Bcl-2 and Ki67 as specific prognostic markers in estrogen receptor positive breast cancers

Objective: Breast cancer is a heterogeneous disease, so many biomarkers can be potential predictors in its outcome. The aim of this study is to test the hypothesis that combinatorial assessment of both Ki-67 proliferation index and B-cell Lymphoma 2 (Bcl-2) protein, would provide prognostic information on occurrence of relapses in breast cancer patients. Method: Immunohistochemical expression of Ki67 and Bcl-2, represented as Ki67/Bcl-2 index, were evaluated in 183 Estrogen Receptor positive breast cancer patients from 2007 to 2012, compared with other clinical-pathologic findings. During the follow up period (45–114 months) recurrences were observed in 36 patients (19.7 %). Results: A significant correlations were notified between Ki67/Bcl-2 index with age, tumour size, nuclear grade, histologic grade, lympho-vascular invasion, progesterone receptor status and expression of p53 protein product (p < 0,05). The occurrence of relapses in the group of low Ki67/high Bcl-2 index was lower, compared with the group of high Ki67/low Bcl-2 index breast cancer patients (p < 0.01). Conclusion: The combination of Ki67 and Bcl-2 biomarkers is useful tool in prediction of relapses in Estrogen positive breast cancer patients

Evaluation of PD-L1 expression in various formalin-fixed paraffin embedded tumour tissue samples using SP263, SP142 and QR1 antibody clones

Background & objectives: Cancer cells can avoid immune destruction through the inhibitory ligand PD-L1. PD-1 is a surface cell receptor, part of the immunoglobulin family. Its ligand PD-L1 is expressed by tumour cells and stromal tumour infltrating lymphocytes (TIL). Methods: Forty-four cancer cases were included in this study (24 triple-negative breast cancers (TNBC), 10 non-small cell lung cancer (NSCLC) and 10 malignant melanoma cases). Three clones of monoclonal primary antibodies were compared: QR1 (Quartett), SP 142 and SP263 (Ventana). For visualization, ultraView Universal DAB Detection Kit from Ventana was used on an automated platform for immunohistochemical staining Ventana BenchMark GX. Results: Comparing the sensitivity of two different clones on same tissue samples from TNBC, we found that the QR1 clone gave higher percentage of positive cells than clone SP142, but there was no statistically significant difference. Comparing the sensitivity of two different clones on same tissue samples from malignant melanoma, the SP263 clone gave higher percentage of positive cells than the QR1 clone, but again the difference was not statistically significant. Comparing the sensitivity of two different clones on same tissue samples from NSCLC, we found higher percentage of positive cells using the QR1 clone in comparison with the SP142 clone, but once again, the difference was not statistically significant. Conclusion: The three different antibody clones from two manufacturers Ventana and Quartett, gave comparable results with no statistically significant difference in staining intensity/ percentage of positive tumour and/or immune cells. Therefore, different PD-L1 clones from different manufacturers can potentially be used to evaluate the PD- L1 status in different tumour tissues. Due to the serious implications of the PD-L1 analysis in further treatment decisions for cancer patients, every antibody clone, staining protocol and evaluation process should be carefully and meticulously validated

Androgen Receptor Expression in Epithelial and Stromal Cells of Prostatic Carcinoma and Benign Prostatic Hyperplasia

BACKGROUND: Prostatic carcinoma (PCa) derives from prostatic epithelial cells. However stromal microenvironment, associated with malignant epithelium, also plays a role in prostatic carcinogenesis. Alterations in prostatic stromal cells contribute to the loss of growth control in epithelial cells that lead to progression of PCa.AIM: To analyse the differences between Androgen Receptor (AR) expression in both epithelial and stromal cells in PCa and the surrounding benign prostatic hyperplasia (BPH) and to compare the results with tumour grade.MATERIAL AND METHODS: Samples from 70 cases of radical prostatectomy specimens were used. The expression and intensity of the signal for AR was analysed in the epithelial and stromal cells of PCa and BPH, and the data was quantified using histological score (H-score).RESULTS: AR showed significantly lower expression in both epithelial and stromal cells of PCa compared to BPH. In PCa a significant positive correlation of AR expression was found between stromal and epithelial cells of PCa. AR expression showed a correlation between the stromal cells of PCa and tumour grade.CONCLUSION: AR expression is reduced in epithelial and stromal cells of PCa. Expression of AR in stromal cells of PCa significantly correlates with tumour grade

TIMP3 Promoter Methylation Represents an Epigenetic Marker of BRCA1ness Breast Cancer Tumours.

Tumours presenting BRCAness profile behave more aggressively and are more invasive as a consequence of their complex genetic and epigenetic alterations, caused by impaired fidelity of the DNA repair processes. Methylation of promoter CpG islands represents an alternative mechanism to inactivate DNA repair and tumour suppressor genes. In our study, we analyzed the frequency of methylation changes of 24 tumour suppressor genes and explored their association with BRCAness profile. BRCA1ness profile and aberrant methylation were studied in 233 fresh frozen breast tumour tissues by Multiplex Ligation-dependent Probe Amplification (MLPA) and Methylation Specific (MS)-MLPA methods, respectively. Our analyses revealed that 12.4% of the breast cancer (BC) patients had tumours with a BRCA1ness profile. TIMP3 showed significantly higher (p = 5.8х10) methylation frequency in tumours with BRCA1ness, while methylation of APC, GSTP1 and RASSF1 promoters was negatively associated with BRCA1ness (р = 0.0017, р = 0.007 and р = 0.046, respectively). TIMP3 methylation was also associated with triple negative (TN) BC. Furthermore, TN tumours showing BRCA1ness showed stronger association with TIMP3 methylation (p = 0.0008) in comparison to TN tumours without BRCA1ness (p = 0.009). In conclusion, we confirmed that TIMP3 methylation is a marker for TN tumours and furthermore we showed for the first time that TIMP3 promoter methylation is an epigenetic marker of BRCA1ness tumours

Y-chromosome haplogroup architecture confers susceptibility to azoospermia factor c microrearrangements: a retrospective study

Aim To assess the association between azoospermia factor c microrearrangements and semen quality, and between Y-chromosome background with distinct azoospermia factor c microrearrangements and semen quality impairment. Methods This retrospective study, carried out in the Research Center for Genetic Engineering and Biotechnology “Georgi D. Efremov,” involved 486 men from different ethnic backgrounds referred for couple infertility from 2002- 2017: 338 were azoospermic/oligozoospermic and 148 were normozoospermic. The azoospermia factor c microrearrangements were analyzed with sequence tagged site and sequence family variant markers, quantitative fluorescent polymerase chain reaction, and multiplex ligation probe amplification analysis. The Y-haplogroups of all participants were determined with direct single nucleotide polymorphism typing and indirect prediction with short tandem repeat markers.Results Our participants had two types of microdeletions: gr/gr and b2/b3; three microduplications: b2/b4, gr/gr, and b2/b3; and one complex rearrangement gr/gr deletion + b2/b4 duplication. Impaired semen quality was not associated with microrearrangements, but b2/b4 and gr/ gr duplications were significantly associated with haplogroup R1a (P < 0.001 and P = 0.003, respectively) and b2/b3 deletions with haplogroup E (P = 0.005). There were significantly more b2/b4 duplication carriers in Albanians than in Macedonians with haplogroup R1a (P = 0.031). Conclusion Even though azoospermia factor c partial deletions/duplications and Y-haplogroups were not associated with impaired semen quality, specific deletions/ duplications were significantly associated with distinct haplogroups, implying that the Y chromosome background may confer susceptibility to azoospermia factor c microrearrangements

Comparative proteomics analysis of human FFPE testicular tissues reveals new candidate biomarkers for distinction among azoospermia types and subtypes.

Understanding molecular mechanisms that underpin azoospermia and discovery of biomarkers that could enable reliable, non-invasive diagnosis are highly needed. Using label-free data-independent LC-MS/MS acquisition coupled with ion mobility, we compared the FFPE testicular proteome of patients with obstructive (OA) and non-obstructive azoospermia (NOA) subtypes hypospermatogenesis (Hyp) and Sertoli cell-only syndrome (SCO). Out of 2044 proteins identified based on ≥2 peptides, 61 proteins had the power to quantitatively discriminate OA from NOA and 30 to quantitatively discriminate SCO from Hyp and OA. Among these, H1-6, RANBP1 and TKTL2 showed superior potential for quantitative discrimination among OA, Hyp and SCO. Integrin signaling pathway, adherens junction, planar cell polarity/convergent extension pathway and Dectin-1 mediated noncanonical NF-kB signaling were significantly associated with the proteins that could discriminate OA from NOA. Comparison with 2 transcriptome datasets revealed 278 and 55 co-differentially expressed proteins/genes with statistically significant positive correlation. Gene expression analysis by qPCR of 6 genes (H1-6, RANBP1, TKTL2, TKTL1, H2BC1, and ACTL7B) with the highest discriminatory power on protein level and the same regulation trend with transcriptomic datasets, confirmed the proteomics results. In summary, our results suggest some underlying pathways in azoospermia and broaden the range of potential novel candidates for diagnosis. SIGNIFICANCE: Using a comparative proteomics approach on testicular tissue we have identified several pathways associated with azoospermia and a number of testis-specific and germ cell-specific proteins that have the potential to pinpoint the type of spermatogenesis failure. Furthermore, comparison with transcriptomics datasets based on genome-wide gene expression analyses of human testis specimens from azoospermia patients identified proteins that could discriminate between obstructive and non-obstructive azoospermia subtypes on both protein and mRNA levels. Up to our knowledge, this is the first integrated comparative analysis of proteomics and transcriptomics data from testicular tissues. We believe that the data from our study contributes significantly to increase the knowledge of molecular mechanisms of azoospermia and pave the way for new investigations in regards to non-invasive diagnosis

The highest frequency of c.3700_3704del detected among Albanians from Kosovo.

BACKGROUND The spectrum of and mutations varies among populations; however, some mutations may be frequent in particular ethnic groups due to the "founder" effect. The c.3700_3704del mutation was previously described as a recurrent variant in Eastern European countries. This study aimed to investigate the frequency of c.3700_3704del mutation in Albanian breast and ovarian cancer patients from North Macedonia and Kosovo. MATERIALS AND METHODS A total of 327 patients with invasive breast and/or ovarian cancer (111 Albanian women from North Macedonia and 216 from Kosovo) were screened for 13 recurrent mutations. Targeted NGS with a panel of 94 cancer-associated genes including and was performed in a selected group of 118 patients. RESULTS We have identified 21 /2 pathogenic variants, 17 (14 and 3 ) in patients from Kosovo (7.9%) and 4 (1 and 3 ) in patients from North Macedonia (3.6%). All mutations were found in one patient each, except for c.3700_3704del mutation which was observed in 14 unrelated families, all except one originating from Kosovo. The c.3700_3704del mutation accounts for 93% of mutation positive cases and is present with a frequency of 6% among breast cancer patients from Kosovo. CONCLUSIONS This is the first report of mutations among breast and ovarian cancer patients from Kosovo. The finding that c.3700_3704del represents a founder mutation in Kosovo with the highest worldwide reported frequency supports the implementation of fast and low-cost screening protocol, regardless of the family history and even a pilot population-based screening in at-risk population

The Spectrum of FANCM Protein Truncating Variants in European Breast Cancer Cases

Germline protein truncating variants (PTVs) in the FANCM gene have been associated with a 2–4-fold increased breast cancer risk in case-control studies conducted in different European populations. However, the distribution and the frequency of FANCM PTVs in Europe have never been investigated. In the present study, we collected the data of 114 European female breast cancer cases with FANCM PTVs ascertained in 20 centers from 13 European countries. We identified 27 different FANCM PTVs. The p.Gln1701* PTV is the most common PTV in Northern Europe with a maximum frequency in Finland and a lower relative frequency in Southern Europe. On the contrary, p.Arg1931* seems to be the most common PTV in Southern Europe. We also showed that p.Arg658*, the third most common PTV, is more frequent in Central Europe, and p.Gln498Thrfs*7 is probably a founder variant from Lithuania. Of the 23 rare or unique FANCM PTVs, 15 have not been previously reported. We provide here the initial spectrum of FANCM PTVs in European breast cancer cases

Rare germline copy number variants (CNVs) and breast cancer risk.

Funder: CIHRGermline copy number variants (CNVs) are pervasive in the human genome but potential disease associations with rare CNVs have not been comprehensively assessed in large datasets. We analysed rare CNVs in genes and non-coding regions for 86,788 breast cancer cases and 76,122 controls of European ancestry with genome-wide array data. Gene burden tests detected the strongest association for deletions in BRCA1 (P = 3.7E-18). Nine other genes were associated with a p-value < 0.01 including known susceptibility genes CHEK2 (P = 0.0008), ATM (P = 0.002) and BRCA2 (P = 0.008). Outside the known genes we detected associations with p-values < 0.001 for either overall or subtype-specific breast cancer at nine deletion regions and four duplication regions. Three of the deletion regions were in established common susceptibility loci. To the best of our knowledge, this is the first genome-wide analysis of rare CNVs in a large breast cancer case-control dataset. We detected associations with exonic deletions in established breast cancer susceptibility genes. We also detected suggestive associations with non-coding CNVs in known and novel loci with large effects sizes. Larger sample sizes will be required to reach robust levels of statistical significance