Pritesh Trivedi

Comparative evaluation of cancer stem cell markers in normal pancreas and pancreatic ductal adenocarcinoma

Chemoresistance and self-renewal of cancer stem cells (CSC), found in many tumors including pancreatic ductal adenocarcinoma (PDAC), are believed to underlie tumor mass regrowth. The distribution of cells carrying the putative stem-cell markers CD133, Nestin, Notch1-4, Jagged1 and 2, ABCG2 and aldehyde dehydrogenase (ALDH1) was assessed immunohistochemically using PDAC and normal pancreas tissue microarrays. The immunoreactivity was semi-quantitatively graded against the normal pancreas and was correlated with the differentiation grade and disease stage. No statistical significant differences were found between normal pancreas and PDAC in the expression of Nestin, Notch1, 3 and 4, ABCG2 or ALDH1. Notch2 and Jagged1 and 2 expression were increased in PDAC. CD133-positive cells were above-normal in PDAC, but the difference was not statistically significant. Nestin, Notch1-4, Jagged1, ABCG2 and ALDH1 immunostaining scores were not correlated with tumor grade or disease stage. CD133 and Notch2 expression was significantly inversely correlated with tumor grade, but not disease stage. Notch3 immunostaining positively correlated with tumor stage, but not with differentiation grade. Jagged2 protein expression correlated inversely with disease stage, but not with tumor grade. From the clinical standpoint, improved delineation of the tumor CSC signature, putatively responsible for tumor initiation and recurrence after initial response to chemotherapy, may offer novel therapeutic targets for this highly lethal cancer.

Intra-tumoral heterogeneity in the expression of programmed-death (PD) ligands in isogeneic primary and metastatic lung cancer: Implications for immunotherapy

ABSTRACT Purpose: There is inconclusive evidence to suggest the expression of programmed cell death (PD) ligand 1 (PD-L1) is a putative predictor of response to PD-1/PD-L1-targeted therapies in lung cancer. We evaluated the heterogeneity in the expression of PD-1 ligands in isogeneic primary and metastatic LC specimens. Experimental Design: From 12,580 post mortem cases, we identified 214 patients with untreated metastatic LC, of which 98 had adequately preserved tissues to construct a syngeneic primary LC/metastasis tissue microarray. Immunostaining for PD-L1 and 2 was evaluated in paired primary and metastatic lesions and correlated with clinicopathologic features. Results: We included 98 patients with non-small cell (NSCLC, n = 65, 66%), small cell histology (SCLC, n = 29, 30%) and four (4%) atypical carcinoids (AC). In total 8/65 (12%) primary PD-L1 positive NSCLC, had discordant matched metastases (14/17, 82%). PD-L1 negative primaries had universally concordant distant metastases. SCLCs were universally PD-L1 negative across primary and metastatic disease. PD-L2 positive NSCLC (n = 11/65, 17%) had high rate of discordant metastases (n = 24/27, 88%) and four cases (6%) had PD-L2 positive metastases with negative primaries. 2/29 SCLC (7%) and 1/4 AC (25%) were PD-L2 positive with discordance in all the sampled metastatic sites (n = 5). We found no correlation between the expression of PD ligands and clinicopathologic features of LC. Conclusions: Intra-tumoral heterogeneity in the expression of PD ligands is common in NSCLC, while PD-L1 is homogeneously undetectable in primary and metastatic SCLC. This holds implications in the clinical development of immune response biomarkers in LC.

Expression profiling and significance of VEGF-A, VEGFR2, VEGFR3 and related proteins in endometrial carcinoma.

BACKGROUND Angiogenesis plays a key role in the progression of various tumors, including endometrial carcinomas. Several cytokines and their associated receptors are shown to be involved, particularly VEGF-A with VEGFR1, -2 and -3. METHODS The expressions of VEGF-A, VEGFR2 and VEGFR3 were studied in by immunohistochemistry in 76 endometrial carcinoma specimens. VEGFR2 and VEGFR3 receptor expression were also studied by qRT-PCR in 17 tumors in comparison to normal endometrium. The expression profiles were correlated with tumor type, grade, stage, lymphovascular invasion, disease free survival, and the expressions of other cytokine receptors (EGFR, CXCR1 and CXCR2). RESULTS Immunohistochemically, 63% of endometrial cancers expressed VEGF-A, 55% VEGFR2 and 26% VEGFR3. VEGFR3 was significantly correlated with tumor stage (p=0.02), with a trend towards poorer disease free survival (p=0.09). VEGF-A was significantly correlated with microvessel density (p<0.01). Using qRT-PCR, increased expression of VEGFR2 (17.2-fold) and VEGFR3 (21.9-fold) was seen in endometrial carcinomas compared with normal endometrium, with significant correlations among the expression levels of VEGFR2, VEGFR3, EGFR, CXCR1 and CXCR2. CONCLUSION Our study suggests that evaluation of VEGFR3 expression in tumors may provide prognostic data, and help identify patients who would best benefit from anti-angiogenic therapeutic agents. This is the first report showing correlations between the expressions levels of the different receptors.

Bcl-6 and c-Myc are rarely co-expressed in adult diffuse large B-cell lymphoma.

Bcl-6 is expressed in germinal centre derived B-cell non-Hodgkin lymphomas including diffuse large B-cell lymphoma (DLBCL) and is likely to play a major role in driving proliferation of a subset of DLBCLs, especially those of germinal centre B-cell subtype, but the role of c-Myc, which is important for proliferation in various lineages is not known. We used the highly standardised staining conditions of a tissue microarray to characterise co-expression of c-Myc and Bcl-6 in DLBCL. We carried out immunohistochemistry of 73 arrayed cases. The majority (62/73) did not express c-Myc, but 11 cases (15%) showed nuclear staining. 5/53 (9%) of Bcl-6 expressing cases co-expressed c-Myc, whereas a much higher proportion, 6/20 (30%), of Bcl-6 negative cases were positive for c-Myc. Overall survival of c-Myc expressing cases was the same as those that had absent expression. There was no significant correlation between c-Myc expression and DLBCL subtype (germinal centre or non-germinal centre subtypes).

Positivity rate of TTF-1 on immunohistochemistry in pancreatic neuroendocrine tumors

To the Editor: TTF-1 (thyroid transcription factor-1) is a homeodomain transcription factor, which is expressed in thyroid and lung epithelial cells, and their malignant counterparts. In lung cancers, TTF-1 expression is found in about 60% of adenocarcinomas and 90 % of poorly differentiated neuroendocrine carcinomas (PD-NEC)/small cell lung carcinomas (SCLC), as well as in 35 % of well-differentiated neuroendocrine tumors (NET). However, TTF-1 expression has been shown in a variety of other tumors, including adenocarcinomas of the kidney, breast, biliary tract, liver, ovary and uterus (ranging from 1 to 2 %). With regard to NET, TTF-1 expression was also reported in those from the gastrointestinal tract, bladder, prostate and cervix uteri. However, in this and other studies, the authors did not find any TTF-1 positivity among pancreatic tumors. In this study we investigated the expression of TTF-1 in 57 pancreatic tumor resections carried out at the Hammersmith Hospital, London, UK. Ethics committee approval was obtained from the institutional review board of the Tissue Bank of The Imperial College Healthcare NHS Trust (ref: R1008). Histopathology reports and slides were first reviewed to confirm the diagnosis and select representative blocks for the study. All NET were positive for CD56, synaptophysin and chromogranin A. Next, the expression of TTF-1 was evaluated by immunohistochemistry, using sections from formalin-fixed, paraffin embedded tissue blocks. Two micron sections were cut from each block, and immunohistochemical staining and detection were performed using an automated Leica Bond 3 machine according to the manufacturer’s protocol. The primary anti-TTF-1 antibody used was (NCL-L-TTF-1, clone: SPT24, Leica, Wetzlar, Germany, dilution 1:100). For positive controls, sections from normal lung were used. As a negative control for each case, the primary antibody was replaced by phosphate buffered saline. We found that of the 57 tumors, 2 had weak positivity in 10–20 % of cells (Fig. 1a,b), and 2 had strong nuclear positivity to TTF-1 in >80 % of cells (Fig. 1c,d). The rest of the tumors were uniformly negative for TTF-1. Therefore in our cohort of cases, 7 % (4/57) of pancreatic NET were TTF-1 positive, with 3.5 % (2/57) of the tumors strongly positive. All four cases were well-differentiated NETwith uniform cells, granular eosinophilic cytoplasm and coarse chromatin pattern,

Programmed cell death ligands expression in phaeochromocytomas and paragangliomas: Relationship with the hypoxic response, immune evasion and malignant behavior

ABSTRACT The hypoxic response underlies the pathogenesis and malignant behavior of PCC/PGL. Regulation of PD-1 receptor-ligand signaling, a therapeutically actionable driver of the anti-tumor immune response, is a hypoxic-driven trait across malignancies. We evaluated the prognostic role of PD ligands in association with biomarkers of hypoxia and angiogenesis in patients with PCC/PGL. Tissue microarrays sections including consecutive cases diagnosed between 1983–2011 were stained for PD-L1 and 2, hypoxia inducible factor 1a (Hif-1a), Carbonic Anhydrase IX (CaIX), Vascular Endothelial Growth Factor-A (VEGF-A). We explored the biologic significance of PD ligands expression using gene set enrichment analysis (GSEA) on The Cancer Genome Atlas (TCGA) for PCC/PGL (n = 184). In total, 100 patients, 10% malignant, 64% PCC, 29% familial with median tumor size of 4.7 cm (range 1–14) were included. Median follow-up was 4.7 y. We found PD-L1 expression in 18% of PCC/PGL, which was independent of adverse pathological features including capsular (CI), vascular invasion (VI), necrosis (N) and expression of biomarkers of hypoxia. PD-L2 expression (16%) strongly correlated with CI, VI, N and malignant behavior (p < 0.05) and was associated with stronger Hif-1a and CaIX immunolabeling (p < 0.01). PD-L2 was predictive of shorter survival (162 versus 309 months, HR 3.1 95%CI 1.1–9.2, p = 0.02). GSEA on TGCA samples confirmed enrichment of transcripts involved in hypoxia and anti-cancer immunity. We report for the first time PD ligands expression in PCC/PGL with a distinctive prognostic, clinico-pathologic and immuno-biologic role. These findings support a potential therapeutic role for PD-1/PD-L1 targeted checkpoint inhibitors in these tumors. KEY MESSAGE The molecular mechanisms underlying immune evasion in malignant phaeochromocytomas and paragangliomas (PCC/PGL) are poorly understood. This study demonstrates for the first time a distinctive immune-biologic and prognostic role of programmed death ligands 1 and 2 (PD-L1, PD-L2), two actionable drivers of the anti-cancer immune response. RNA-sequencing of tumor tissues reveals enrichment of transcripts relating to hypoxia and immune-exhaustion to explain the adverse clinical course observed in PD-L2 overexpressing tumors. These findings provide a rationale for the development of anti PD-1 therapies in malignant PCC/PGL.

The status of epidermal growth factor receptor in borderline ovarian tumours

The majority of borderline ovarian tumours (BOTs) behave in a benign fashion, but some may show aggressive behavior. The reason behind this has not been elucidated. The epidermal growth factor receptor (EGFR) is known to contribute to cell survival signals as well as metastatic potential of some tumours. EGFR expression and gene status have not been thoroughly investigated in BOTs as it has in ovarian carcinomas. In this study we explore protein expression as well as gene mutations and amplifications of EGFR in BOTs in comparison to a subset of other epithelial ovarian tumours. We studied 85 tumours, including 61 BOTs, 10 low grade serous carcinomas (LGSCs), 9 high grade serous carcinomas (HGSCs) and 5 benign epithelial tumours. EGFR protein expression was studied using immunohistochemistry. Mutations were investigated by Sanger sequencing exons 18-21 of the tyrosine kinase domain of EGFR. Cases with comparatively higher protein expression were examined for gene amplification by chromogenic in situ hybridization. We also studied the tumours for KRAS and BRAF mutations. Immunohistochemistry results revealed both cytoplasmic and nuclear EGFR expression with variable degrees between tumours. The level of nuclear localization was relatively higher in BOTs and LGSCs as compared to HGSCs or benign tumours. The degree of nuclear expression of BOTs showed no significant difference from that in LGSCs (mean ranks 36.48, 33.05, respectively, p=0.625), but was significantly higher than in HGSCs (mean ranks: 38.88, 12.61 respectively, p< 0.001) and benign tumours (mean ranks: 35.18, 13.00 respectively, p= 0.010). Cytoplasmic expression level was higher in LGSCs. No EGFR gene mutations or amplification were identified, yet different polymorphisms were detected. Five different types of point mutations in the KRAS gene and the V600E BRAF mutation were detected exclusively in BOTs and LGSCs. Our study reports for the first time nuclear localization of EGFR in BOTs. The nuclear localization similarities between BOTs and LGSCs and not HGSCs support the hypothesis suggesting evolution of LGSCs from BOTs. We also confirm that EGFR mutations and amplifications are not molecular events in the pathogenesis of BOTs.