminating the retinoblastic oncogene (Rb) phosphorylation. In the cells infected with oncogenic HPV, overexpression of p16 ink is determined. However, despite this, cells infected with HPV continue to proliferate because Rb is inactivated by the oncoprotein E7 of HPV. Since the immunohistochemical response to Ki-67 and p16 ink highly correlates with HPV-infection, the detection of these markers is used to confirm the diagnosis in cases of suspicion of intraepithelial damage of high oncogenic risk [3]. But in the case of metastatic carcinoma of women, p16 ink acquires a differential diagnostic value. According to the literature, the overexpression of p16 ink is an absolute indicator of cervical carcinoma is found partially in adenoarcinomas of the ovaries, up to 95% of pancreatic carcinoma, as well as in squamous cell carcinoma of the oropharynx with HPV-dependent carcinogenesis [4,6].
The aim is to investigate the complex of morphological, morphometric and immunohistochemical characteristics of cases of isolated metastases in the inguinal lymph nodes of female and male patients separately for the improvement of algorithms for diagnostics of the primary source; to find out the frequency of expression of p16 ink in metastases without primary site in females.

RESULTS AND DISCUSSION
According to the algorithm for the diagnosis of tumors without primary site after the primary immunohistochemical panel all observations were divided into 4 groups, which showed differences in age and gender ( According to Table 2, except lymphoproliferative lesions, immunophenotypes of other tumors were considered as metastases, the origin of which became a diagnostic task. Analysis of metastatic carcinomas of women revealed the positivity of the marker p16 ink in 6 out of 7 (85.71%) observations. Among those, 3 (42.86%) had moderate squamous cell differentiation (CKHMW +), 1 -low squamous cell (Fig. A-B), which, together with positive p16 ink , made the primary localization of these observations in the cervix probable, and 2 more cases had signs of serous moderately differentiated adenocarcinoma of the ovary, which also apart from p16 ink + demonstrated positivity of CA125, WT-1 and CK7 ( Fig. Г-Д). The only case of p16 ink negative metastasis into inguinal lymph nodes in women was the carcinoma from Merkel cells that had specific features of the CK20 + (paranuclear), chromogran +/-, synaptophysin +/-, CD117 +, TTF1- (Fig. E-Ж). Differences in the histological structure of p16 ink (+) and p16 ink (-) of metastatic carcinomas were reflected in the indicators of their morphometric study with the ImageJ program (Table 3).
Immunohistochemical profile of metastatic carcinomas of men with isolated lesion of the inguinal lymph nodes determined the similar distribution of tumor nosologies, but the negativity of the marker p16 ink in all observations. Namely: 1 out of 4 (25%) observations had low squamous cell differentiation (CKHMW +), without positive organ-specific markers, 1 -low-differentiated neuroendocrine cancer, 1 -metastasis of carcinoma from Merkel cells, 1seminal carcinoma (CD117 +, PLAP +, vitmentin) ( Table 4). 19/ Vol. XXIV / 2 T a b l e 3 In the group of melanoma metastases, women dominated (6:1), but it should be noted that, along with the standard immunohistochemical responses on melanoma S100 +, Vimentin +, such lesions of the inguinal lymph nodes also had partial cyto-plasmic expression of p16 ink , but significantly lower, compared to nevuses which proves the pathway for carcinogenesis of loss or translocation of the CDKN2A gene. Morphometric indices of melanoma metastases are presented in Table. 5 N o t e . ꭓ±SD -mean value ± standard deviation, statistically significant difference was considered in p<0.05.

Coefficient of «roundness» (parameter ImageJ) ꭓ±SD
The third subgroup of mesenchymal metastatic tumors included 2 cases: 1 metastasis of leiomyosarcoma in woman and 1 -mesenchymal tumor with undetermined malignant potential in man, both cases had p16 ink (-) negative status. Morphometric indicators are listed in Table 6.