Genetic Heterogeneity, Tumor Microenvironment and Immunotherapy in Triple-Negative Breast Cancer.

Heterogeneity of triple-negative breast cancer is well known at clinical, histopathological, and molecular levels. Genomic instability and greater mutation rates, which may result in the creation of neoantigens and enhanced immunogenicity, are additional characteristics of this breast cancer type. Clinical outcome is poor due to early age of onset, high metastatic potential, and increased likelihood of distant recurrence. Consequently, efforts to elucidate molecular mechanisms of breast cancer development, progression, and metastatic spread have been initiated to improve treatment options and improve outcomes for these patients. The extremely complex and heterogeneous tumor immune microenvironment is made up of several cell types and commonly possesses disorganized gene expression. Altered signaling pathways are mainly associated with mutated genes including p53, PIK3CA, and MAPK, and which are positively correlated with genes regulating immune response. Of note, particular immunity-associated genes could be used in prognostic indexes to assess the most effective management. Recent findings highlight the fact that long non-coding RNAs also play an important role in shaping tumor microenvironment formation, and can mediate tumor immune evasion. Identification of molecular signatures, through the use of multi-omics approaches, and effector pathways that drive early stages of the carcinogenic process are important steps in developing new strategies for targeted cancer treatment and prevention. Advances in immunotherapy by remodeling the host immune system to eradicate tumor cells have great promise to lead to novel therapeutic strategies. Current research is focused on combining immune checkpoint inhibition with chemotherapy, PARP inhibitors, cancer vaccines, or natural killer cell therapy. Targeted therapies may improve therapeutic response, eliminate therapeutic resistance, and improve overall patient survival. In the future, these evolving advancements should be implemented for personalized medicine and state-of-art management of cancer patients.

Mitochondriopathies as a Clue to Systemic Disorders-Analytical Tools and Mitigating Measures in Context of Predictive, Preventive, and Personalized (3P) Medicine.

The mitochondrial respiratory chain is the main site of reactive oxygen species (ROS) production in the cell. Although mitochondria possess a powerful antioxidant system, an excess of ROS cannot be completely neutralized and cumulative oxidative damage may lead to decreasing mitochondrial efficiency in energy production, as well as an increasing ROS excess, which is known to cause a critical imbalance in antioxidant/oxidant mechanisms and a "vicious circle" in mitochondrial injury. Due to insufficient energy production, chronic exposure to ROS overproduction consequently leads to the oxidative damage of life-important biomolecules, including nucleic acids, proteins, lipids, and amino acids, among others. Different forms of mitochondrial dysfunction (mitochondriopathies) may affect the brain, heart, peripheral nervous and endocrine systems, eyes, ears, gut, and kidney, among other organs. Consequently, mitochondriopathies have been proposed as an attractive diagnostic target to be investigated in any patient with unexplained progressive multisystem disorder. This review article highlights the pathomechanisms of mitochondriopathies, details advanced analytical tools, and suggests predictive approaches, targeted prevention and personalization of medical services as instrumental for the overall management of mitochondriopathy-related cascading pathologies.

Colposcopic scoring indexes in the evaluation of cervical lesions with the cytological result of atypical squamous cells, cannot exclude high-grade lesion.

AIM: Colposcopic indexes including Reid index and Swede score were developed to make the colposcopy more objective. The aim of our study was to evaluate the significance of colposcopic indexes in the evaluation of ASC-H cervical lesions. METHODS: We carried out a cross-sectional study in the Clinic of Obstetrics and Gynecology between January 2013 and December 2018. The study included 535 women, from which 66 women had a cytological result ASC-H. Scoring of all colposcopic findings was assessed according to Reid modified index and Swede score and a composite score was determined. Frequency distributions were compared using χ2 /Fisher exact test. Spearman rank correlation coefficient was computed between RCI and Swede score. RESULTS: Sensitivity, specificity, positive and negative predictive value and positive likelihood ratio of modified Reid colposcopic index at a cutoff of ≥4 for the detection of HSIL+ lesions were: 86.11% (95% CI: 70.5-95.3), 83.33% (95% CI: 65.3-94.4), 86.11% (95% CI: 69.7-94.8), 83.33% (95% CI: 64.5-93.7) and 5.17 (95% CI: 2.3-11.6). Swede score with the cutoff value ≥5 showed comparable results to modified Reid index with the increased sensitivity: 94.44% (95% CI: 81.3-99.3). CONCLUSION: ASC-H category represents the trickiest cytological diagnosis as it is underlined with the high risk of severe cervical dysplasia. Evaluating the cervical lesion by the use of colposcopic indices helps the gynecologist to objectively evaluate all the pathologies of uterine cervix. Swede score with the cutoff value 8 also enables a 'see and treat' option in management of atypical squamous cells, cannot exclude high-grade lesions.

miRNA Expression Profiles in Luminal A Breast Cancer-Implications in Biology, Prognosis, and Prediction of Response to Hormonal Treatment.

Breast cancer, which is the most common malignancy in women, does not form a uniform nosological unit but represents a group of malignant diseases with specific clinical, histopathological, and molecular characteristics. The increasing knowledge of the complex pathophysiological web of processes connected with breast cancercarcinogenesis allows the development of predictive and prognostic gene expressionand molecular classification systems with improved risk assessment, which could be used for individualized treatment. In our review article, we present the up-to-date knowledge about the role of miRNAs and their prognostic and predictive value in luminal A breast cancer. Indeed, an altered expression profile of miRNAs can distinguish not only between cancer and healthy samples, but they can classify specific molecular subtypes of breast cancer including HER2, Luminal A, Luminal B, and TNBC. Early identification and classification of breast cancer subtypes using miRNA expression profilescharacterize a promising approach in the field of personalized medicine. A detection of sensitive and specific biomarkers to distinguish between healthy and early breast cancer patients can be achieved by an evaluation of the different expression of several miRNAs. Consequently, miRNAs represent a potential as good diagnostic, prognostic, predictive, and therapeutic biomarkers for patients with luminal A in the early stage of BC.

Pathway Analysis of Selected Circulating miRNAs in Plasma of Breast Cancer Patients: A Preliminary Study.

MicroRNAs in the circulation of breast cancer (BC) patients have great potential for the early diagnosis, treatment and monitoring of breast cancer. The aim of this preliminary study was to obtain the expression profile of selected miRNAs in the plasma of BC patients that could discriminate BC patients from healthy volunteers and may be useful in early detection of BC. Significantly deregulated miRNAs were evaluated by pathway analysis with the prediction of potential miRNA targets. The study enrolled plasma samples from 65 BC patients and 34 healthy volunteers. Selected miRNAs were screened in pilot testing by the real-time PCR (qPCR) method, and the most appropriate reference genes were selected for normalisation by the geNorm algorithm. In the final testing, we detected miR-99a, miR-130a, miR-484 and miR-1260a (p < 0.05) as significantly up-regulated in the plasma of BC patients. Kyoto Encyclopaedia of Genes and Genomes (KEGG) pathway analysis revealed that all significantly deregulated miRNAs are involved in the Hippo and Transforming Growth Factor-beta (TGF-beta) signalling pathways. Our study confirmed a different profile of selected circulating miRNAs in the plasma of BC patients with an emphasis on some critical points in the analysis process.

Novel Biomarkers of Early Atherosclerotic Changes for Personalised Prevention of Cardiovascular Disease in Cervical Cancer and Human Papillomavirus Infection.

Cervical cancer is associated with a causative role of human papillomavirus (HPV), which is a highly prevalent infection. Recently, women with a genital HPV infection were found to have increased incidence of cardiovascular diseases (CVD), including severe cardiovascular events such as myocardial infarction and stroke. The pathomechanisms of this relation are not yet fully understood, and may significantly affect the health of a large part of the population. Accelerated atherosclerosis is assumed to play a key role in the pathophysiology of this relationship. To identify high-risk groups of the population, it is necessary to stratify the CVD risk. Current algorithms, as widely used for the estimation of CVD risk, seem to be limited by the individual misclassification of high-risk subjects. However, personalised prediction of cardiovascular events is missing. Regarding HPV-related CVD, identification of novel sensitive biomarkers reflecting early atherosclerotic changes could be of major importance for such personalised cardiovascular risk prediction. Therefore, this review focuses on the pathomechanisms leading to HPV-related cardiovascular diseases with respect to atherosclerosis, and the description of potential novel biomarkers to detect the earliest atherosclerotic changes important for the prevention of CVD in HPV infection and cervical cancer.

Why the Gold Standard Approach by Mammography Demands Extension by Multiomics? Application of Liquid Biopsy miRNA Profiles to Breast Cancer Disease Management.

In the global context, the epidemic of breast cancer (BC) is evident for the early 21st century. Evidence shows that national mammography screening programs have sufficiently reduced BC related mortality. Therefore, the great utility of the mammography-based screening is not an issue. However, both false positive and false negative BC diagnosis, excessive biopsies, and irradiation linked to mammography application, as well as sub-optimal mammography-based screening, such as in the case of high-dense breast tissue in young females, altogether increase awareness among the experts regarding the limitations of mammography-based screening. Severe concerns regarding the mammography as the "golden standard" approach demanding complementary tools to cover the evident deficits led the authors to present innovative strategies, which would sufficiently improve the quality of the BC management and services to the patient. Contextually, this article provides insights into mammography deficits and current clinical data demonstrating the great potential of non-invasive diagnostic tools utilizing circulating miRNA profiles as an adjunct to conventional mammography for the population screening and personalization of BC management.

Different amplification patterns of 3q26 and 5p15 regions in cervical intraepithelial neoplasia and cervical cancer.

PURPOSE: The aim of this study was to evaluate and correlate the amplification of chromosomal regions 3q26 and 5p15 in different cytological and histological subgroups of patients and to compare the sensitivity and specificity of amplification tests with cytology, colposcopy and HPV status. METHODS: The work was conducted at the Department of Obstetrics and Gynaecology in cooperation with the Institute of Pathological Anatomy, JFM CU in Martin and UNM during years 2013-2016. Prospective longitudinal study included 131 patients. We focused on the FISH diagnosis of the amplification of regions encoding the components of telomerase enzyme (3q26, 5p15) in cytology specimens. We manually evaluated 100 atypical cells per slide and analysed the amplification patterns. Correlations between cytological, histological, HPV DNA results and amplification patterns of chromosomal regions 3q26 and 5p15 were analysed by chi-squared test and non-parametric Man - Whitney U test. RESULTS: The results showed that the amplification of chromosomal regions increases with the degree of dysplasia toward the invasive disease (p < 0.001). Whereas the increase in the amplification of 3q26 is noticeable already at CIN 2 + lesions (p < 0.01), 5p15 amplification is shifted up toward CIN 3/CIS (p < 0.001) and cervical cancer. Amplification of selected regions correlated with each other and also with hrHPV-positive status (p < 0.01). CONCLUSION: The analysis of the amplification of 3q26 and 5p15 regions may serve in the future for the differential diagnosis of cervical lesions and to determine their malignant potential. High specificity of these tests can improve the excellent sensitivity of HPV DNA test.

DNA methylation as mechanism of apoptotic resistance development in endometrial cancer patients.

DNA methylation is a significant epigenetic modification which plays a key role in regulation of gene expression and influences functional changes in endometrial tissue. Aberrant DNA methylation changes result in deregulation of important apoptotic proteins during endometrial carcinogenesis and apoptosis resistance development. Evading apoptosis is still a major problem in the successful treatment of endometrial cancer patients. The aim of our study was to examine the promoter DNA methylation changes in 22 apoptosis-associated genes in endometrioid endometrial cancer patients, precancerous lesions and healthy tissue from various normal menstrual cycle phases using a unique pre-designed methylation platform. We observed as the first a significant difference in promoter DNA methylation status in genes: BCL2L11 (p < 0.001), CIDEB (p < 0.03) and GADD45A (p < 0.05) during endometrial carcinogenesis and BIK gene (p < 0.03) in different phases of normal menstrual cycle. The results of our study indicate that deregulation of mitochondrial apoptotic pathway can considerably contributes to the apoptosis resistance development and may be helpful in identifying of new potent biomarkers in endometrial cancer.

Active cigarette smoking and the risk of breast cancer at the level of N-acetyltransferase 2 (NAT2) gene polymorphisms.

The aim of our study was to assess the correlation between the tobacco exposure and NAT2 gene (rs1041983 C/T, rs1801280 T/C, rs1799930 G/A) polymorphisms in association with breast cancer development. We wanted to determine the prognostic clinical importance of these polymorphisms in association with smoking and breast cancer. For the detection of possible association between smoking, NAT2 gene polymorphisms, and the risk of breast cancer, we designed a case-controlled study with 198 patients enrolled, 98 breast cancer patients and 100 healthy controls. Ten milliliters of peripheral blood from the cubital vein was withdrawn from every patient. The HRM (high resolution melting) analysis was used for the detection of three abovementioned NAT2 gene polymorphisms. When comparing a group of women smoking more than 5 cigarettes a day with the patients smoking fewer than 5 cigarettes a day, we found out that if women were the carriers of aberrant AA genotype for rs1799930, the first group of women had higher risk of breast carcinoma than the second group. If patients were the carriers of aberrant TT genotype for rs1041983, for rs1801280CC genotype, and rs1799930AA genotype and they smoked more than 5 cigarettes a day, they had higher risk of malignant breast disease than never-smoking women. Our results confirm the hypothesis that NAT2 gene polymorphisms (rs1041983 C/T, rs1801280 T/C, and rs1799930 G/A) in association with long-period active smoking could be the possible individual risk-predicting factors for breast cancer development in the population of Slovak women.

Arteriovenous malformation of vein of Galen as a rare non-hypoxic cause of changes in fetal heart rate pattern during labor.

The aim of this case report is to describe a rare non-hypoxic cause of pathological changes in fetal heart rate pattern during labor, and to determine management, including a description of important prenatal aspects when pathologic cardiotocographic recording is performed during labor. A fetus with rare arteriovenous malformation of the vein of Galen, which represents less than 1% of all intracranial arteriovenous malformations, was monitored by intrapartum external cardiotocography in the 37 + 5 gestational week. The baby was born by cesarean section because of signs of imminent intrauterine hypoxia on cardiotocography. However, metabolic acidosis was not confirmed in umbilical cord blood sampling. Despite intensive neonatal care management, the newborn died 31 h after delivery because of progressive cardiac decompensation, hypotension and multi-organ failure. Precise diagnosis of the abovementioned pathology, a pre-labor plan for delivery and postnatal prognosis assessment can significantly contribute to the avoidance of a misdiagnosis of fetal hypoxia and unnecessary operative delivery with marked medico-legal consequences.

Survival and risk factors associated with uterine sarcomas and carcinosarcomas in stage I and II.

OBJECTIVE: Uterine sarcomas are rare mesodermal malignant tumors with an incidence between 0.5 and 3.3 cases per 100,000 females per year. Most sarcomas are aggressive tumors leading to poor overall survival rates and only limited therapeutic options. The aim of this study was to evaluate the risk factors for uterine sarcomas and carcinosarcomas, and to identify the factors influencing the survival rate. SUBJECTS AND METHODS: We conducted a retrospective study with twenty-nine patients who were diagnosed with uterine sarcoma and thirty-four patients with carcinosarcoma between the years 1990 and 2006 at the Oncogynecologic center at the University Hospital in Martin, Slovakia. We focused on the analysis of the risk factors and survival rate of early stages I and II. RESULTS: We confirmed highly statistically significant values for the inverse correlation between survival and tumor size, positive lymph nodes, high mitotic activity, vascular invasion, positive peritoneal cytology, elevated CA-125, smoking and BMI in sarcoma and carcinosarcoma group (p<0.001 for all factors). The use of lymphadenectomy had no effect on survival of all patients. DISCUSSION: Sarcomas and carcinosarcomas are aggressive tumors leading to poor overall survival rates and only limited therapeutic options. As there is no consensus on specific treatment, an individual approach based on evaluation of known risk factors is essential.

Amplification of 3q26 and 5p15 regions in cervical intraepithelial neoplasia.

OBJECTIVE: To analyze different amplification patterns of 3q26 and 5p15 regions in low-grade and high-grade cervical intraepithelial neoplasia. DESIGN: Experimental research. SETTING: Department of Obstetrics and Gynecology at a medical faculty in Slovakia. POPULATION: A group of 83 patients referred for colposcopic examination. METHODS: Amplification of 3q26 and 5p15 regions was analyzed on the 100 most atypical cells from a cervical cytology slide by fluorescent in situ hybridization using a multicolor hybridization probe. Chi-squared and Man-Whitney U-tests were used for statistical analysis. MAIN OUTCOME MEASURES: Liquid-based cytology samples and biopsy samples obtained during colposcopic examination correlated with high-risk human papillomavirus status and with amplification patterns of selected regions analyzed by fluorescent in situ hybridization. RESULTS: The number of cells with 3q26 and 5p15 gain rises with the severity of the lesion p < 0.01. The sensitivity of 3q26 amplification for CIN2+ lesions was 72.1% (95% confidence interval 56.3-84.7) and specificity was 90.0% (95% confidence interval 76.3-97.1). The sensitivity of 5p15 amplification for CIN2+ lesions was 69.8% (95% confidence interval 53.9-82.8) and specificity was 85.0% (95% confidence interval 70.2-94.3). CONCLUSION: Evaluation of telomerase components can help in differential diagnosis of low-grade and high-grade cervical lesions and in individualized management of these patients.

Amplification of TERT and TERC genes in cervical intraepithelial neoplasia and cervical cancer.

OBJECTIVES: Telomerase is activated in various stages of oncogenesis. For cervical cancer, telomerase is already active in precancerous lesions. In our study we focused on the analysis of the amplification patterns of telomerase genes TERT and TERC. DESIGN AND SETTING: We included 39 patients in our study between January 2012 and April 2013. Each patient underwent a classical gynaecological examination and a colposcopy. During the colposcopic examination we collected material for a Pap smear, HPV DNA test (HC2) and LBC (LiquiPrep™), and performed punch biopsies for histopathological evaluation. Residual cytologic sample was hybridized with the FISH probe and telomerase genes were analysed. RESULTS: The amplification of the TERT gene showed us a very similar amplification pattern as TERC and gradually corresponded with both histolopathological (p<0.001) and cytopathological findings (p<0.001). The specificity and sensitivity of TERC gene amplification for the detection of CIN2+ lesions (cut off value 2.3) was 88.2% and 95.5% respectively (PPV 91.3%, NPV 93.8%). CONCLUSIONS: We identified increasing amplification pattern of telomerase genes in cervical lesions. According to our results telomerase genes could help in the future to determine the malignant potential of cervical lesions and could be tested together with cytology and HPV DNA in order to obtain the highest combined sensitivity and specificity for CIN2+ lesion detection.

Detection of methylation of the promoter region of the MAL and CADM1 genes by pyrosequencing in cervical carcinoma.

OBJECTIVE: Cervical cancer is the second most common cancer disease affecting the female population. A key factor in development of the disease is the human papillomavirus infection (HPV). The disease is also impacted by epigenetic changes such as DNA methylation, which causes activation or exclusion of certain genes, and simultaneously the hypermethylation of cytosines in the promoters and turn-off of previously active genes occur. In this study, we focused on the introduction of pyrosequencing for the detection of DNA methylation of the selected CADM1 and MAL genes. METHODS: DNA was isolated from cytological cervical smear of patients with different types of dysplasia [L-SIL (n=14), ASC-US (n=15), H-SIL (n=1)] and four control samples from healthy women. Prepared samples were further analyzed by bisulfite conversion and subsequent pyrosequencing (Pyromark Q96 ID, Qiagen, Germany). We examined the extent of methylation of CpG islands and as control samples of this method we used a fully methylated and unmethylated DNA. Methylation level (Met level) from each sample was quantified as the mean value [sum of all methylated CpG islands in %/total number of CpG islands (MAL n=4; CADM1 n=3)]. RESULTS: In total, 30 clinical samples and 4 control samples from healthy women were analyzed. By means of the analysis of the CADM1promoter region, the values of the Met level were obtained [fully methylated DNA (94.83 and 88); completely unmethylated DNA (0 and 0); and control samples from healthy patients (6.825 and 0.825), L-SIL (2.107 and 2.778), ASC-US (7.313 and 3.626), H-SIL (0 and 0)]. By means of the analysis of the MAL promoter region, the values of Met level were obtained [fully methylated DNA (53.25); completely unmethylated DNA (0.875); and control samples from healthy patients (2.925), L-SIL (1.517), ASC-US (2.833), and H-SIL (4)]. CONCLUSION: We introduced a pyrosequencing method for quantification of methylation of CADM1, MAL promoter regions, and detected methylations in clinical samples and also some basal methylation in healthy women.

mRNA expression in cervical specimens for determination of severe dysplasia or worse in HPV-16/18-positive squamous lesions.

OBJECTIVES: The objective of current study was to determine the p16 mRNA level in cervical cells by relative quantification (RQ) and to test viral E6 expression in human papillomavirus (HPV) -16 or -18-positive specimens by widely used methods. We targeted the pivotal mRNA level associated with severe dysplasia or worse. MATERIALS AND METHODS: Cervical specimens were taken from 134 women with cervical disease and 132 women with normal cytologic results. The presence of HPV was analyzed by sequencing. The results of p16 and E6 analyses were statistically processed in receiver operating characteristic curve analysis to predict severe dysplasia or worse. RESULTS: The HPV DNA was detected in 81.4% (109/134) of women with cervical disease and in 27.3% (36/132) of women with normal cytologic results. HPV-16 or -18 were present in 59.7% (80/134) of abnormal specimens. p16 and E6 mRNA expression was increasing with severity of cervical dysplasia. p16 mRNA expression was found 4.35-fold and 13.15-fold increased in high-grade squamous intraepithelial lesions and squamous cell carcinomas, respectively. E6 mRNA expression was significantly increased (p = .0038) in severe dysplasias or worse. The RQ method achieved better sensitivity (82.6%), and E6 mRNA got better specificity (80.6%) for the prediction of severe dysplasia or worse. CONCLUSIONS: An increasing level of p16 and E6 mRNA transcripts could mean the potential of cervical dysplasia progression to cancer, but further studies should be done to confirm this proposition. Nevertheless, we consider using both tests to improve the sensitivity and specificity for prediction of severe dysplasia or worse.