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Background: Vulvar lichen sclerosus (VLS) is a chronic and recurrent dermatosis of an inflammatory nature with severe focal atrophy of the skin. Connective tissue changes are polymorphic and are still not taken into account in histological diagnostics due to the difficulty of interpreting routine histological methods. In this work, we use multiphoton microscopy (MPM) as a new imaging technique that provides detailed information about the organization of collagen fibers in the dermis based on a non-linear second harmonic generation (SHG) process., Objective: To determine the degree of connective tissue damage in lichen sclerosus using standard histological techniques and to reveal the diagnostic capabilities of multiphoton microscopy., Material and Methods: We studied 42 biopsies with a histopathological diagnosis of VLS and 10 biopsies of normal vulvar skin. Histological, histochemical and immunohistochemical evaluation was used in comparison with MPM data. Quantitative analysis included the determination of the thickness, length of collagen fibers and the average intensity of the SHG signal., Results: A comprehensive study of the skin showed 4 groups of changes that can be regarded as the degree of the dermis damage: initial, mild, moderate, severe. The affected area at the initial and mild degree has subtle changes, however, it is reliably identified by quantitative analysis of the SHG signal. So, the initial degree is characterized by thin (1.3-1.8 µm) long (56-69 µm) collagen fibers, with a moderate degree, the fibers are thickened (3.4-4.3 µm) and fragmented (22-37 µm). The affected area in moderate and severe cases undergoes homogenization, which is associated with the deposition of extremely thin (0.6-0.9 μm) short (16-28 μm) collagen fibers and the expression of type V collagen., Conclusion: Multiphoton microscopy in the second harmonic generation mode is a reliable method for identifying collagen fibers in tissues. The study made it possible to identify 4 degrees of the dermis damage in vulvar lichen sclerosus.

Mutations in the gene DHH are an extremely rare cause of disorders of sex development 46,XY (DSD,46XY). The article describes the clinical cases of two unrelated patients with gonadal dysgenesis 46,XY with female phenotype. By using a next generation sequencing method, in both cases the same biallelic variant substitution c. 419T>G in the DHH gene was revealed. Taking into account the data on the role of DHH in the formation of the nervous system, the diagnosis of minifascicular polyneuropathy at the preclinical stage was confirmed in both cases. These cases demonstrate the value of using NGS, which allows simultaneous analysis of a wide range of candidate genes in DSD and the diagnosis of comorbidities before the development of the clinical picture. These are the first descriptions of patients with mutations in the DHH gene in the Russian population.

Inflammatory myofibroblastic tumors of the uterus (uIMT) are rare and difficult to diagnose neoplasms, since the morphological characteristics of this tumor are not specific and are found in other pathological changes. In addition, until recently, specific uIMT markers have not been identified and their diagnostic standards not defined. However, in recent years, there have been more and more studies aimed to identify characteristic morphological, immunohistochemical, and molecular genetic features for the differential diagnosis of uIMT. Recent papers studying uIMT indicate anaplastic lymphoma kinase (ALK) as a potentially reliable marker of uIMT. This communication describes a clinical case of uIMT in a 40-year-old woman who has been preoperatively diagnosed with a large subserous interstitial myomatous nodule. The final diagnosis was made, by analysing a combination of morphological and immunohistochemical signs. This clinical case with a literature review is indicated to consider ALK as a key criterion in the diagnosis of uIMT, as well as the relationship between subsequent treatment and the presence of ALK in the studied tissues.

Objective: To investigate the expression of proliferation and apoptotic factors (Ki-67, Bcl-2), inflammatory factors (NF-kβ p65, COX-2), adhesion factors (β-catenin), estrogen (ER-α) and progesterone receptors (PR-α) in ovarian endometrioma (OE) in patients with recurrent OE by an immunohistochemical assay., Subject and Methods: This investigation enrolled 48 reproductive-aged patients with OE. According to the course of the disease during a follow-up period of 1.5 years after surgical treatment, the biomaterial obtained from the examined patients was divided into two groups: 1) an OE capsule from 19 patients with recurrent OE (a study group); 2) an OE capsule from 28 patients without recurrent OE (a comparison group). This investigation used histological and immunohistochemical examinations. The histological analysis of the OE capsule was performed following a standard procedure. Their immunohistochemical analysis was carried out using the Tissue-Tek Quick-Ray kit that allows the preparation of paraffin blocks with a large number of tissue samples (tissue microarrays). Antibodies to Ki-67 (clone 30-9, VENTANA), Bcl-2 (clone 124, VENTANA), NF-kβ p65 (clone p65, 'Spring Bioscience Corp.'), COX-2 (clone CX-294, Agilent), β-catenin (clone 14, VENTANA), ER-α (clone SP1, VENTANA), and PR-α (clone 1E2, VENTANA) were also employed in the investigation. The specimens were prepared according to a standard protocol using a Ventana Ultra immunohistostainer. Positive and negative controls were used to correctly carry out immunohistochemical tests. Statistical analysis was performed using the applied statistical analysis programs Statistica 10.0 and Microsoft Excel., Results: The patients with recurrent OE had a significantly decreased expression of Ki-67 (2.86% vs. 9.69%; р=0.044) in the epithelial component of the OE capsule; a significantly lower expression of NF-kβ p65 (2.54 vs. 3.5; р=0.0082) and СОХ-2 (0.231 vs. 1.381; р=0.0025) in the stromal component of the OE capsule, a significantly increased expression of β-catenin (2.5 vs. 1.59; р=0.017) in the stromal component of the OE capsule; a significantly increased expression of PR-α (188.46 vs. 71.15; р=0.028) in the epithelial component of an OE capsule. The expression of ER-α (stromal component, 266 vs. 256.84; p=0.48; epithelial component, 251.54 vs. 233.85, p=0.82) and Bcl-2 (stromal component, 0.33 vs. 0.25; p=0.85; epithelial component, 0.944 vs. 0.625; p=0.31) in the OE capsule is not statistically significantly different between the study patient groups., Conclusion: The immunohistochemical difference in the expression of a number of the markers under study can serve as the basis for a further investigation of these markers as predictors of recurrent OE after surgical treatment. Further investigations of these factors will also be able to examine the molecular mechanisms underlying the pathogenesis of recurrent OE, which will make it possible to affect these mechanisms in order to eliminate the fundamental causes of a recurrence of this disease.

Aim: to investigate the frequency of the types of fallopian tubal secretory cell expansion (SCE) in diseases of the reproductive organs and to determine the immunophenotype and biological role of the cells in the early stages of the pathogenesis of high-grade ovarian serous carcinomas (HGOSC)., Subjects and Methods: The investigation enrolled 287 patients with extraovarian diseases and ovarian serous tumors varying in grade, whose fallopian tubes were morphologically and immunohistochemically examined using p53, Ki-67, PAX2, Bcl-2, beta-catenin, and ALDH1 markers. The material was statistically processed applying the Mann-Whitney test and χ2 test., Results: The rate of secretory cell proliferation (SCP) (more than 10 consecutive secretory cells) and that of secretory cell overgrowth (SCO) (more than 30 consecutive secretory cells) increase with age in all investigated reproductive system diseases. The rate of SCP in the corpus fimbriatum of the patients with HGOSC was 5.9 times higher than that in those with extraovarian disease (p<0.01); when comparing the same patient groups, that of SCO was 3.4 times higher (p<0.05). The immunohistochemical characteristics of the investigated lesions (in scores) were as follows: PAX2 was expressed in the intact epithelium (2.8), in SCP (1.3), in SCO (1.2), in serous tubal intraepithelial carcinoma (STIC) (1.0), and in HGOSC (0.9); Bcl-2 was in the intact epithelium (2.2), in SCP (2.1), STIC (0.9), and in HGOSC (0.6), β-catenin was in the intact epithelium (0.5), in SCP (2.85), in SCO (2.95), in STIC (0.6), and in HGOSC (0.5); ALDH1 was in the intact epithelium (0.5), in SCP (2.91), in SCO (2.92), in STIC (1.2), and in HGOSC (0.6). There were statistically significant differences with a 95% confidence interval (p<0.05) for: 1) PAX2 between the intact epithelium and pathology (fallopian tube lesions and HGOSC); 2) Bcl-2 between the intact epithelium and SCE (SCP and SCO) and between SCE and HGOSC; 3) beta-catenin between the intact epithelium and SCE (SCP and SCO) and between SCE and HGOSC; 4) ALDH1 between the intact epithelium and SCE, between and SCE and STIC, and between STIC and HGOSC., Conclusion: SCE was shown to be an independent intraepithelial lesion. The incidence of this abnormality increased with age and significantly differed in the patients with fallopian tubal lesions in extraovarian diseases from that in those with malignant ovarian serous tumors (by 5.3 times), while these groups showed a three-fold difference in SCO. Thus, SCP may serve as a more sensitive marker for the early stages of the pathogenesis of ovarian serous carcinoma. The studied types of SCE demonstrated multiple molecular events (loss of PAX2 expression and increased Bcl-2, beta-catenin, and ALDH1 expressions), some of which underwent considerable changes, by increasing the severity of a pathological process (loss of ALDH1, and beta-catenin, and bcl-2 expressions). Thus, therapeutic exposure in the early stages of pathogenesis may have a few points of application and just several molecules can serve as independent markers for early pathological changes in the fallopian tubal epithelium.

Stratified mucin-producing intraepithelial lesion (SMILE) is a rare premalignant cervical lesion that combines the structural features of cervical intraepithelial neoplasia (CIN) and endocervical adenocarcinoma in situ (AIS)., Aim: to analyze archival materials for the detection of SMILE and its subsequent morphological and immunohistochemical characterization., Material and Methods: Cervical cone specimens from 53 women with histologically verified CIN3 were examined. The diagnosis of SMILE was based on the positive mucicarmine staining and weak focal expression of p63. The samples containing SMILE were further immunohistochemically examined using the biomarkers P16, Ki-67, Oct4, CD117, CD34, p53, EMA, and CK15. SMILE was detected in 2 of the 53 patients and concurrent with CIN3 in both cases. SMILE was characterized by the stratified arrangement of atypical cells containing mucin, the positive mucicarmine staining of the entire layer of the atypical epithelium, weak focal p63 expression, high Ki-67 expression, and diffuse р16Іnk4а staining. Both SMILE samples showed weak diffuse p53 expression in the presence of single cells with the pronounced nuclear staining pattern for p53 in one female patient. Weak focal CK15 expression was visualized in SMILE. The expression of the stem cell markers Oct4 and CD117 and the angiogenic marker CD34 was absent in the examined cervical epithelial preparations. The diffuse and intense expression of the marker EMA, which was not different from that in the endocervical and stratified squamous epithelium, and CIN3 was established in SMILE., Results: The findings suggest that SMILE is morphologically and immunohistochemically similar to CIN3. In this investigation, these abnormalities differed only in the mucicarmine staining and expression of p63. This may be indicative of the underdiagnosis of SMILE, attendant СIN3 in the routine practice of a clinical pathologist, as the diagnosis of CIN3 is primarily based on the results of assessment of only the preparations stained with hematoxylin and eosin; the expression of p16 and Ki-67 is evaluated in some cases, which fails to differentiate SMILE and CIN3 in a number of preparations., Conclusion: The diagnosis of SMILE can be made only by immunohistochemical examination.

Aim: to study the incidence of fallopian tube lesions (secretory cell proliferations (SCP), p53 signature, serous tubal intraepithelial lesions (STIL), and serous tubal intraepithelial carcinomas (STIC) in ovarian epithelial tumors and to propose their pathogenetic association with a certain histotype of the ovarian tumor., Material and Methods: The investigation enrolled 136 patients with ovarian epithelial tumors, whose fallopian tubes were morphologically and immunohistochemically (IHC) examined using p53, Ki-67, and PAX2. Statistical analysis was carried out applying the Mann-Whitney test and χ(2) test., Results: Lesions meeting the STIC criteria were found in 14.7% of cases (only in ovarian serous carcinoma (OSC)), those suspecting STICs were in 25.7%, and those without signs of STICs were in 59.6%. IFC examination diagnosed STIC in 10% of cases (only in OSC), STIL in 13.3%, p53 signature in 11.7% (only in serous tumors), and the normal/reactively changed tubal epithelium in 65%. The incidence of STILs correlated with the malignant potential of serous tumors significantly (p<0.05). There were significant differences in the incidence of STILs in patients with different histotypes of ovarian carcinomas (p<0.05). PAX2-negative SCP was detected in 75% of OSC, 60% of serous borderline tumors, and 40% of serous cystadenomas. The differences in the incidence of SCP between serous tumors of varying grade, between serous tumors and non-serous carcinomas, between OSC and non-serous carcinomas were significant (p<0.05)., Conclusion: The investigation has shown that IHC examination should be used for the accurate diagnosis of STILs. It has also provided evidence for the pathogenetic association between STIC and high-grade OSC and revealed significant differences in the incidence of other fallopian tubal intraepithelial lesions in serous cystadenomas, borderline tumors, and OSC, in different ovarian carcinomas. The findings may suggest that the earliest stage in the pathogenesis of OSC is the development of SCP, followed by the formation of p53 signatures that may further give rise to STIL, and finally STC (due to the acquisition of additional mutations).