Markers of Lymphocytic-Macrophagal Infiltration and Their Association With the Receptor Phenotype and Proliferative Activity of Tumor Tissue in Various Molecular-Biological Types of Endometrial Cancer

Background and Aims: The last years were characterized by a shift from the former subdivision of endometrial cancer (EC) into two main types [1, 2] to modern molecular biological classifications of this disease [3-5]. The purpose of this investigation was an attempt to compare such prognostic indicators for EC as features of lymphocytic [6] and macrophage infiltration of tumor tissue with markers of its hormonal sensitivity (receptor phenotype) and the proliferation index Ki-67 [7], taking into account the molecular biological type of the disease. Materials and Methods: The study involved material from untreated patients with endometrial cancer (a total of 219 people). The average age of patients was close to 55-60 years. Using classification of Talhouk et al. [5] allowed to perform a search for POLE mutations, evaluate by IHC the expression of the oncoprotein p53 and MMR (mismatch-repair) proteins /MLH1, MSH2, MSH6 and PMS2/, and also identify the type of disease without a characteristic molecular profile (WCMP). The IHC method was also used to study the rate of estrogen (ER) and progesterone (PR) receptors, Ki-67 proliferative activity index, as well as the severity of macrophage-lymphocytic tissue infiltration of EC based on the analysis of the macrophage (CD68) and lymphocytic cells (cytotoxic CD8 and regulatory FoxP3) markers using reagents from Ventana and Dako. Statistical assessment of the relationships of the studied indicators was carried out by the Spearman rank correlation coefficient. Results: FoxP3 (in contrast to CD8 and CD68) positively and significantly correlates (ρ varies from 0.2895 to 0.3477) more often with ER, but not with PR. Ki-67 index in EC tissue positively and reliably correlates with FoxP3 both in the MMR-D and WCMP groups and in the combined cohort of EC patients. In the latter case, a similar relationship with Ki-67 extends to other studied markers of lymphocytic-macrophage infiltration, namely CD8 and CD68 (ρ 0,1746-0,3294). Only in the entire group of EC patients there is a positive rank correlation (0.4119!) between ER and PR expression. Conclusions: In patients with certain types of EC the connection between the estrogenic signal and PR induction is lost; it is especially noticeable in the MMR-D group, as exemplified by the negative correlation (-0.2951) of FoxP3 and PR expression. Taken together with existing data this indicates an important role of the endocrine component for differentiating separate groups of patients with EC, that may also be of practical importance. References: 1. Bokhman JV. Gynecol Oncol 1983; 15: 10-17. 2. Suarez AA et al. Gynecol Oncol 2017;144(2):243-249. 3. Murali R et al. Lancet Oncol 2014; 15: e268-278. 4.Berstein LM et al. Future Oncol. 2017 13(28):2593-2605. 5. Talhouk A. et al. Cancer. 2017;123(5):802-813. 6. Gargiulo P. et al. Cancer Treat Rev. 2016;48:61-8. 7. Kitson S. et al. Mod Pathol. 2017; 30(3): 459-468.

Results of treatment of patients with endometrial polyps using a differentiated approach

The problem of treatment of endometrial polyps remains relevant, because it has high risks of malignancy and a steady tendency to increase the frequency of relapses and is inherent from 26 to 78% in women of different age categories. The objective: is to reduce the frequency of relapses of endometrial polyps by introducing a differentiated treatment strategy for patients with endometrial polyps based on the study of new pathogenesis links. Materials and methods. Clinical and laboratory examinations and treatment of 66 women diagnosed with endometrial polyp at the age of 24–43 years were carried out, which were further divided into two statistically equivalent groups: A (n=34) and B (n=32). All women received treatment according to the 4-stage algorithm of the current order of the Ministry of health of Ukraine No. 676 dated 31.12.2004. The difference in the management of group A patients was that their treatment was supplemented by immunomodulate therapy. At the first stage of the study, additional hysteroresectoscopy was performed, endometrial samples were obtained to determine its type of pathology, develop an immunohistochemical profile of the endometrium with the establishment of a receptor phenotype, identify the inflammatory process, and determine the state of the apoptosis system and the APUD system of the endometrium. Results. Active histological screening of the endometrial condition showed that signs of chronic endometritis (positive reaction of CD-138 and CD-68 markers) in women with endometrial polyp with physiological background endometrium were observed in 26.5% of group A patients after 3 months of treatment, which required a repeat course of treatment based on the results of viral and bacteriological examination. In group B, this rate was 46.9%. All the examined women, against the background of the use of gestagens, showed changes in the endometrial receptor phenotype, which were manifested by a decrease in the number of progesterone receptors in the glandular epithelium by an average of 1.4 times. However, the expression levels of Bcl-2 protein and EC cells had no statistical differences, since there were no signs of proliferation in the background endometrium, including local ones. Viral-bacterial screening of the study showed a sharp decrease in the number of pathogens in the endometrium, but in studies of patients of both groups, viral-bacterial associations were determined. Bacterial screening revealed the presence of anaerobic microflora. The study of the level of tumor necrosis factor in flushes from the uterine cavity indicated a unidirectional trend with the dynamics of the CD-138 index. The results of treatment of patients with endometrial polyp with physiological endometrium showed that the full effect of treatment in group A (antibacterial therapy with gestagens + immunomodulatory therapy) was determined after a year in 85.2% of patients, 14.8% of women had a relapse of endometrial polyp, in the group where patients received only antibacterial therapy with gestagens – a relapse was diagnosed in 37.5% of patients. Conclusion. Determining the tactics of differentiated treatment of endometrial polyps, it is necessary to take into account the results of immunohistochemical research and viral-bacterial control of the endometrial condition, which allows improving the results of treatment of endometrial polyps from 62.5% to 85.2 %, and is a prevention of relapse of endometrial polyp. Key words: endometrial polyps, hysteroscopy, endometrial immunohistochemical examination, apoptosis system, endometrial APUD system, viral-bacterial screening.

Gain-of-function GABRB3 variants identified in vigabatrin-hypersensitive epileptic encephalopathies

Variants in the GABRB3 gene encoding the β3-subunit of the γ-aminobutyric acid type A ( receptor are associated with various developmental and epileptic encephalopathies. Typically, these variants cause a loss-of-function molecular phenotype whereby γ-aminobutyric acid has reduced inhibitory effectiveness leading to seizures. Drugs that potentiate inhibitory GABAergic activity, such as nitrazepam, phenobarbital or vigabatrin, are expected to compensate for this and thereby reduce seizure frequency. However, vigabatrin, a drug that inhibits γ-aminobutyric acid transaminase to increase tonic γ-aminobutyric acid currents, has mixed success in treating seizures in patients with GABRB3 variants: some patients experience seizure cessation, but there is hypersensitivity in some patients associated with hypotonia, sedation and respiratory suppression. A GABRB3 variant that responds well to vigabatrin involves a truncation variant (p.Arg194*) resulting in a clear loss-of-function. We hypothesized that patients with a hypersensitive response to vigabatrin may exhibit a different γ-aminobutyric acid A receptor phenotype. To test this hypothesis, we evaluated the phenotype of de novo variants in GABRB3 (p.Glu77Lys and p.Thr287Ile) associated with patients who are clinically hypersensitive to vigabatrin. We introduced the GABRB3 p.Glu77Lys and p.Thr287Ile variants into a concatenated synaptic and extrasynaptic γ-aminobutyric acid A receptor construct, to resemble the γ-aminobutyric acid A receptor expression by a patient heterozygous for the GABRB3 variant. The mRNA of these constructs was injected into Xenopus oocytes and activation properties of each receptor measured by two-electrode voltage clamp electrophysiology. Results showed an atypical gain-of-function molecular phenotype in the GABRB3 p.Glu77Lys and p.Thr287Ile variants characterized by increased potency of γ-aminobutyric acid A without change to the estimated maximum open channel probability, deactivation kinetics or absolute currents. Modelling of the activation properties of the receptors indicated that either variant caused increased chloride flux in response to low concentrations of γ-aminobutyric acid that mediate tonic currents. We therefore propose that the hypersensitivity reaction to vigabatrin is a result of GABRB3 variants that exacerbate GABAergic tonic currents and caution is required when prescribing vigabatrin. In contrast, drug strategies increasing tonic currents in loss-of-function variants are likely to be a safe and effective therapy. This study demonstrates that functional genomics can explain beneficial and adverse anti-epileptic drug effects, and propose that vigabatrin should be considered in patients with clear loss-of-function GABRB3 variants.

CLINICAL AND IMMUNOHISTOCHEMICAL FEATURES OF PRIMARY BREAST CANCER AND METACHRONOUS OVARIAN AND ENDOMETRIAL TUMORS

The aim of the study was to assess the patterns of development of metachronous cancer (endometrial cancer, EC, and ovarian cancer, OC) in breast cancer (BC) patients dependent of receptor phenotype of breast tumors. Materials and Methods: In the study, 63 patients with ВС, who developed metachronous EC (n = 47) or OC (n = 16) were enrolled. Expression of estrogen receptor (ER), progesterone receptor (PR), HER/2neu was assessed using immunohistochemical approach. Results: BC in patients with metachronous EC and OC was characterized by a different frequency of molecular subtypes with the dominance of luminal A (36%) and B (43%) subtypes. In primary BC, we have established a correlation between ER expression and regional lymph nodes status (r = −0.50, p < 0.05); negative correlation between HER2/neu expression and tumor stage (r = −0.48, p < 0.05); between the molecular subtype of BC and its size (r = −0.33, p <0.05), the molecular subtype of primary BC and metastases in regional lymph nodes (r = 0.27, p <0.05). In the patients with luminal subtype BC metachronous tumors developed with the highest frequency (OC — 50%, EC — 50%). After treatment of primary BC metachronous tumors developed at different period: EC (22.2%) — most often in 3–5 years, OC (11.0%) — after 10 years and more. Conclusion: Our data evidence on the clinical significance of the individual characteristics of the BC, especially its molecular subtype, and the need to calculate the personalized risk of development of metachronous tumors of the reproductive system in patients with the BC.