Immunohistochemical analysis of a panel of cancer stem cell markers and potential therapeutic markers in pancreatic ductal adenocarcinoma.

PURPOSE: Pancreatic Ductal Adenocarcinoma (PDAC) is the most common type of pancreatic malignancies. It is known for its aggressive nature and high mortality rate. This calls for an urgent need of new prognostic and therapeutic markers that can be targeted for personalized treatment of the patient. METHODS: Among 142 patients diagnosed with pancreatic cancers at Aga Khan University Hospital, a total of 62 patients were selected based on their confirmed diagnosis of PDAC. Immunohistochemistry was performed on Formalin-Fixed Paraffin-Embedded (FFPE) sections using selected antibodies (CD44, CD133, L1CAM, HER2, PD-L1, EGFR, COX2 and cyclin D1). All the slides were scored independently by two pathologists as per the set criteria. RESULTS: Expression of all cancer stem cell markers was found to be significantly associated with one or more potential therapeutic markers. CD44 expression was significantly associated with HER2 (p = 0.032), COX2 (p = 0.005) and EGFR expression (p = 0.008). CD133 expression also showed significant association with HER2 (p = 0.036), COX2 (p = 0.004) and EGFR expression (p = 0.018). L1CAM expression was found to be associated with expression of COX2 (p = 0.017). None of the proteins markers showed association with overall survival of the patient. On the other hand, among the clinicopathological characteristics, histological differentiation (p = 0.047), lymphovascular invasion (p = 0.021) and perineural invasion (p = 0.014) were found to be significantly associated with patient's overall survival. CONCLUSION: Internationally, this is the first report that assesses the selected panel of cancer stem cell markers and potential therapeutic targets in a single study and evaluates its combined expression. The study clearly demonstrates association between expression of cancer stem cell markers and therapeutic targets hence paves a way for precision medicine for pancreatic cancer patients.

Immunohistochemical Detection and Prognostic Significance of p53, Epidermal Growth Factor Receptor, Murine Double Minute 2, and Isocitrate Dehydrogenase 1 in Glioblastoma Multiforme Patients of Pakistan.

Introduction: Glioblastoma multiforme (GBM) is one of the deadliest cranial tumors occurring in adults. Various biomarkers have been tested for their significance in diagnosis, prognosis, and treatment of GBM. Some well-studied markers in GBM are Isocitrate dehydrogenase 1 (IDH1), Murine double minute 2 (MDM2), Epidermal Growth Factor Receptor (EGFR), and p53. The aim of this study was to investigate the protein expression of these markers in GBM patients of Pakistan. Methods: A total of 102 surgically resected formalin-fixed paraffin-embedded specimens from patients diagnosed and treated at Aga Khan University Hospital were included in this study. Immunohistochemistry (IHC) for IDH1, MDM2, EGFR, and p53 was performed using Dako EnVision System and respective monoclonal antibodies. Survival analysis was performed to check association of markers protein expression with prognosis in GBM patients. Results: There were 73 males and 29 females in this study, with a median age of 49 years at the time diagnosis. Overexpression of molecular markers was as follows: 52% for EGFR, 26% for p53, 72% for IDH1, and 83% for MDM2. We did observe that EGFR was significantly associated with increased age of our patients and with worse survival. Age > 40 years was a predictor for worse prognosis as well. Conclusion: EGFR overexpression and advanced age were worse prognostic indicators.

Hormone receptors AR, ER, PR and growth factor receptor Her-2 expression in oral squamous cell carcinoma: Correlation with overall survival, disease-free survival and 10-year survival in a high-risk population.

Oral squamous cell carcinoma (OSCC) comprises most of head and neck neoplasms and is one of the highest-ranking and lethal cancers in Pakistan due to prevailing mouth habits. Several types of receptors act as prognostic markers and targets for therapy in some cancers, but their application in OSCC is largely unexplored. This study aimed to evaluate the expression of hormonal receptors and Her-2 in OSCC patients and correlate it with 10-year, overall and disease-free survival. To achieve this objective, immunohistochemistry for Her-2, AR, ER and PR was performed on 100 formalin-fixed paraffin-embedded primary OSCC specimens. Receptor expression was correlated with mouth habits and clinicopathological features and patient survival was analyzed using Kaplan-Meier method and Cox regression univariate analysis. We observed that in 100 patients, there were 57 males and 43 females. Immunopositive Her-2 expression was observed in 21% of patients, AR in 13%, ER in 3% and 0% for PR. Patients with betel quid/areca nut mouth habits had significantly absent Her-2 expression (P = 0.035). Also, Her-2 negative patients were also negative for AR expression (P = 0.002). Her-2 positive patients had poor 10-year survival (P = 0.041). A trend of low survival and high recurrence rate was observed in AR positive patients, but this was not significant (P = 0.072). No statistically relevant correlations were seen in the case of ER and PR. In conclusion, Her-2 may be a valuable marker for predicting long-term prognosis of OSCC patients.

Body Mass Index and Diabetes Mellitus May Predict Poorer Overall Survival of Oral Squamous Cell Carcinoma Patients: A Retrospective Cohort From a Tertiary-Care Centre of a Resource-Limited Country.

Introduction: Oral squamous cell carcinoma (OSCC) is one of the highest-ranking cancers among both genders in Pakistan. Obesity is linked to a much higher risk for developing multiple cancer types. Individuals with Diabetes mellitus (DM) face an increased risk for developing oral cancer. Hence, the objective of this study was to identify the effect of obesity and DM on the prognosis of OSCC patients. Methods: This retrospective cohort analysis was conducted on 386 patients diagnosed and treated for OSCC at The Aga Khan University Hospital, Karachi, Pakistan. Patient information was obtained from hospital medical records. Obesity was defined as having a body mass index (BMI) of ⩾25 kg/m2 according to the WHO Asian cut-offs for BMI. Patient BMI was correlated with diabetes status, clinicopathological features and overall survival. Kaplan-Meier survival analysis was performed, along with univariate and multivariate cox regression analysis to test the effect of obesity and diabetes on overall survival. Results: In a set of 386 patients, there were 296 males (76.7%) and 90 females (23.3%). The mean BMI was 24.4 (SD ± 5.25) and 42.7% of patients were found to be obese (⩾25 BMI). 64 patients (16.6%) were diabetic. The risk of death was significantly higher in underweight patients (P = .035) compared with normal weight individuals. Diabetics had a higher mean BMI compared with non-diabetics. However, DM was not a statistically valid predictor of survival. Conclusion: Underweight OSCC patients were at a higher risk of death compared with normal weight OSCC patients.

High CD44 Immunoexpression Correlates with Poor Overall Survival: Assessing the Role of Cancer Stem Cell Markers in Oral Squamous Cell Carcinoma Patients from the High-Risk Population of Pakistan.

Oral squamous cell carcinoma (OSCC) is a top-ranked cancer in the Pakistani population, and patient survival has remained unchanged at ∼50% for several decades. Recent advances have claimed that a subset of tumour cells, called cancer stem cells (CSCs), are responsible for tumour progression, treatment resistance, and metastasis, which leads to a poor prognosis. This study investigated the impact of CSC markers expression on overall survival (OS) and disease-free survival (DFS) of OSCC patients. Materials and Methods. Immunohistochemistry was used to evaluate CD44, CD133, L1CAM, and SOX2 expression in a well-characterized cohort of 100 Pakistani patients with primary treatment naïve OSCC. The immunoreactivity for each marker was correlated with patient clinicopathologic characteristics, oral cancer risk chewing habits, and survival. The minimum follow-up time for all patients was five years, and survival estimates were calculated using the Kaplan-Meier method and Cox proportional hazards model. Results. In this cohort of 100 patients, there were 57 males and 43 females. The median OS and DFS time durations observed were 64 and 52.5 months, respectively. Positive expression for CD44, CD133, L1CAM, and SOX2 was observed in 33%, 23%, 41%, and 63% of patients. High CD44 expression correlated with decreased OS (P=0.047) but did not influence DFS. However, CD133, L1CAM, and SOX2 had no effect on either OS or DFS. Tonsils, nodal involvement, and AJCC stage were independent predictors of worse OS and DFS both. Conclusion. Of the CSC markers investigated here, only CD44 was a predictor for poor OS. CD44 was also associated with advanced AJCC and T stages. Interestingly, CD133 was significantly lower in patients who habitually consumed oral cancer risk factors.

Genetic landscape of pancreatic adenocarcinoma patients: a pilot study from Pakistan.

BACKGROUND: Pancreatic adenocarcinoma is one of the most aggressive malignancies with extremely low survival rate. Studies have shown that the exploration of key genes can provide a basis for targeted treatment of these patients. The genomic architecture of the Pakistani pancreatic adenocarcinoma patients remains unexplored. Keeping the scenario in place, the current study aims to analyse 88 cancer related genes in Pakistani pancreatic adenocarcinoma patients in order to elucidate candidate gene(s) for targeted molecular therapy. METHODS AND RESULTS: A total 18 patients were included in the study initially and FFPE tumor samples were obtained. After confirmation of diagnosis and appropriate tumor content, DNA was extracted. Based on the quality and quantity of the extracted DNA, six pancreatic adenocarcinoma tumor samples were selected. Following to this, all the samples were subjected to targeted sequencing (Axen Cancer Panel 1). Variant detection was done and clinical significance of identified variants was assessed using ClinVar database. Targeted sequencing of tumor samples revealed a total of 29 alterations in the coding region of various genes. Among these five pathogenic variants were found in KRAS, BRCA1, TP53 and APC genes. CONCLUSION: This is the first study that explores genes involved in pancreatic adenocarcinoma from the Pakistani population. Results obtained from the pilot study can guide us about the key genetic players in the Pakistani pancreatic adenocarcinoma population. This can lead to our better understanding of the molecular targeted therapies for these patients and designing future researches on larger sample size.