Significant Association of PD-L1 With CD44 Expression and Patient Survival: Avenues for Immunotherapy and Cancer Stem Cells Downregulation in Pancreatic Cancers.

Background: Pancreatic cancers are known for their aggressive nature. This aggressiveness may be attributed to the presence of cancer stem cells (CSCs), which promote relapse, metastasis, and resistance to chemotherapy. Targeting CSCs is essential to reverse this aggressiveness in pancreatic malignancies. Literature highlights the association of PD-L1 expression with CSCs in various cancers, suggesting immunotherapy as a promising therapeutic approach. This study is aimed at investigating the potential of immunotherapy in pancreatic cancers by examining its association with selected CSC marker expression. Method: A retrospective cohort study was conducted involving 56 patients with confirmed diagnoses of pancreatic cancers at Aga Khan University Hospital from January 2015 to October 2022. After exclusions, based on refusal to provide consent or incomplete follow-up data, 38 patients were enrolled in the study. Immunohistochemistry was performed on formalin-fixed paraffin-embedded (FFPE) tumor tissue samples to assess the expression of CSC markers (CD133, CD44, and L1CAM) and immune checkpoint inhibitor marker (PD-L1). Statistical analysis was employed to determine associations between marker expression, clinical factors, and overall survival. Results: The study revealed that 86.8% of pancreatic cancer cases exhibited positive PD-L1 expression. Moreover, a significant association of PD-L1 expression was observed with the presence of CD44 protein (p = 0.030), as well as with the overall survival of patients (p = 0.023). Conclusion: Our findings show a significant association of PD-L1 with CD44 marker expression as well as patient survival. This research shows the potential to serve as the foundation for investigating the efficacy of immunotherapy in reducing CD44-expressing CSCs in pancreatic cancer, potentially enhancing patient outcomes.

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.

Germline Mutation Analysis in Sporadic Breast Cancer Cases With Clinical Correlations.

Demographics for breast cancers vary widely among nations. The frequency of germline mutations in breast cancers, which reflects the hereditary cases, has not been investigated adequately and accurately in highly-consanguineous Pakistani population. In the present discovery case series, germ-line mutations in twenty-seven breast cancer candidate genes were investigated in eighty-four sporadic breast cancer patients along with the clinical correlations. The germ-line variants were also assessed in two healthy gender-matched controls. The clinico-pathological features were evaluated by descriptive analysis and Pearson χ2 test (with significant p-value <0.05). The most frequent parameters associated with hereditary cancer cases are age and ethnicity. Therefore, the analyses were stratified on the basis of age (≤40 years vs. >40 years) and ethnicity. The breast cancer gene panel assay was carried out by BROCA, which is a genomic capture, massively parallel next generation sequencing assay on Illumina Hiseq2000 with 100bp read lengths. Copy number variations were determined by partially-mapped read algorithm. Once the mutation was identified, it was validated by Sanger sequencing. The ethnic analysis stratified on the basis of age showed that the frequency of breast cancer at young age (≤40 years) was higher in Sindhis (n = 12/19; 64%) in contrast to patients in other ethnic groups. Majority of the patients had stage III (38.1%), grade III (50%), tumor size 2-5 cm (54.8%), and invasive ductal carcinoma (81%). Overall, the analysis revealed germ-line mutations in 11.9% of the patients, which was not significantly associated with younger age or any particular ethnicity. The mutational spectrum was restricted to three genes: BRCA1, BRCA2, and TP53. The identified mutations consist of seven novel germ-line mutations, while three mutations have been reported previously. All the mutations are predicted to result in protein truncation. No mutations were identified in the remaining twenty-four candidate breast cancer genes. The present study provides the framework for the development of hereditary-based preventive and treatment strategies against breast cancers in Pakistani population.

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.

Absence of Glutathione S-Transferase Theta 1 Gene Is Significantly Associated With Breast Cancer Susceptibility in Pakistani Population and Poor Overall Survival in Breast Cancer Patients: A Case-Control and Case Series Analysis.

PURPOSE: Deletion of Glutathione S-Transferase Theta 1 (GSTT1) encoding gene is implicated in breast cancer susceptibility, clinical outcomes, and survival. Contradictory results have been reported in different studies. The present investigation based on a representative Pakistani population evaluated the GSTT1-absent genotype in breast cancer risk and prognosis. METHODS: A prospective study comprising case-control analysis and case series analysis components was designed. Peripheral blood samples were collected from enrolled participants. After DNA extraction, GSTT1 genotyping was carried out by a multiplex PCR with ß-globin as an amplification control. Association evaluation of GSTT1 genotypes with breast cancer risk, specific tumor characteristics, and survival were the primary endpoints. RESULTS: A total of 264 participants were enrolled in the molecular investigation (3 institutions). The study included 121 primary breast cancer patients as cases and 143 age-matched female subjects, with no history of any cancer, as controls. A significant genetic association between GSTT1-absent genotype and breast cancer susceptibility (p-value: 0.03; OR: 2.13; 95% CI: 1.08-4.29) was reported. The case-series analysis showed lack of association of GSTT1 genotypes with menopause (p-value: 0.86), tumor stage (p-value: 0.12), grade (p-value: 0.32), and size (p-value: 0.07). The survival analysis revealed that GSTT1-absent genotype cases had a statistically significant shorter overall survival (OS) than those with the GSTT1-present genotype cases (mean OS: 23 months vs 33 months). The HR (95% CI) for OS in patients carrying GSTT1-absent genotype was 8.13 (2.91-22.96) when compared with the GSTT1-present genotype. CONCLUSIONS: The present study is the first report of an independent significant genetic association between GSTT1-absent genotype and breast cancer susceptibility in a Pakistani population. It is also the foremost report of the association of this genotype with OS in breast cancer cases. Upon further validation, GSTT1 variation may serve as a marker for devising better population-specific strategies. The information may have translational implications in the screening and treatment of breast cancers.