BRCA1 promoter methylation & its immunohistochemical correlation in sporadic breast cancer

Background & objectives: Studies have shown that apart from hereditary breast carcinomas, breast cancer susceptibility gene 1 (BRCA1) mutations conferring to its loss are seen in sporadic breast carcinomas (SBC) as well. The aim of the present study was to assess BRCA1 methylation in females presenting at King George’s Medical University, Lucknow, with SBC by both immunohistochemistry (IHC) and methylation PCR with respect to hormonal profile and various morphological prognostic parameters. The primary objective was to look for the association between BRCA1 protein expression and DNA promoter methylation. Methods: 81 mastectomy specimens from SBC of invasive breast carcinoma (no special type) were included in this study. After a detailed morphological assessment, formalin fixed paraffin embedded tissue from a representative tumour area was selected for BRCA1 IHC by heat-mediated antigen retrieval under high pH and DNA extraction and further bisulphate treatment. BRCA1 was studied for methylation by methylated and unmethylated PCR-specific primers. Results: BRCA1 promoter methylation was present in 42/81 (51.9%) participants, with significant BRCA1 protein loss (72.7%; P=0.002). A significant association between BRCA1 loss and hormonal profile was found (P=0.001); maximum in triple negative breast carcinoma (TNBC) (72%; 18/25). Most of the TNBC also harboured methylation (68%). Although not significant grade II and III tumours, lymph vascular invasion, ductal carcinoma in situ, and nodal metastasis (?3) were seen in a higher percentage in methylated tumours. Mortality in SBC was significantly associated with BRCA1 loss (30.3%; P=0.024). Interpretation & conclusions: Study results highlight the concept of “BRCAness” in SBC as well. Hence, we can confer that identification of BRCA1 loss in SBC can make it a perfect candidate for poly ADP- ribose polymerase inhibitors or cisplatin-based therapy like hereditary ones.

Diagnostic utility of cytospin, cell block and immunocytochemistry in pleural effusion cytology

Background: With the introduction of cytospin, the sensitivity of diagnosing malignancies has increased mainly due to the increase in cellular yield. Cell block also gives the advantage of ancillary testing and allows for retrospective studies. Immunocytochemical markers are used to differentiate and subtype various malignancies in body effusions.Aim of the study was to compare the morphological features of both technique and to assess the diagnostic utility of cell block methods in the cytodiagnosis of pleural effusions.Methods: This was a Prospective observational comparative study of two cytopreparatory techniques. All samples were examined and processed by cytospin and cell block techniques. Continuous data were expressed as Mean±SD (standard deviation) while categorical data were expressed in number, percentage and compared by chi-square (χ2) test.Results: The final diagnosis of both cytospin (147 cases) and cell block (150 cases) techniques was divided into four broad categories: Inadequate, Benign, Suspicious and Malignant. The significant diagnostic cytospin (AUC=0.857, p<0.001) in discriminating positive and negative malignant cases with 75.00% sensitivity (95% CI=53.3-90.2) and 100.00% specificity (95% CI=86.7-100.0) and with 100.0% positive predictive value and 81.2% negative predictive value. In contrast, cell block also showed significant diagnostic but with higher accuracy (AUC=1.000, p<0.001) and sensitivity 100.00% (95% CI=86.7-100.0) and specificity 100.00% (95% CI=86.7-100.0) and 100.0% positive predictive value and 100.0% negative predictive value than cytospin technique.Conclusions: Cell block as a technique should be used in routine practice as it not only increases the diagnostic yield but ancillary test can also be done.

Expression and cellular distribution of insulin-like growth factor 1 receptor during window of implantation in infertile women with intramural fibroids

Background: The “window of implantation” (WOI) is a transient but well defined period during which the hostile endometrial lining is transformed to a surface receptive to accept the embryo. Recently, data are beginning to accumulate suggesting negative influence of non-cavity distorting intramural uterine fibroids (NCD-IMF) on endometrial receptivity that may have implications for implantation failure. However, molecular mechanisms underlying infertility associated with NCD-IMF remain unclear. The aim of present study was to examine the expression and cellular distribution of insulin-like growth factor 1 receptor (IGF1R) during WOI in infertile women with NCD-IMF and fertile controls. While, reports are available that support role of IGF1R in mediating adhesive interaction with the implanting blastocyst, the effect of NCD-IMF on IGF1R expression during the WOI is not defined.Methods: Quantitative real-time polymerase chain reaction and immunohistochemistry were used to evaluate messenger RNA (mRNA) and protein expression of IGF1R in midsecretory endometrial biopsies obtained from infertile women with NCD-IMF (n=20) and healthy fertile controls (n=10).Results: As compared to fertile controls, significantly higher IGF1R: i) mRNA levels (1.59 fold up regulation; p=0.044) and ii) immunoscore in the luminal epithelium (8.94±3.13 versus 6.31±1.49; p=0.009) were observed in infertile women with NCD-IMF.Conclusions: Over expression of IGF1R in infertile women with NCD-IMF, during the window of receptivity, may result in altered ability of uterine epithelial cells for blastocyst adhesion and subsequent implantation, which might lead to poor reproductive outcome in these women.

Cellular homeostasis, implantation window and unexplained infertility: role of phosphatase and tensin homolog deleted on chromosome 10

Background: Balance between endometrial cell proliferation and apoptosis is crucial for successful embryo implantation. PTEN (phosphatase and tensin homolog deleted on chromosome 10), a pro-apoptotic factor, is proposed to be one of the signaling proteins through which estrogen and progesterone act to affect cellular homeostasis. Although reports in literature have suggested role of PTEN in regulating endometrial cell proliferation and apoptosis during window of implantation, its involvement in women with unexplained infertility is not clear. In the present study, we examined expression, cellular distribution and activation status of PTEN, cell proliferation, and apoptosis in midsecretory endometrium from women with unexplained infertility as compared to fertile controls.Methods: Endometrial biopsies from infertile (n=11) and fertile women (n=22) were used for immunohistochemical evaluation of PTEN, phospho-PTEN and Ki67. Terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate nick end labeling assay was performed for detection of apoptotic cells.Results: Biopsies from infertile women as compared to fertile controls demonstrated statistically significant: i) decrease in nuclear PTEN (P < 0.001), increase in nuclear phospho-PTEN (P < 0.05), increase in nuclear and cytoplasmic phospho-PTEN/PTEN ratio (P < 0.001 and P < 0.05 respectively) in endometrial stroma, ii) increase in cytoplasmic phospho-PTEN (P < 0.001) and phospho-PTEN/PTEN ratio (P < 0.05) in glandular epithelium (GE), iii) increase in Ki67 labeling in GE (P < 0.01) and stroma (P < 0.05) and, iv) decrease in (P < 0.001) apoptosis.Conclusions: Altered PTEN expression and associated modulation in cellular homeostasis during the implantation window might contribute to mechanism underlying unexplained infertility.

Computed tomography- guided percutaneous core needle biopsy for diagnosis of intathoracic mass lesions: experience at a tertiary care centre of North India

Background: CT guided core needle biopsy is a less invasive method for initial diagnostic workup in the assessment of intrathoracic masses. This study was conducted to evaluate the diagnostic yield of the procedure as well as to demonstrate the spectrum of various disease in our population.Methods: Present study was conducted in a tertiary care hospital for a study period of two years. Patients with intrathoracic mass were included and CT guided biopsies were performed following a protocol. The CT guided biopsies received were examined for histological diagnosis. Immunohistochemistry was carried out where ever routine histopathology was not sufficient for diagnosis. Relevant immunohistochemical panels were applied for lung, mediastinal and pleural tumours according to the histological differential diagnosis. Detailed demographic and clinical profiles along with radiological findings were noted.Results: Total of 138 cases were taken for CT guided FNAC procedure and 123 (89.1%) cases yielded diagnostic biopsy. Lung was the most commonly involved organ followed by mediastinum. Bronchogenic carcinoma was the most common lesion reported in lung and Non-Hodgkin Lymphoma was the most common mediastinal lesion. Lung collapse was most common radiological feature.Conclusions: CT guided percutaneous biopsy is a valuable diagnostic technique providing for early accurate diagnosis and being minimally invasive procedure. Care should be taken while tissue processing and section cutting of intrathoracic biopsies as the biopsies are small and tissue loss should be prevented so that sufficient material is available for immunohistochemistry.

Clinicopathological and immunohistochemical analysis of Sarcomatoid carcinoma of head and neck mucosal region: a retrospective analysis

Background: Sarcomatoid carcinoma is a biphasic tumour comprising both of malignant epithelial and mesenchymal elements derived monoclonally from same stem cells. These are unusual variants of squamous cell carcinoma and constitute less than 1% of the head and neck mucosal tumors. Only few studies have been published and needs more understanding to establish treatment guidelines. The aim of this study was to review the cases of carcinosarcoma arising from mucosal sites of head and neck and study their clinical, histological and Immunohistochemical features.Methods: Retrospective data and slides of histologically proven sarcomatoid carcinoma over a period of thirty -four months between January 2016 - October 2018 were retrieved and evaluated for various clinical and histopathological parameters.Results: Total of 22 cases were included in the study and the mean age of presentation was 58years with male: female ration 2:1. Most of the patients (81.8%) presented with a mass lesion of less than 6 months duration. The most common site was oral cavity (68.1%) followed by larynx (22.7%). Clinical stage was known in eleven cases. One case presented with pulmonary metastasis. Histopathologically, epithelial differentiation was identified in nine cases (41%) on morphology and in thirteen cases could be highlighted by cytokeratin positivity. The Mesenchymal component was arranged in sheets (63.7%) and fascicles (31.8%). Marked anaplasia and brisk mitosis wereseen in 54.5% and 19.3% respectively.  On immunohistochemistry all 22 cases were positive for Vimentin, twenty cases were positive for cytokeratin/EMA and aberrant mesenchymal markers were expressed in 10% of cases.  Follow up was available in eighteen cases out of which fourteen cases died within one year of diagnosis.Conclusions: Diagnosis of sarcomatoid carcinoma is challenging especially on small biopsy because of overlapping features with other spindle cell tumors. Understanding the clinicopathological features facilitates their diagnosis and effective clinical management.

PI3K/Akt/mTOR signaling & its regulator tumour suppressor genes PTEN & LKB1 in human uterine leiomyomas.

Background & objectives: Despite their high occurrence and associated significant level of morbidity manifesting as spectrum of clinical symptoms, the pathogenesis of uterine leiomyomas (ULs) remains unclear. We investigated expression profile of tumour suppressor genes PTEN (phosphatase and tensin homolog deleted on chromosome ten) and LKB1 (liver kinase B1), and key signaling components of P13K (phosphatidylinositol 3-kinase)/Akt (protein kinase B)/mTOR (mammalian target of rapamycin) pathway in leiomyomas and adjacent normal myometrium in women of reproductive age, to explore the possibility of targeting this pathway for future therapeutic implications. Methods: Real time PCR (qPCR) was used to quantify relative gene expression levels of PTEN, Akt1, Akt2, mTOR, LKB1 and VEGFA (vascular endothelial growth factor A) in leiomyoma as compared to adjacent normal myometrium. Immunohistochemistry was subsequently performed to analyze expression of PTEN, phospho-Akt, phospho-mTOR, phospho-S6, LKB1 and VEGFA in leiomyoma and adjacent normal myometrium. Results: Significant upregulation of PTEN (2.52 fold; P=0.03) and LKB1 (3.93 fold; P=0.01), and downregulation of VEGFA (2.95 fold; P=0.01) genes were observed in leiomyoma as compared to normal myometrium. Transcript levels of Akt1, Akt2 and mTOR did not vary significantly between leiomyoma and myometrium. An increased immunoexpression of PTEN (P=0.015) and LKB1 (P<0.001) and decreased expression of VEGFA (P=0.01) was observed in leiomyoma as compared to myometrium. Immunostaining for activated (phosphorylated) Akt, mTOR and S6 was absent or low in majority of leiomyoma and myometrium. Interpretation & conclusions: Upregulation of PTEN and LKB1 in concert with negative or low levels of activated Akt, mTOR and S6 indicates that PI3K/Akt/mTOR pathway may not play a significant role in pathogenesis of leiomyoma.

Expression and clinicopathological significance of antiapoptotis protein survivin in gallbladder cancer.

Background: Clinical significance of survivin (antiapoptosis protein) in gallbladder cancer is not yet established. Aims: This study was performed to assess the expression pattern of survivin in benign and malignant gallbladder lesions using immunohistochemistry (IHC), and to assess its clinicopathological significance. Settings and Design: Prospective study from July 2012 to July 2014 was performed as a part of intramural research project. Materials and Methods: Tissue samples from resected gallbladder for cholelithiasis (n = 27) and carcinoma gallbladder (n = 24) were evaluated for survivin expression by IHC using a scoring system. Their expression was correlated with different clinicopathological parameters. Statistical Analysis: Fisher’s exact test, Student’s t‑test, and Chi‑square test were used as appropriate for data analysis. Kaplan–Meier methods were used to calculate overall and disease‑free survival rates among different groups. Two‑sided P < 0.05 was considered as significant. Results: Benign group (19 females, age [mean ± standard deviation [SD]] 45 ± 14 years) and malignant group (20 females, age [mean ± SD] 48.9 ± 13.4 years) were comparable with respect to menopausal status, presence, size and types of stones. However, survivin expression was significantly higher (66.7%, 95% confidence interval [CI] 24–75) in gallbladder cancer than in cholelithiasis group (33%, CI 46–83), P = 0.025). Its expression did not correlate with gender, age, menopausal status, presence of gallstones or their size, number and type, tumor differentiation, and tumor stage. Conclusions: Significantly higher expression of survivin protein in gallbladder cancer as compared to cholelithiasis group suggests its role in gallbladder carcinogenesis though it may not have prognostic value.

CD3+, CD4+ & CD8+ tumour infiltrating lymphocytes (TILs) are predictors of favourable survival outcome in infiltrating ductal carcinoma of breast.

Background & objectives: Tumour infiltrating lymphocytes (TILs) represent the host immune response against cancer cells associated with good or bad prognosis in different tumour types. This study was undertaken to evaluate the significance of CD3+, CD4+ and CD8+ TILs in breast cancer tissues in relation to clinico-pathological variables and survival outcome. Methods: Immunohistochemistry (IHC) was performed with antibodies against CD3, CD4 and CD8 antigens on formalin-fixed paraffin-embedded tissue sections of 150 breast cancer patients. Intratumoural and stromal TIL counting was performed semiquantitatively. Results: The higher CD3+, CD4+ and CD8+ intratumoural and stromal counts showed independent and direct association with good prognosis. The prognostic predictor value of intratumoural counts was higher than stromal counts. The independent associations of intratumoural and stromal counts became more prominent when adjusted with stage and grade, respectively. Among intratumoural counts, the high (++/+++) CD4+ count (OR=3.85, 95% CI=3.28-16.71, P<0.001) showed the highest survival followed by CD3+ (OR=2.70, 95% CI=1.76-8.30, P=0.001) and CD8+ (OR=2.58, 95% CI=1.55-5.86, pP=0.001) the least when compared to respective low (+) counts. In contrast, among stromal counts, the high CD8+ count (OR=3.13, 95% CI=2.20-9.57, pP<0.001) showed the highest survival followed by CD4+ (OR=3.02, 95% CI=2.07-8.89, 0.001) and CD3+ (OR=2.45, 95% CI=1.53-6.73, 0.002) the least. Interpretation & conclusions: Our results suggest that intratumoural CD4+ and stromal CD8+ counts by immunohistochemistry may serve as an independent prognosticator for favourable outcome in breast cancer.

Prognostic Impact of CD3 Tumor Infiltrating Lymphocytes in Triple-negative Breast Cancer.

Background: Aim of the present study was to evaluate the prognostic significance of CD3+ tumor infiltrating lymphocyte (TILs) in triple-negative breast cancer (TNBC). Methods: Immunohistochemistry was done with antibodies to CD3 TIL, estrogen receptors (ERs), progesterone receptor (PR) and C-erbB2 in tissue sections of 49 TNBC patients. CD3+ intratumoral and stromal TILs were counted in relation to known clinicopathological factors. Results: Intratumoral CD3+ TILs were significantly associated with stage (p = 0.05) with insignificant association with age, menopausal status, family history, grade and lymph node status. Higher counts of stromal CD3+ TILs were significantly associated with stage (p = 0.05), whereas grade, lymph node status, age, menopausal status and family history were insignificant with CD3+ count. The higher CD3 intratumoral and stromal counts both showed significant association with good prognosis (p 0.05). Conclusion: CD3+ TILs may serve as good prognostic marker in TNBC. The results of present study need further validation on larger sample size.

Molecular and phenotypic expression of decorin as modulator of angiogenesis in human potentially malignant oral lesions and oral squamous cell carcinomas.

Background: Decorin is an extracellular matrix, multifunctional small proteoglycan molecule in tumor stroma that has been shown to be modulator of angiogenesis. No clinical data is available so far on decorin expression and survival outcome of oral cancer. Aim: The aim of the present study was to examine molecular and phenotypic expression of two angiogenesis modulators viz. decorin and vascular endothelial growth factor-A (VEGF-A) in human potentially malignant oral lesions (PMOLs) and oral squamous cell carcinomas (OSCC) in relation to clinico-pathological variables and survival outcome. Materials and Methods: Tissue biopsies were obtained from 72 PMOLs, 108 OSCC and 52 healthy controls. The PMOLs included cases of leukoplakias and oral submucous fi brosis. Immunohistochemistry was performed using antibodies against decorin, VEGF-A and CD-31. Messenger-ribonucleic acid (mRNA) expression was analyzed by using real-time polymerase chain reaction. Results: Cytoplasmic staining of decorin was observed in the basal layer of epithelium in 53 (73.61%) cases of PMOLs and in peritumoral stroma in 55 (50.92%) cases of OSCC. None of the cases showed nuclear expression of decorin. Decorin expression both at phenotypic and molecular level was found to be down-regulated from PMOLs to OSCC. Lymph node metastasis and reduced decorin expression independently correlated with overall survival in OSCC. VEGF-A expression had no signifi cant impact on survival outcome. Conclusion: Micro vessel density and VEGF-A expression were signifi cantly associated with reduced decorin expression in tumor stroma suggesting, decorin as angiogenic modulator in OSCC. Down-regulation of decorin expression and the presence of lymph node metastasis were adverse factor independently affecting overall survival in OSCC.

Immunohistochemical expression of MIB-1 and PCNA in precancerous and cancerous lesions of uterine cervix.

BACKGROUND AND OBJECTIVE: The present study was done to analyze the immunoexpression of diagnostic markers (MIB-1: molecular immunology borstel and PCNA: proliferating cell nuclear antigen) in grading cervical intraepithelial lesion (CIN) and squamous cell carcinoma (SCC) in cervix. SETTING AND DESIGN: Total 150 cervical biopsies were divided into four groups respectively; Group I-Normal (n = 32), Group II- CIN (n = 60), Group III- SCC (n = 44), Group IV- CA cervix (n = 14) respectively. MATERIALS AND METHODS: These biopsies were stained with monoclonal antibodies by streptavidin-- biotin method. Mean labeling index was calculated and grading was performed using the I--III scoring system. STATISTICAL ANALYSIS: Findings were correlated with age and menopausal status. Statistical analysis was done by using student sample‘t’ test and analysis of variance (ANOVA) by SPSS 10 package. RESULTS: MIB-1 immunostaining was positive in 112/150 (74.6%) cases and PCNA in 118 /150 (78.6%) cases. Labeling indices showed linear progression from normal to CIN to SCC to cancer lesion. Few cases of low-grade CIN lesion had high proliferative index. A significant positive correlation was found between age and PCNA and MIB-1 values (P < 0.05) when comparison was made for all the cases. CONCLUSION: These markers may be useful in identifying low-grade CIN lesion with high proliferative index. These cases should be kept for follow up studies so that proper intervention can be taken at an early stage. This method is simple and cost effective and can easily be done in formaline-fixed paraffin embedded tissues in a clinical laboratory for grading CIN and SCC lesions in cervix.

Performance characteristics of bisulfite conversion and SYBR green based quantitative PCR for DNA methylation analysis.

Genomic DNA methylation is one of the most important epigenetic modifications in eukaryotes play vital role in development of severe disease like cancer. Many techniques used for assessment of DNA methylation, bisulfite treatment followed by methylation specific polymerase reaction (MSP) are one of them, which introduce conversion of unmethylated cytosine into uracil. The significant level of bisulfite treated DNA degradation results in the failure of methylation detection. Therefore, this step is to be properly controlled to avoid the degradation of DNA. In the present study, an attempt has been made to access the incubation time of DNA with bisulfate treatment at three time points i.e. 2.5, 4 and 16 hrs to get complete conversion of cytosine to uracil. Currently, the experiments were undertaken using oral cancer tissue, with varying incubation time of bisulfite treatment and 2 representative genes viz MGMT and p16 were selected for the quantitative assessment of methylation by real time PCR. Both genes are frequently methylated at promoter region in carcinogenesis. The short term incubation for 4hrs indicated better real time threshold value for p16 and MGMT gene methylation (Ct 25.55, 27.25) and unmethylation (Ct 18.82, 25.84) in tissue whereas it was 28.16, 37.35 and 21.98, 26.19 in blood sample, respectively as compared to other incubation time which shows less degradation of full length DNA.

Hepsin immunohistochemical expression in prostate cancer in relation to Gleason's grade and serum prostate specific antigen.

Background: Hepsin (HPN), a type II trans-membrane serine protease, has been reported to be one of the most up regulated genes in prostate cancer. The aim of the present study was to find out immuno-histochemistry based phenotypic expression of HPN in formalin fixed paraffin embedded sections of prostate cancer compared with that in benign prostatic hyperplasia, in a prospective clinical setting, to know the differential status of HPN expression in benign and malignant prostatic disease. Materials and Methods: Tissue biopsies of histologically proven cases of prostatic cancers (48), benign prostatic hyperplasia (42), benign prostatic hyperplasia with prostatic intraepithelial neoplasia (7) and 4 cases of benign prostatic hyperplasia with prostatitis, were subjected to immunohistochemical staining with HPN antibody by strepavidin biotin method. Results: Hepsin expression was 100% in prostate carcinoma, 11.9% (5/42) in benign prostatic hyperplasia, 57.14% (4/7) in benign prostatic hyperplasia with prostatic intraepithelial neoplasia (PIN), and none in benign prostatic hyperplasia with prostatitis. Hepsin staining showe higher expression in high grade tumor in comparison to low grade tumor. Conclusions: Positive immunohistochemical expression of hepsin in cent percent cases of prostate cancer cases is intriguing, underscoring the significance of hepsin gene expression in prostate cancer.

Immunohistochemical localization of mycobacterium tuberculosis complex antigen with antibody to 38 kDa antigen versus Ziehl Neelsen staining in tissue granulomas of extrapulmonary tuberculosis.

BACKGROUND: Extra-pulmonary tuberculosis often presents a diagnostic challenge because of its diverse clinical manifestations and low yield of acid fast bacilli in tissue sections. AIM: The aim of the present study is immuno-histochemical localization of tuberculous bacilli or their components that persist in the granulomas but have lost the property of staining with acid fast stains and to assess the advantage of immuno-staining over conventional Ziehl Neelsen staining. MATERIAL AND METHODS: Immuno-histochemical staining using species-specific monoclonal anti-body to 38 kDa protein of Mycobacterium tuberculosis complex and Ziehl-Neelsen staining for acid fast bacilli (AFB) was done on 69; 36 cases of confirmed extra-pulmonary tuberculosis and 33 non-tuberculous cases, in archival formalin fixed paraffin embedded tissue sections OBERVATIONS: AFB positivity was observed in only 36.1% of tuberculous granulomas while immuno-histochemical staining was positive in 100% of tuberculous granulomata with zero false positivity and negativity. CONCLUSIONS: The immuno-hiostochemical localization of tuberculous bacilli and their components in tissue sections may be an efficient diagnostic adjunct to conventional ZN staining for the diagnosis of granulomas of extra-pulmonary tuberculosis. The technique is simple, sensitive and specific. It can be standardized and performed by trained technicians in routine laboratory. This will also help in clinical decision-making and in reducing the usual practice of prescribing empirical anti-tubercular treatment based on clinical suspicion alone in the absence of demonstrable evidence of tuberculous infection.