Predominance of MGMT promoter methylation among Pakistani glioblastoma patients.

BACKGROUND: Glioblastoma multiforme (GBM), the most prevalent subgroup of neuroepithelial tumors, is characterized by dismal overall survival (OS). Several studies have linked O6-methylguanine-DNA-methyltransferase (MGMT) promoter methylation to OS in GBM patients. However, MGMT methylation frequencies vary geographically and across ethnicities, with limited data for South Asian populations, including Pakistan. This study aimed to analyze MGMT promoter methylation in Pakistani GBM patients. METHODS: Consecutive primary GBM patients diagnosed ≥ 18 years-of-age, with no prior chemotherapy or radiotherapy history, were retrospectively selected. DNA was isolated from formalin-fixed-paraffin-embedded tissues. MGMT promoter methylation was analyzed using methylation-specific PCR. Clinical, pathological, and treatment data were assessed using Fisher's exact/Chi-squared tests. OS was calculated using Kaplan-Meier analysis in SPSS 27.0.1. RESULTS: The study included 48 GBM patients, comprising 38 (79.2%) males and 10 (20.8%) females. The median diagnosis age was 49.5 years (range 18-70). MGMT methylation was observed in 87.5% (42/48) of all cases. Patients with MGMT methylation undergoing radiotherapy or radiotherapy plus chemotherapy exhibited significantly improved median OS of 7.2 months (95% CI, 3.7-10.7; P < 0.001) and 16.9 months (95% CI, 15.9-17.9; P < 0.001), respectively, compared to those undergoing surgical resection only (OS: 2.2 months, 95% CI, 0.8-3.6). CONCLUSION: This is the first comprehensive study highlighting a predominance of MGMT methylation in Pakistani GBM patients. Furthermore, our findings underscore the association of MGMT methylation with improved OS across diverse treatment modalities. Larger studies are imperative to validate our findings for better management of Pakistani GBM patients.

Contribution of constitutional BRCA1 promoter methylation to early-onset and familial breast cancer patients from Pakistan.

PURPOSE: Constitutional BRCA1 promoter methylation has been identified as a potential risk factor for breast cancer (BC) in the Caucasian population. However, this data is lacking for BC patients of Asian origin. Therefore, we assessed the contribution of constitutional BRCA1 promoter methylation in Pakistani BC patients. METHODS: A total of 385 BRCA1/2-negative index BC patients (197 early-onset BC (≤ 30 years), 152 familial BC, 17 familial BC and ovarian cancer, 19 male BC) and 107 healthy controls were screened for the constitutional BRCA1 promoter methylation by methylation-sensitive high-resolution melting assay. Overall, 131 patients displayed triple-negative BC (TNBC) and 254 non-TNBC phenotypes. The prevalence of BRCA1 promoter methylation was calculated based on clinicopathological characteristics using univariable and multivariable logistic regression models. RESULTS: Constitutional BRCA1 promoter methylation was identified in 19.5% (75/385) of BC patients and 13.1% (14/107) of controls. The frequency of methylation was higher in early-onset BC (23.4% vs. 13.1%, P = 0.035) and TNBC patients (29.0% vs. 13.1%, P = 0.004) compared to controls. Methylation was also more prevalent in patients with high-grade than low-grade tumors (21.7% vs. 12.2%, P = 0.034) and progesterone receptor (PR)-negative than PR-positive tumors (26.0% vs. 13.9%, P = 0.004). Constitutional BRCA1 promoter methylation remained independently associated with TNBC phenotype (odds ratio 1.99; 95% CI 1.12-3.54; P = 0.02) after adjusting for BC diagnosis age, tumor grade, ER, and PR status. CONCLUSION: Constitutional BRCA1 promoter methylation is associated with TNBC and can serve as a non-invasive blood-based biomarker for Pakistani TNBC patients.

A digital score of tumour-associated stroma infiltrating lymphocytes predicts survival in head and neck squamous cell carcinoma.

The infiltration of T-lymphocytes in the stroma and tumour is an indication of an effective immune response against the tumour, resulting in better survival. In this study, our aim was to explore the prognostic significance of tumour-associated stroma infiltrating lymphocytes (TASILs) in head and neck squamous cell carcinoma (HNSCC) through an AI-based automated method. A deep learning-based automated method was employed to segment tumour, tumour-associated stroma, and lymphocytes in digitally scanned whole slide images of HNSCC tissue slides. The spatial patterns of lymphocytes and tumour-associated stroma were digitally quantified to compute the tumour-associated stroma infiltrating lymphocytes score (TASIL-score). Finally, the prognostic significance of the TASIL-score for disease-specific and disease-free survival was investigated using the Cox proportional hazard analysis. Three different cohorts of haematoxylin and eosin (H&E)-stained tissue slides of HNSCC cases (n = 537 in total) were studied, including publicly available TCGA head and neck cancer cases. The TASIL-score carries prognostic significance (p = 0.002) for disease-specific survival of HNSCC patients. The TASIL-score also shows a better separation between low- and high-risk patients compared with the manual tumour-infiltrating lymphocytes (TILs) scoring by pathologists for both disease-specific and disease-free survival. A positive correlation of TASIL-score with molecular estimates of CD8+ T cells was also found, which is in line with existing findings. To the best of our knowledge, this is the first study to automate the quantification of TASILs from routine H&E slides of head and neck cancer. Our TASIL-score-based findings are aligned with the clinical knowledge, with the added advantages of objectivity, reproducibility, and strong prognostic value. Although we validated our method on three different cohorts (n = 537 cases in total), a comprehensive evaluation on large multicentric cohorts is required before the proposed digital score can be adopted in clinical practice. © 2021 The Authors. The Journal of Pathology published by John Wiley & Sons Ltd on behalf of The Pathological Society of Great Britain and Ireland.

Prognostic significance of minor high grade component in non-invasive papillary urothelial carcinoma of urinary bladder: (A study of 273 consecutive cases over a period of 3 years).

PURPOSE: Our study aimed to determine the prognostic significance of minor high-grade components (HGC) in non-invasive papillary urothelial carcinomas compared with pure low-grade and high-grade tumors. MATERIAL AND METHODS: We retrospectively retrieved 273 in-house cases of non-invasive papillary urothelial carcinomas (pTa) from 2016 to 2018 for which follow up data was available in hospital archives. We stratified our data into four main groups (G). G1, pure low-grade (n = 164); G2, HGC ≤5 % (n = 17); G3, HGC >5 % to ≤25 % (n = 14); and G4, pure high-grade (n = 78). Prognosis was assessed in terms of recurrence, grade and stage of progression, metastasis, and death. The mean follow up duration was 34.72 ± 20 months (range 20-60 months). RESULTS: All four groups showed no difference in tumor recurrence (G1 81.7 %, G2 88.2 %, G3 92.9 %, G4 92.3 % p-value 0.183). In terms of grade progression, there was no significant difference in G2 35.3 % and G3 35.7 % and both groups showed worst prognosis compared to G1 16.5 % p-value 0.04. Regarding stage progression (G1 6.7 %, G2 23.5 %, G3 28.6 %, G4 41% p-value 0.001), metastasis (G1 5.5 %, G2 5.9 %, G3 7.1 %, G4 17.9 % p-value 0.01) and death (G1 4.3 %, G2 5.9 %, G3 7.1 %, G4 15.4 % p-value 0.02) there was no significant difference in G2 and G3 and both groups showed worst prognosis than G1 and better than G4. CONCLUSION: Urothelial carcinomas with minor high-grade component ≤25 % behaved worst than pure low grade and better than pure high grade and should be treated as distinct grade entity.

Application Of New WHO Classification On Malignancies Of Sinonasal Tract: Correlation With Epidemiology And Survival In Pakistani Population.

Objectives: To analyse the clinicopathological characteristics of sinonasal malignancies in the light of the updates regarding head and neck tumours. METHODS: The retrospective study was conducted at the Shaukat Khanum Memorial Cancer Hospital and Research Centre, Lahore, Pakistan, and comprised data of patients diagnosed with primary malignant tumours of the sinonasal tract between 2015 and 2020. Slides related to biopsies and resection specimens were retrieved from the institutional database and reviewed by two pathologists. Follow-up data was also obtained. Data was analysed using SPSS 20. RESULTS: Of the 245 samples, 144(58.7%) were epithelial tumours, 46(18.7%) neuroectodermal tumours, 41(16.7%) haematolymphoid tumours and 14(5.7%) were malignant soft tissue tumours. A heavy reliance was placed on immunohistochemical stains to diagnose poorly-differentiated tumours. Survival was dismal, especially with early and frequent spread to the brain (33.3% in cases of Sinonasal Undifferentiated Carcinoma). CONCLUSIONS: A wide array of sinonasal malignancies was seen. Updated knowledge of the malignancies prevalent in the region is imperative for timely diagnosis and treatment.

Cytological and histopathological correlation of thyroid lesions.

OBJECTIVE: To determine accuracy of cytological diagnosis in comparison with the corresponding histopathological diagnosis of thyroid lesions. METHODS: The retrospective study was conducted at the Shaukat Khanum Memorial Cancer Hospital, Lahore, Pakistan, and comprised data from January to December 2017 of all in-patient cases of thyroid cytology with their histopathological diagnosis. Both Haematoxylin and Eosin stain slides and cytological smears were reviewed. True negative, true positive, false negative and false positive cases were marked using the criteria defined in Table-1. RESULTS: Of the total 36 cases, 5(13.9%) were non-diagnostic or unsatisfactory for cytological assessment. Cytological diagnosis achieved sensitivity of 82.3%, specificity 64.3%, positive predictive value 73.6%, negative predictive value 75%, false positive rate 35.7% and false negative rate 17.6%. The diagnostic accuracy of cytological diagnosis was 63.9%. CONCLUSIONS: There was significant cytological and histopathological concordance of thyroid lesions.

PD-L1 expression in sebaceous carcinomas.

BACKGROUND: Traditional systemic treatments for unresectable, recurrent, and/or advanced sebaceous carcinoma (SC) are ineffective. Tumoral immune microenvironment characterization is essential for considering immune checkpoint inhibitors as a treatment option. METHODS: A total of 173 resected SCs were reviewed. Clinical information, lesion size, and location were collected. Microscopic examination documented histopathologic features and expression of immunohistochemical markers PD-L1 and CD8. PD-L1 percentage was assessed amongst tumor (PD-L1 + Tu) and immune infiltrating cells (PD-L1 + Inf). Each case was attributed a combined positive score (CPS) following Head and Neck squamous cell carcinoma recommendations. PD-L1 expression was evaluated according to clinicopathologic parameters. Human Papilloma Virus presence (HPV) was analyzed using PCR microarray scanning. RESULTS: A therapeutically relevant CPS was seen in 51.4% of cases. Higher PD-L1 + Tu, PD-L1 + Inf, and CPSs were positively associated with greater lesion size and an extraocular location. No association was seen with patient age or gender. 9.2% of SCs showed PD-L1 + Tu ≥ 1, while 52.0% showed PD-L1 + Inf ≥ 1. A higher CD8 + T-lymphocyte density was significantly associated with a higher CPS, PD-L1 + Tu, and PD-L1 + Inf. Tumor-associated T-cell infiltrate's density was higher along tumor periphery. HPV-16, HPV-43, HPV-52, and HPV-66 were detected in 8.4% of SCs. There was no significant association between HPV status, PD-L1 expression, and CPS. A significant number of SCs express PD-L1 at therapeutic levels. Nevertheless, PD-L1 expression shows a higher intertumoral heterogeneity, in extraocular than in biologically distinct periocular cases. CONCLUSION: Our data support the need for large-scale prospective studies evaluating anti-PD-L1 immunotherapy mainly in extraocular SC treatment.

Prognostic outcomes of treatment naïve oral tongue squamous cell carcinoma (OTSCC): a comprehensive analysis of 14 years.

OBJECTIVES: To analyze the factors predicting survival outcomes in treatment naïve oral tongue squamous cell carcinoma (OTSCC). MATERIALS AND METHODS: A comprehensive review of 531 oral tongue carcinoma patients treated with upfront surgery followed by adjuvant radiotherapy or chemoradiotherapy was conducted from 2004-2018. RESULTS: The mean age of presentation was 53 years (11-86) with a male to female ratio of 1.3:1. The associated risk factors were smoking (21%), betel nut (16%), naswar (9%) and alcohol (1%). Most of the cases were either well (45.1%) or moderately (46.2%) differentiated. Surgery was performed in 164 patients alone while 368 were treated with surgery in combination with adjuvant modalities. Overall (OS) and disease free survival (DFS) were 66 and 71%, respectively, with a median follow up of 2.5 years. Cox regression analysis showed nodal positivity, increased depth of invasion (DOI) and higher lymph node ratio (LNR) as significant prognosticators impacting OS and DSS. CONCLUSION: Nodal volume, DOI and LNR are the most consistent predictors of poor outcome in OTSCC. Nodal positivity, depth of invasion > 5 mm and lymph node ratio > 0.04 adversely affect OS and DSS.

NKX3.1 a useful marker for mesenchymal chondrosarcoma: An immunohistochemical study.

INTRODUCTION: Mesenchymal chondrosarcoma is a rare subtype of chondrosarcoma. The tumor has a characteristic bimorphic pattern with areas of poorly differentiated small round cell component and interspersed islands of well differentiated hyaline cartilage. Histological diagnosis of mesenchymal chondrosarcoma is very challenging especially in small biopsies when tumor presents with little cartilaginous component. In such cases, it is very difficult to distinguish mesenchymal chondrosarcoma from other round blue cell tumors like Ewing's sarcoma, rhabdomyosarcoma, small cell osteosarcoma and desmoplastic round blue cell tumor. Immunohistochemically, mesenchymal chondrosarcoma stains positive for NKX2.2, CD99, S100 and SOX9. This immunoprofile is non-specific and overlaps with other round blue cell tumors. Till recently, there was no reliable immunohistochemical marker to differentiate mesenchymal chondrosarcoma from other round blue cell tumors. NKX3.1, though widely used as a diagnostic biomarker for prostatic adenocarcinoma, has been recently proposed by Yoshida et al. (2020) as a unique marker of mesenchymal chondrosarcoma and EWSR1-NFATC2 sarcoma. OBJECTIVE: The aim of our study was to further explore utility of NKX3.1 as a diagnostic marker of mesenchymal chondrosarcoma. MATERIAL & METHODS: We applied NKX3.1 immunohistochemistry to 21 cases of mesenchymal chondrosarcoma and 32 cases of other round blue cell tumors. RESULTS: 14 out of 21 cases (66.7%) of mesenchymal chondrosarcoma stained positive for NKX3.1 with nuclear expression in small round component. Cartilaginous component was predominantly negative. All other round blue cell tumors showed negative results. CONCLUSION: Based on our study results we suggest that NKX3.1 is a useful immunohistochemical marker in differentiating mesenchymal chondrosarcoma from its histological mimics.

Clinical, microscopic and molecular presentation in pediatric versus adult old world cutaneous Leishmaniasis.

BACKGROUND: Cutaneous leishmaniasis (CL) is an emerging uncontrolled tropical parasitic disease in endemic and nonendemic areas with a high prevalence in the pediatric age group. METHOD: A total of 382 individuals from Lebanon, Saudi Arabia, Pakistan, and Syria diagnosed with CL by punch biopsy/scrapings were grouped into adults (>18 years) and pediatrics (≤18 years). Data recorded included clinical features [number, location, type, size, and extensiveness (size larger than 3 cm, more than 5 lesions per patient, lesion present for more than 12 months, special types, disfiguring lesion or closeness to vital sensory organs) of lesions] and microscopic findings [Ridley's Parasitic Index and Ridley's Pattern]. In addition, molecular confirmation and speciation were performed. RESULTS: In comparison with adults, patients in the pediatric group (n = 158, 41.4%) showed significantly higher number of lesions, more facial involvement, and more extensive disease (P < .05). Microscopically, a more advanced Ridley's pattern was observed. The other variables did not show statistical difference between the two groups. CONCLUSION: Historically, CL has been known to be a neglected tropical disease of poverty and pediatric predilection. In our pediatric group, CL manifests with more extensive disease clinically mirrored by more advanced lesions microscopically.

Prevalence and spectrum of MLH1, MSH2, and MSH6 pathogenic germline variants in Pakistani colorectal cancer patients.

BACKGROUND: Pathogenic germline variants in MLH1, MSH2 and MSH6 genes account for the majority of Lynch syndrome (LS). In this first report from Pakistan, we investigated the prevalence of pathogenic MLH1/MSH2/MSH6 variants in colorectal cancer (CRC) patients. METHODS: Consecutive cases (n = 212) were recruited at the Shaukat Khanum Memorial Cancer Hospital and Research Centre (SKMCH&RC), between November 2007 to March 2011. Patients with a family history of > 3 or 2 HNPCC-associated cancers were classified as HNPCC (n = 9) or suspected-HNPCC (n = 20), respectively (group 1; n = 29). Cases with no family history were designated as non-HNPCC (group 2; n = 183). MLH1/MSH2/MSH6 genes were comprehensively screened in group 1. Pathogenic/likely pathogenic variants identified in group 1 were subsequently evaluated in group 2. RESULTS: Eight distinct pathogenic/likely pathogenic MLH1/MSH2 variants were found in group 1 (10/29; 34.5%), belonging to HNPCC (5/9; 55.6%) and suspected-HNPCC (5/20; 25%) families and in group 2 (2/183; 1.1%) belonging to non-HNPCC. Overall, three recurrent variants (MSH2 c.943-1G > C, MLH1 c.1358dup and c.2041G > A) accounted for 58.3% (7/12) of all families harboring pathogenic/likely pathogenic MLH1/MSH2 variants. Pathogenic MSH6 variants were not detected. CONCLUSION: Pathogenic/likely pathogenic MLH1/MSH2 variants account for a substantial proportion of CRC patients with HNPCC/suspected-HNPCC in Pakistan. Our findings suggest that HNPCC/suspected-HNPCC families should be tested for these recurrent variants prior to comprehensive gene screening in this population.

Spectrum and prevalence of BRCA1/2 germline mutations in Pakistani breast cancer patients: results from a large comprehensive study.

BACKGROUND: Pathogenic germline mutations in BRCA1 and BRCA2 (BRCA1/2) account for the majority of hereditary breast and/or ovarian cancers worldwide. To refine the spectrum of BRCA1/2 mutations and to accurately estimate the prevalence of mutation in the Pakistani population, we studied 539 breast cancer patients selected for family history and age of diagnosis. METHODS: Comprehensive screening for BRCA1/2 germline mutations was performed using state-of-the-art technologies. RESULTS: A total of 133 deleterious mutations were identified in 539 families (24.7%), comprising 110 in BRCA1 and 23 in BRCA2. The prevalence of BRCA1/2 small-range mutations and large genomic rearrangements was 55.4% (36/65) for families with breast and ovarian cancer, 27.4% (67/244) for families with two or more cases of breast cancer, 18.5% (5/27) for families with male breast cancer, and 12.3% (25/203) for families with a single case of early-onset breast cancer. Nine mutations were specific to the Pakistani population. Eighteen mutations in BRCA1 and three in BRCA2 were recurrent and accounted for 68.2% (75/110) and 34.8% (8/23) of all identified mutations in BRCA1 and BRCA2, respectively. Most of these mutations were exclusive to a specific ethnic group and may result from founder effects. CONCLUSIONS: Our findings show that BRCA1/2 mutations account for one in four cases of hereditary breast/ovarian cancer, one in five cases of male breast cancer, and one in eight cases of early-onset breast cancer in Pakistan. Our study suggests genetic testing of an extended panel of 21 recurrent BRCA1/2 mutations for appropriately selected patients and their families in Pakistan.

Diagnostic accuracy of glypican-3 in differentiating hepatocellular carcinoma from metastatic liver tumours.

OBJECTIVE: To determine the diagnostic accuracy of Glypican-3 in differentiating hepatocellular carcinoma from metastatic liver tumours while taking histopathology as the gold standard.. METHODS: The cross-sectional study was conducted at Shaukat Khanum Memorial Cancer Hospital, Lahore, Pakistan, from January 1 to June 30, 2016, and comprised cases of malignant liver tumours. Samples were collected from the pathology department. Glypican-3was applied on them. Tumours were classified as positive when they showed >5% positivity and negative when showing <5% positivity. Frequencies and percentages of cases showing GPC3 positivity and negativity along with frequency and percentages of hepatocellular carcinoma and metastatic tumours were calculated. RESULTS: Of the 240 patients, 143(59.58%) were males and 97 (40.42%) were females. Overall mean age was 54.65 ± 13.46 years. On histopathology, 134 cases were hepatocellular carcinoma (55.83%) and 106 (44. 17%) cases turned out to be metastatic carcinoma. Glypican-3staining was positive in 116 (48.33%) cases and negative in 124(51.67%). Sensitivity was 82%, Specificity 94.33%, positive predictive value 94.82% and negative predictive value 80.64%. Diagnostic accuracy was 87.5%. CONCLUSIONS: Glypican-3 was found to be a highly sensitive and specific Immunohistochemistry stain distinguishing hepatocellular carcinoma from the clear majority of metastatic carcinomas of the liver.

Contribution of BRCA1 large genomic rearrangements to early-onset and familial breast/ovarian cancer in Pakistan.

BACKGROUND: Germline mutations in BRCA1 and BRCA2 (BRCA1/2) account for the majority of hereditary breast and/or ovarian cancers. Pakistan has one of the highest rates of breast cancer incidence in Asia, where BRCA1/2 small-range mutations account for 17% of early-onset and familial breast/ovarian cancer patients. We report the first study from Pakistan evaluating the prevalence of BRCA1/2 large genomic rearrangements (LGRs) in breast and/or ovarian cancer patients who do not harbor small-range BRCA1/2 mutations. MATERIALS AND METHODS: Both BRCA1/2 genes were comprehensively screened for LGRs using multiplex ligation-dependent probe amplification in 120 BRCA1/2 small-range mutations negative early-onset or familial breast/ovarian cancer patients from Pakistan (Group 1). The breakpoints were characterized by long-range PCR- and DNA-sequencing analyses. An additional cohort of 445 BRCA1/2 negative high-risk patients (Group 2) was analyzed for the presence of LGRs identified in Group 1. RESULTS: Three different BRCA1 LGRs were identified in Group 1 (4/120; 3.3%), two of these were novel. Exon 1-2 deletion was observed in two unrelated patients: an early-onset breast cancer patient and another bilateral breast cancer patient from a hereditary breast cancer (HBC) family. Novel exon 20-21 deletion was detected in a 29-year-old breast cancer patient from a HBC family. Another novel exon 21-24 deletion was identified in a breast-ovarian cancer patient from a hereditary breast and ovarian cancer family. The breakpoints of all deletions were characterized. Screening of the 445 patients in Group 2 for the three LGRs revealed ten additional patients harboring exon 1-2 deletion or exon 21-24 deletion (10/445; 2.2%). No BRCA2 LGRs were identified. CONCLUSIONS: LGRs in BRCA1 are found with a considerable frequency in Pakistani breast/ovarian cancer cases. Our findings suggest that BRCA1 exons 1-2 deletion and exons 21-24 deletion should be included in the recurrent BRCA1/2 mutations panel for genetic testing of high-risk Pakistani breast/ovarian cancer patients.

High prevalence and predominance of BRCA1 germline mutations in Pakistani triple-negative breast cancer patients.

BACKGROUND: Women harboring BRCA1/2 germline mutations have high lifetime risk of developing breast/ovarian cancer. The recommendation to pursue BRCA1/2 testing is based on patient's family history of breast/ovarian cancer, age of disease-onset and/or pathologic parameters of breast tumors. Here, we investigated if diagnosis of triple-negative breast cancer (TNBC) independently increases risk of carrying a BRCA1/2 mutation in Pakistan. METHODS: Five hundred and twenty-three breast cancer patients including 237 diagnosed ≤ 30 years of age and 286 with a family history of breast/ovarian cancer were screened for BRCA1/2 small-range mutations and large genomic rearrangements. Immunohistochemical analyses were performed at one center. Univariate and multiple logistic regression models were used to investigate possible differences in prevalence of BRCA1/2 mutations according to patient and tumor characteristics. RESULTS: Thirty-seven percent of patients presented with TNBC. The prevalence of BRCA1 mutations was higher in patients with TNBC than non-TNBC (37 % vs. 10 %, P < 0.0001). 1 % of TNBC patients were observed to have BRCA2 mutations. Subgroup analyses revealed a larger proportion of BRCA1 mutations in TNBC than non-TNBC among patients 1) diagnosed at early-age with no family history of breast/ovarian cancer (14 % vs. 5 %, P = 0.03), 2) diagnosed at early-age irrespective of family history (28 % vs. 11 %, P = 0.0003), 3) had a family history of breast cancer (49 % vs. 12 %, P < 0.0001), and 4) those with family history of breast and ovarian cancer (81 % vs. 28 %, P = 0.0005). TNBC patients harboring BRCA1 mutations were diagnosed at a later age than non-carriers (median age at diagnosis: 30 years (range 22-53) vs. 28 years (range 18-67), P = 0.002). The association between TNBC status and presence of BRCA1 mutations was independent of the simultaneous consideration of family phenotype, tumor histology and grade in a multiple logistic regression model (Ratio of the probability of carrying BRCA1/2 mutations for TNBC vs. non-TNBC 4.23; 95 % CI 2.50-7.14; P < 0.0001). CONCLUSION: Genetic BRCA1 testing should be considered for Pakistani women diagnosed with TNBC.

A novel deleterious c.2656G>T MSH2 germline mutation in a Pakistani family with a phenotypic overlap of hereditary breast and ovarian cancer and Lynch syndrome.

BACKGROUND: Hereditary breast and ovarian cancer syndrome (HBOC) and Lynch syndrome (LS) account for a significant proportion of inherited gynecologic malignancies, mainly caused by pathogenic germline mutations in the BRCA1 and BRCA2 genes or in mismatch repair (MMR) genes, such as MLH1 and MSH2. Women harboring deleterious mutations in these genes have increased life-time risks of developing a number of malignancies including ovarian cancer. Since there is a phenotypic overlap of HBOC and LS, timely identification of individuals at-risk of a particular syndrome is crucial in order to optimize cancer risk management. CASE PRESENTATION: We report a novel pathogenic MSH2 mutation, c.2656G > T, which was identified in a 67-year-old female patient with breast cancer, who had previously tested negative for a deleterious mutation in the breast cancer susceptibility genes BRCA1, BRCA2, CHEK2 or RAD51C. The patient reported a personal history of endometrial cancer diagnosed at age 48, and a strong family history of breast and ovarian cancer, as well as several other malignancies within the spectrum of LS. The novel mutation was also found in the index patient's daughter and a niece, who were diagnosed with endometrial and ovarian cancer, respectively. Breast and endometrial tumors from c.2656G > T mutation carriers showed loss of MSH2 and MSH6 protein expression. The mutation was absent in the control population. CONCLUSIONS: Our finding suggests that testing for MMR genes may be of benefit to BRCA1/2 negative families with overlapping HBOC and LS phenotype in Pakistan. It is clinically significant to identify individuals harboring mutations in genes linked with a particular syndrome so that they can benefit from targeted life-saving cancer surveillance and preventive strategies.

Fine-Needle Aspiration Cytology in the Diagnosis of Pancreatic Neuroendocrine Tumors: A Single-Center Experience of 25 Cases.

OBJECTIVE: Pancreatic neuroendocrine tumors (Pan NETs) are rare but are being increasingly diagnosed. The objective of this retrospective study was to share our experience of fine-needle aspiration (FNA), including endoscopic ultrasound-guided FNA with rapid on-site evaluation (ROSE), with the use of immunohistochemical (IHC) markers in the diagnosis of Pan NET. STUDY DESIGN: A total of 25 cases of Pan NET diagnosed on pancreatic FNA between 2008 and 2013 were identified from our hospital database. Clinical history, radiology, cytomorphological features, and IHC performed were reviewed. RESULTS: The mean age of our patient group was 52 years; 15/25 were male. Most presented with abdominal pain and the majority of the lesions were in the pancreatic body, the largest being 14 cm in size. Based on the 2010 World Health Organization criteria, cases were further graded as follows: 21 cases were grade 1, 2 cases were grade 2 and 2 cases were grade 3. Proliferation marker Ki-67 was utilized in 6 cases for definitive grading. Of the 25 cases, 23 were diagnosed as nonfunctional while 2 were functional; 1 patient had MEN-1 syndrome and 1 had von Recklinghausen's syndrome. CONCLUSIONS: Our data suggests that FNA, with ROSE and IHC markers, is highly sensitive and specific for diagnosing Pan NET.

Patterns of cancer cell sphere formation in primary cultures of human oral tongue squamous cell carcinoma and neck nodes.

Recently a sub-population of cells with stem cell characteristics, reported to be associated with initiation, growth, spread and recurrence, has been identified in several solid tumors including oral tongue squamous cell carcinoma (OTSCC). The aim of our pilot study was to isolate CD44+ cancer stem cells from primary cultures of OTSCC and neck node Level I (node-I) biopsies, grow cell spheres and observe their characteristics in primary cultures. Parallel cultures of hyperplastic lesions of tongue (non-cancer) were set up as a control. Immunohistochemistry was used to detect CD44/CD24 expression and magnetic activated cell sorting to isolate CD44+ cell populations followed by primary cell culturing. Both OTSCC and node-I biopsies produced floating spheres in suspension, however those grown in hyperplastic and node-I primary cultures did not exhibit self-renewal properties. Lymph node metastatic OTSCC, express higher CD44/CD24 levels, produce cancer cell spheres in larger number and rapidly (24 hours) compared to node negative OTSCC (1 week) and non-cancer specimens (3 weeks). In addition, metastatic OTSCC have the capacity for proliferation for up to three generations in primary culture. This in vitro system will be used to study cancer stem cell behavior, therapeutic drug screening and optimization of radiation dose for elimination of resistant cancer cells.