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Heterogeneity within the tumor-infiltrating lymphocytes (TIL) population limits immunotherapeutic efficacy against cancer. Between two subpopulations of exhausted CD8+ TILs (progenitor-exhausted; TPEX, terminally exhausted; TTEX), TTEX cells remain unresponsive to anti–programmed cell death protein 1(PD-1) therapy. Deciphering whether and how PD-1–resistant TTEX cells engage in tumor promotion could improve the response to immunotherapy. Here, we report that TTEX cells actively participate in tumor progression by modulating cancer stem cells (CSC). TTEX cells strongly correlated with elevated CSC frequency in poorly immune-infiltrated (CD8+ TIL low) advanced human breast and ovarian carcinomas. TTEX directly upregulated CSC frequency in vitro, which was not affected by anti–PD-1 treatment. The TTEX-influenced CSCs were highly clonogenic and exhibited a multidrug-resistant phenotype, overexpressing drug efflux pumps like ABCC1 and ABCB1. These CSCs were highly invasive, displaying increased invadopodia development and elevated cofilin, CXCR4, and matrix metalloproteinase 7 (MMP7) expression. The invasive properties along with epithelial–mesenchymal plasticity of TTEX-educated CSCs increased metastasis in vivo. TTEX increased cell surface levels and activation of VEGFR2 in CSCs, and silencing or inhibition of VEGFR2 reversed the CSC-stimulatory effects of TTEX. LAMP3 and NRP1 on the surface of TTEX stimulated VEGFR2 in CSCs to promote aggressiveness. Cumulatively, these findings suggest that screening patients with carcinoma for both CD8+ TILs and TTEX frequency prior to anti–PD-1 therapy could improve patient outcomes. In addition, targeting the LAMP3/NRP1–VEGFR2 axis could be a therapeutic strategy in advanced patients with carcinoma with limited CD8+ T-cell infiltration and high TTEX frequency. Significance: Cross-talk with TTEX CD8+ T cells mediated by the VEGFR2 axis induces aggressive properties in cancer stem cells to promote tumor progression.

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Heterogeneity within the tumor-infiltrating lymphocytes (TIL) population limits immunotherapeutic efficacy against cancer. Between two subpopulations of exhausted CD8+ TILs (progenitor-exhausted; TPEX, terminally exhausted; TTEX), TTEX cells remain unresponsive to anti–programmed cell death protein 1(PD-1) therapy. Deciphering whether and how PD-1–resistant TTEX cells engage in tumor promotion could improve the response to immunotherapy. Here, we report that TTEX cells actively participate in tumor progression by modulating cancer stem cells (CSC). TTEX cells strongly correlated with elevated CSC frequency in poorly immune-infiltrated (CD8+ TIL low) advanced human breast and ovarian carcinomas. TTEX directly upregulated CSC frequency in vitro, which was not affected by anti–PD-1 treatment. The TTEX-influenced CSCs were highly clonogenic and exhibited a multidrug-resistant phenotype, overexpressing drug efflux pumps like ABCC1 and ABCB1. These CSCs were highly invasive, displaying increased invadopodia development and elevated cofilin, CXCR4, and matrix metalloproteinase 7 (MMP7) expression. The invasive properties along with epithelial–mesenchymal plasticity of TTEX-educated CSCs increased metastasis in vivo. TTEX increased cell surface levels and activation of VEGFR2 in CSCs, and silencing or inhibition of VEGFR2 reversed the CSC-stimulatory effects of TTEX. LAMP3 and NRP1 on the surface of TTEX stimulated VEGFR2 in CSCs to promote aggressiveness. Cumulatively, these findings suggest that screening patients with carcinoma for both CD8+ TILs and TTEX frequency prior to anti–PD-1 therapy could improve patient outcomes. In addition, targeting the LAMP3/NRP1–VEGFR2 axis could be a therapeutic strategy in advanced patients with carcinoma with limited CD8+ T-cell infiltration and high TTEX frequency.Significance:Cross-talk with TTEX CD8+ T cells mediated by the VEGFR2 axis induces aggressive properties in cancer stem cells to promote tumor progression.

Vasculogenic mimicry (VM), an endothelial cell–independent alternative mechanism of blood supply to the malignant tumour, has long been considered as an adverse prognostic factor in many cancers. The correlation of VM with laminin‐5γ2 and the assessment of their harmonized expression as an independent risk factor have not been elucidated yet in oral squamous cell carcinoma (OSCC). CD31/PAS staining stratified 116 clinically diagnosed OSCC specimens into VM+ and VM− cohorts. The expression pattern of laminin‐5γ2 and its upstream modulator MMP2 was evaluated by immunohistochemistry and Western blot. The Kaplan–Meier and Cox regression analyses were performed to assess the survival and prognostic implications. The presence of VM demonstrated a significant correlation with the expression of laminin‐5γ2 (p

Regulator-of-G-protein-signaling-5 (RGS5), a pro-apoptotic/anti-proliferative protein, is a signature molecule of tumor-associated pericytes, highly expressed in several cancers, and is associated with tumor growth and poor prognosis. Surprisingly, despite the negative influence of intrinsic RGS5 expression on pericyte survival, RGS5(high)pericytes accumulate in progressively growing tumors. However, responsible factor(s) and altered-pathway(s) are yet to report. RGS5 binds with Gαi/q and promotes pericyte apoptosis in vitro, subsequently blocking GPCR-downstream PI3K-AKT signaling leading to Bcl2 downregulation and promotion of PUMA-p53-Bax-mediated mitochondrial damage. However, within tumor microenvironment (TME), TGFβ appeared to limit the cytocidal action of RGS5 in tumor-residing RGS5(high)pericytes. We observed that in the presence of high RGS5 concentrations, TGFβ–TGFβR interactions in the tumor-associated pericytes lead to the promotion of pSmad2–RGS5 binding and nuclear trafficking of RGS5, which coordinately suppressed RGS5–Gαi/q and pSmad2/3–Smad4 pairing. The RGS5–TGFβ–pSmad2 axis thus mitigates both RGS5- and TGFβ-dependent cellular apoptosis, resulting in sustained pericyte survival/expansion within the TME by rescuing PI3K-AKT signaling and preventing mitochondrial damage and caspase activation. This study reports a novel mechanism by which TGFβ fortifies and promotes survival of tumor pericytes by switching pro- to anti-apoptotic RGS5 signaling in TME. Understanding this altered RGS5 signaling might prove beneficial in designing future cancer therapy.

A high incidence of oral squamous cell carcinoma (OSCC) is observed in South‐East Asian countries due to addictions such as chewing tobacco. Local invasion and distant metastases are primary causes of poor prognosis in OSCC. This study aimed to understand the alterations in metastasis biomarkers, such as stromal cell–derived factor‐1α (SDF‐1 or SDF1α) and its receptor C‐X‐C chemokine receptor type 4 (CXCR4), in OSCC patient samples that were stratified based on the history of addiction to chewing tobacco. Targeted immunohistochemical staining and Western blotting were performed on primary tumour and metastatic lymph node (LN) tissues in parallel. Overexpression of hepatocyte growth factor (HGF), activated form of its cognate receptor tyrosine kinase, c‐Met (p‐Met), GRB2‐associated‐binding protein 1 (Gab1), phospho‐protein kinase B (pAkt), nuclear factor kappa B (NF‐κB) and cyclooxygenase‐2 (COX‐2) were observed in primary tumour and metastatic lymph nodes in both chewer and non‐chewer cohorts. Variance analysis showed significant positive correlation between them (P

Vasculogenic mimicry (VM), defined as an endothelial cell independent alternative mechanism of blood and nutrient supply by dysregulated tumor cells, is associated with poor prognosis in oral squamous cell carcinoma (OSCC). Here we aim to investigate the underlying molecular mechanism of the synergistic effect of phytochemical Lupeol and standard microtubule inhibitor Paclitaxel in reversing the hypoxia induced VM formation in OSCC. The results demonstrated that the hypoxia induced upregulation of HIF-1α led to augmentation of signaling cascade associated with extracellular matrix remodeling and EMT phenotypes that are mechanistically linked to VM. Induction of HIF-1α altered the expression of EMT/CSC markers (E-Cadherin, Vimentin, Snail, Twist and CD133) and enhanced the ability of cell migration/invasion and spheroid formation. Subsequently, the targeted knockdown of HIF-1α by siRNA led to the perturbation of matrigel mediated tube formation as well as of Laminin-5γ2 expression with the down-regulation of VE-Cadherin, total and phosphorylated (S-897) EphA2, pERK1/2 and MMP2. We also observed that Lupeol in association with Paclitaxel resulted to apoptosis and the disruption of VM associated phenotypes in vitro. We further validated the impact of this novel interventional approach in a patient derived tumor explant culture model of oral malignancy. The ex vivo tumor model mimicked the in vitro anti-VM potential of Lupeol-Paclitaxel combination through down-regulating HIF-1α/EphA2/Laminin-5γ2 cascade. Together, our findings elucidated mechanistic underpinning of hypoxia induced Laminin-5γ2 driven VM formation highlighting that Lupeol-Paclitaxel combination may serve as novel therapeutic intervention in perturbation of VM in human OSCC.

The aim of this study is to understand the association of HPV infection and wnt-β-catenin self-renewal pathway in development of head and neck squamous cell carcinoma (HNSCC). For this reason, the molecular profiles (methylation/deletion/expression) of antagonists (SFRP1/2 and DKK1), agonists (FZD7 and LRP6) and effector protein β-catenin of the pathway were analyzed in HPV positive/negative oral epithelium at first, followed by its changes during development of the tumor along with correlations with different clinico-pathological parameters. HPV infection alone or in combination with tobacco habit could activate p- β-catenin expression in basal/parabasal layers of oral epithelium through high expression of FZD7 and significant down regulation of SFRP1/2 through promoter hypermethylation due to over expression of DNMT1 with ubiquitous down regulation of DKK1 and up-regulation of LRP6. This phenomenon has been seen in respective HPV positive and negative HNSCC tumors with additional deletion/microsatellite size alterations in the antagonists. Overall alterations (methylation/deletion) of SFRP1/2, DKK1 gradually increased from Group I (HPV-/Tobacco-) to Group IV(HPV+/Tobacco+) tumors, leading to the worst prognosis of the patients. Thus, the transmission of differentially activated wnt-β-catenin pathway from HPV positive/negative basal/parabasal layers of oral epithelium to HNSCC tumors determines differences in molecular pathogenesis of the disease.

Aberrant expression of mTOR signaling pathway is significantly associated with gastric cancer. However, the effect of smoking on mTOR expression and its downstream signaling molecules in gastric cancer has not been explored. Our study aims to investigate the effect of smoking on p-mTOR and its correlation with various downstream targets and survival of the smoker and never-smoker in advanced gastric cancer patients. Forty-one smokers and 41 never-smokers patient sample with the advanced gastric carcinoma were chosen for this study. Immunohistochemistry and western blot analysis were performed to check the expression of p-mTOR and its downstream targets. The correlation of p-mTOR with its downstream targets was analyzed by linear regression analysis in Graph Pad Prism software. Survivability analysis was examined by Kaplan–Meier method with log rank test in SPSS. High expression of p-mTOR and its downstream targets were observed in advanced gastric cancer smoker patients as compared to never-smokers by immunohistochemistry and western blot analysis. Results revealed that over expressed p-mTOR in smoker patients were positively correlated with its downstream targets (P

The study was aimed to understand the importance of the hedgehog signaling pathway in development of head and neck squamous cell carcinoma (HNSCC).The molecular profiles of the key regulatory genes of the pathway were analysed in the adjacent normal epithelium and tumor samples. The findings were validated in HNSCC cell line.In the bioinformatical analysis, severe reduction in the expression of HHIP was evident in the datasets. The protein and mRNA expression studies in our sample pool revealed interplay of various isoforms of PTCH1 gene (PTCH1-1 and 1B) together with high/medium expression of GLI, SHH, SMO and HHIP in the basal/parabasal layers of the normal epithelium. As the disease progressed, severe downregulation of HHIP coupled with upregulation of GLI1 and differential expression pattern of various PTCH1 gene isoform was evident. Promoter methylation analysis of PTCH1 gene revealed the involvement of more than one promoter of PTCH1 in regulating the expression of different isoform of this gene during tumorigenesis. Treating the FaDu cell line with the demethylating agent 5-aza-2'-deoxycytidine reversed the methylation effects of HHIP and PTCH1 and de-activated the pathway. Also, reduced expression of HHIP-AS1 was observed in our sample pool suggesting multiple ways of regulation of the HHIP gene. Lastly, the patients with under expression of HHIP, HHIP-AS1, high expression of GLI1 showed worse five-year over-all survival trend.Dynamic promoter switching of PTCH1 and frequent inactivation of HHIP are the key regulatory events of hedgehog pathway activation in HNSCC.

Triple negative breast cancer (TNBC) originates from a less differentiated ductal cell of breast, which is less sensitive to chemotherapy. The chemotolerance mechanism of TNBC has not yet been studied in detail. For this reason, molecular profiles (expression/genetic/epigenetic) of Y654-p-β-catenin (active) and its kinase epidermal growth factor receptor (EGFR) along with SH3GL2 (regulator of EGFR homeostasis) were compared between neoadjuvant chemotherapy treated (NACT) and pretherapeutic TNBC samples. Reduced nuclear expression of Y654-p-β-catenin protein with low proliferation index and CD44 prevalence showed concordance with reduced expression of EGFR/Y1045-p-EGFR proteins in the NACT samples than the pretherapeutic TNBC samples. Infrequent messenger RNA expression, gene amplification (10-32.5%), and mutation (1%) of EGFR were seen in the TNBC samples irrespective of therapy, suggesting the importance of EGFR protein stabilization in this tumor. The upregulation of SH3GL2 seen in the NACT samples in contrast to the pretherapeutic samples might be due to its promoter hypomethylation, as seen in the quantitative methylation assay. A similar trend of upregulation of SH3GL2 and downregulation of EGFR, Y1045-p-EGFR, Y654-p-β-catenin were seen in the MDA-MB-231 cell line using antharacycline antitumor drugs (doxorubicin/nogalamycin). The NACT patients with reduced expression of Y654-p-β-catenin and/or EGFR and high expression of SH3GL2 showed comparatively better prognosis than the pretherapeutic patients. Thus, our study showed that reduced nuclear expression of Y654-p-β-catenin in NACT samples due to downregulation of EGFR protein through promoter hypomethylation-mediated upregulation of SH3GL2, resulting in low proliferation index/CD44 prevalence with better prognosis of the NACT patients, might have an important role in the chemotolerance of TNBC.

Background: Periampullary cancer (PC) is a global medical burden. Less than 5% of patients experience an overall survival of 5 years or more. The study aims to analyze the importance of epidermal growth factor receptor (EGFR)/insulin-like growth factor 1 receptor (IGF1R)–beta-catenin (β-catenin)–CD44 pathway in the development of periampullary cancer of Indian patients. Subjects and Methods: Expression profile of EGFR, IGF1R, β-catenin, and CD44 was verified by immunohistochemical analysis in primary tumor samples (N = 14) and respective adjacent normal tissues. Results: In periampullary carcinoma patients, a high level of EGFR expression was seen in 64% of the samples along with co-expression of IGF1R in 77.8% of samples. Furthermore, the high expression of EGFR and IGF1R significantly correlated with the increased expression of β-catenin along with CD44 expression of the tumors. Conclusion: The EGFR/IGF1R–β-catenin–CD44 pathway seems to be important in the development of PC with clinical importance.

The occurrence of vasculogenic mimicry (VM) and EphA2-mediated tumour progression are associated with poor prognosis in various solid tumours. Here, we aimed to investigate the prognostic implications of VM and its association with phosphorylated EphA2 receptor in invasive carcinoma of the breast. The patients were stratified based on CD-31/PAS dual staining and subsequently the expression status of phospho-EphA2 (S897), FAK, phospho-ERK1/2 and Laminin 5Ƴ2 was analysed by immunohistochemistry. Survival of patients was correlated within the stratified cohort. The pathologically defined VM phenotype and phospho-EphA2 (S897) expression status were significantly associated with lower disease-free survival (DFS) and overall survival (OS). Both the features were also found to be significantly associated with higher nodal status, poor Nottingham Prognostic Index (NPI) and were more prevalent in the triple-negative breast cancer (TNBC) group. Incidentally, there were no significant association between age of the patient, grade and size of the tumour with VM and phospho-EphA2 (S897). The effector molecules of phospho-EphA2 (S897) viz., Focal Adhesion Kinase (FAK), phospho-ERK1/2 and Laminin 5Ƴ2 were significantly upregulated in the VM-positive cohort. Survival analysis revealed that the VM and phospho-EphA2 (S897) dual-positive cohort had poorest DFS [mean time = 48.313 (39.992–56.633) months] and OS [mean time = 56.692 (49.055–64.328) months]. Individually, VM-positive [Hazard Ratio (HR) 6.005; 95% confidence interval (CI) 2.002–18.018; P = 0.001 for DFS and HR 11.654; 95% CI 3.195–42.508; P

The use of chewing tobacco is a severe risk factor for oral mucosa related diseases including cancer in India as well as USA, although its relationship with Oral Leukoplakia (OL) or related carcinogenicity is still not clear. This work chose two oncogenic pathway proteins- the Epidermal Growth Factor Receptor and the WNT pathway among leukoplakia patients and established their correlation with the individuals' tobacco chewing habit. 89 fresh patients with OL were selected for the work. The samples were classified based on the individual's tobacco chewing habit. The divided samples were then immunostained with antibodies for both of the EGFR as well as WNT pathway proteins. The samples were further classified based on their proliferation status and the expression of these oncoproteins was also observed. In order to compare the cytological data with histological data, 30 OL patients undergoing biopsy were chosen and immunohistological analysis was performed for the same pathways. Results showed overexpressing EGFR and WNT pathway proteins in all OL samples. Structurally atypic cells had a tendency to overexpress these oncoproteins. However the immunocytochemistry data could not confirm any positive effect of chewing tobacco on the OL's proliferative state. Statistical data from the immunfluorescence finally revealed the overexpression of both EGFR and WNT pathway proteins on the proliferative population establishing chewing tobacco as a positive risk factor for the onset of OL. Data from biopsy samples followed the same trend of protein expression seen in the cytological samples. Dysplastic zones showed huge overexpression of EGFR and WNT pathway proteins among tobacco chewers. In conclusion, this is the first time report showing the effect of chewing tobacco on the EGFR and WNT pathway in OL and its possible role as a potential risk factor for its proliferative type.

Background and objectives The aim of the study was to understand the importance of mismatch repair genes MLH1 and MSH2 in chemotolerance and prognosis of breast carcinoma (BC). Methods First, the alterations (deletion/methylation/expression) of MLH1 and MSH2 were analyzed in 45 neoadjuvant chemotherapy (NACT)-treated and 133 pretherapeutic BC samples. The chemotolerant BC cells were characterized by treating two BC cell lines MCF-7 and MDA MB 231 with two anthracycline antitumor antibiotics, doxorubicin and nogalamycin. Results The deletion frequencies were 32% to 38% in MLH1/MSH2 genes and promoter methylation frequencies were 49% to 62% in MLH1 and 41% to 51% in MSH2 in both NACT-treated and pretherapeutic samples. The overall alteration of MLH1 and MSH2 was 58% to 71% in the samples. Reduced messenger RNA (mRNA) and protein expression were found in both the genes and it showed concordance with the molecular alterations. NACT-treated patients showed better prognosis. The chemotherapeutic drug induced increased mRNA/protein expression of the genes in BC cell lines was due to their promoter hypomethylation, as analyzed by quantitative methylation assay. This phenomenon was also evident in NACT-treated BC samples. Conclusion MLH1/MSH2 genes play a critical role in the development of BC. Hypomethylation of MLH1/MSH2 genes might be important in chemotolerance of the disease.

The aim of the study was to understand the role of homologous recombination repair (HRR) pathway genes in development of chemotolerance in breast cancer (BC). For this purpose, chemotolerant BC cells were developed in MCF-7 and MDA MB 231 cell lines after treatment with two anthracycline anti-tumor antibiotics doxorubicin and nogalamycin at different concentrations for 48 h with differential cell viability. The drugs were more effective in MCF-7 (IC50: 0.214-0.242 µM) than in MDA MB 231 (IC50: 0.346-0.37 µM) as shown by cell viability assay. The drugs could reduce the protein expression of PCNA in the cell lines. Increased mRNA/protein expression of the HRR (BRCA1, BRCA2, FANCC, FANCD2, and BRIT1) genes was seen in the cell lines in the presence of the drugs at different concentrations (lower IC50, IC50, and higher IC50) irrespective of the cell viability (68-41%). Quantitative methylation assay showed an increased percentage of hypomethylation of the promoters of these genes after drug treatment in the cell lines. Similarly, chemotolerant neoadjuvant chemotherapy (NACT) treated primary BC samples showed significantly higher frequency of hypomethylation of the genes than the pretherapeutic BC samples. The drugs in different concentrations could reduce m-RNA and protein expression of DNMT1 (DNA methyltransferase 1) in the cell lines. Similar phenomenon was also evident in the NACT samples than in the pretherapeutic BC samples. Thus, our data indicate that reduced DNMT1 expression along with promoter hypomethylation and increased expression of the HRR genes might have importance in chemotolerance in BC.

Aims The cytotoxic response of an intermediate metabolite glyoxylate (Glx) on colon carcinoma has been evaluated in vitro. Main methods The anti-proliferative effect of Glx was assessed on HT-29 and HCT-116 cells by performing MTT assay as well as beta-hexosaminidase assay. Evaluation of apoptotic event of Glx treated cells was measured by flow cytometry using annexin-V/PI staining. The mitochondrial membrane potential and level of ROS were estimated using DiOC6(3)/CCCP and DCFH-DA method, respectively. The assessment of catalase, LDH and IDH were performed. Key findings The results of MTT assay indicated that treatment with Glx significantly inhibited the proliferation of HT-29 and HCT-116 cells. Beta-hexosaminidase assay also confirmed the inhibition of cellular viability. The dose-dependent Glx treatment indicated lowering the colony forming ability of HT-29 and HCT-116 cells. Flow cytometric data demonstrated the significant increment of late apoptotic event after Glx treatment. In addition, substantial LDH activity was noticed in both the colon cancer cells whereas the IDH activity was unaltered after extra-cellular addition of Glx. Further, dissipation of mitochondrial membrane potential and subsequently elevated ROS generation was also detected in the Glx treated colon cancer cells. However, gradual elevation of catalase activities indicated that Glx treatment on colon cancer cells exhibit oxidative stress. Significance This study depicts that supra-physiological concentration of Glx inhibits the proliferation of colon cancer cells due to oxidative stress.

To address the ambiguity of different modes of human papillomavirus (HPV) transmission in breast, the immunohistochemical expression of two oncoproteins E6/E7 of HPV16 was analyzed in primary breast cancer (BC) and adjacent normal skin of 4 samples. The patients were of 35–55 years old having no previous history of cancer. The E6/E7 expressions were evident in both skin and BC. In skin, high/moderate cytoplasmic expressions of E6/E7 proteins were seen in all samples, whereas in BC, high/moderate cytoplasmic expressions of the proteins were observed in 2–3 samples. Thus, it seems that HPV infection in the breast may occur through the skin.

Background The frequency of colorectal carcinoma is inadequate in India compared to the western world. The carcinoma of colon and rectum is usually disturbing among the individuals of older age group whereas it is less frequent in younger age group. This study was based on age, gender, site of primary tumor and histopathological type of colorectal cancer cases. Methods A retrospective study of 420 colorectal carcinoma cases admitted to Chittaranjan National Cancer Institute hospital, Kolkata, India during 2012–2016 was carried out. All the clinicopathological data was collected from the medical records of the patients. The detailed information was entered into tabular sheet and statistical analysis was performed. Results During these five years out of total cases in the Department of Surgical Oncology, 5.09% patients with colorectal carcinoma were admitted for colectomy or hemicolectomy. The percentage of younger age group (≤40year) with colorectal cancer was rise up sharply in context of older age group (>60year). The ratio of male and female affected in colorectal cancer was 1.6:1. Rectum was the most common site 46.2% among the total cancer lesions. From histopathological data, mucinous adenocarcinoma cases were identified with (23.6%) high frequency and mostly detected at younger age group (65.7%). It was found that 18.8% patients with synchronous liver metastasis and 16.7% of patients with Type 2 diabetes mellitus were likely to develop colorectal cancer. Conclusion The concise overview has documented an increased incidence of colorectal carcinoma patients amongst younger individuals with more aggressive forms like mucinous adenocarcinoma and also development of synchronous liver metastasis.

In approximately 30% of triple-negative breast cancer (TNBC) patients a complete pathological response is achieved. However, after neo-adjuvant chemotherapy treatment (NACT) residual tumour cells can be intrinsically resistant to chemotherapy. In this study, associations of the WNT/beta-catenin pathway with chemo-tolerance of NACT treated TNBC patients were compared to that of pre-treatment TNBC patients. Expression analyses were performed in both pre-treatment and NACT treated TNBC samples using immunohistochemistry and qRT-PCR, along with DNA copy number variation (CNV) and promoter methylation analyses to elucidate the mechanism(s) underlying chemo-tolerance. In addition, in vitro validation experiments were performed in TNBC cells followed by in vivo clinicopathological correlation analyses. A reduced expression (41.1%) of nuclear beta-catenin together with a low proliferation index was observed in NACT samples, whereas a high expression (59.0%) was observed in pre-treatment samples. The reduced nuclear expression of beta-catenin in the NACT samples showed concordance with reduced expression levels (47-52.9%) of its associated receptors (FZD7 and LRP6) and increased expression levels (35.2–41.1%) of its antagonists (SFRP1, SFRP2, DKK1) compared to those in the pre-treatment samples. The expression levels of the receptors showed no concordance with its respective gene copy number/mRNA expression statuses, regardless treatment. Interestingly, however, significant increases in promoter hypomethylation of the antagonists were observed in the NACT samples compared to the pre-treatment samples. Similar expression patterns of the antagonists, receptors and beta-catenin were observed in the TNBC-derived cell line MDA-MB-231 using the anthracyclines doxorubicin and nogalamycin, suggesting the importance of promoter hypomethylation in chemotolerance. NACT patients showing reduced receptor and/or beta-catenin expression levels and high antagonist expression levels exhibited a comparatively better prognosis than the pre-treatment patients. Our data suggest that reduced nuclear expression of beta-catenin in NACT TNBC samples, due to downregulation of its receptors and upregulation of its antagonists through promoter hypomethylation of the WNT pathway, plays an important role in chemo-tolerance.

Background: Periampullary cancer (PC) is a global medical burden, although infrequent but less than 5% of patients experience an overall survival of five years or more. The study aims to analyze the importance of Epidermal Growth Factor Receptor (EGFR)/Insulin-Like Growth Factor 1 Receptor (IGF1R) - Beta-catenin (β-catenin)- CD44 pathway in the development of periampullary cancer of Indian patients. Materials and Methods: Expression profile of EGFR, IGF1R, β-catenin, and CD44 was verified by immunohistochemical analysis in primary tumor samples (N=14) and respective adjacent normal tissues. Results: In periampullary carcinoma patients, a high level of EGFR expression was seen in 64% of the samples along with co-expression of IGF1-R in 77.8% samples. Furthermore, the high expression of EGFR and IGF1R significantly correlated with the increased expression of β-catenin along with CD44 expression of the tumors. Conclusion: The EGFR/IGF1R - β-catenin- CD44 pathway seems to be important in the development of PC with clinical importance. Key-words: Periampullary cancer; EGFR; IGF1R; β-catenin; CD44 Key Messages: Interplay of EGFR, IGF1R, β-Catenin and CD44 expressions is important phenomena in the development of PC. Citation Format: Suvendu Maji, Biswabandhu Bankura, Chinmay Kumar Panda, Neyaz Alam. Activation of EGFR/IGF1R - beta catenin - CD44 pathway in periampullary cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 1985.

The tumor suppressor protein p53 is known to control cell cycle arrest and apoptosis. Lupeol is a phytochemical that has been found to induce apoptosis in different cancer types through the extrinsic pathway. As yet, however, its role in the induction of cell cycle arrest and apoptosis through the intrinsic pathway in head and neck cancer has not been investigated. Here, we aimed at understanding the mechanism underlying the antitumor effect of Lupeol in head and neck cancer. The antitumor effect of Lupeol on oral and laryngeal carcinomas was assessed using two in vitro 2D cell line models (HEp-2, UPCI:SCC-131) and, subsequently, an ex vivo 3D tumor explant culture platform that maintains key features of the native tumor microenvironment. The mechanism underlying Lupeol-mediated antitumor responses was delineated using MTT, colony formation, flow cytometry, immunofluorescence, Western blotting and immunohistochemistry assays. We found that Lupeol induced an enhanced expression of p53 in both cell line models tested and, subsequently, cell cycle arrest at the G1 phase. In addition we found that, following Lupeol treatment, p53 induced Bax expression and activated the intrinsic apoptotic pathway (as measured by Caspase-3 cleavage). Interestingly, Lupeol was also found to trigger G1 cell cycle arrest through up-regulation of the expression of CDKN2A, but not p21, resulting in inhibition of CyclinD1. In an ex vivo platform Lupeol was found to impart a potent antitumor response as defined by inhibition of Ki67 expression, decreased cell viability and concomitant activation (cleavage) of Caspase-3. Finally, we found that Lupeol can re-sensitize primary head and neck squamous cell carcinoma (HNSCC) tumor samples that had clinically progressed under a Cisplatin treatment regimen. Together, our data indicate that Lupeol may orchestrate a bifurcated regulation of neoplastic growth and apoptosis in head and neck cancers and may serve as a promising agent for the management of tumors that have progressed on a platinum-based treatment regimen.

Transcriptional activation of β-catenin is a hallmark of Wnt/β-catenin pathway activation. The MCC (Mutated in colorectal cancers) and CTNNBIP1 (catenin, beta interacting protein 1) are two candidate genes which inhibit the transcriptional activity of nuclear β-catenin. The importance of MCC and CTNNBIP1 in breast cancer (BC) development has not yet been studied in detail. For this reason, in present study, the alterations (deletion/methylation/mutation/expression) of MCC and CTNNBIP1 were analyzed in BC of Indian patients (N=120) followed by expression/mutation analysis of β-catenin. Then transcriptional activity of β-catenin was checked by expression analysis of its target genes (EGFR, C-MYC and CCND1) in the same set of samples. Frequent methylation (44-45%) than deletion (20-32%) with overall alterations of 52-55% was observed in MCC/CTNNBIP1 in the BC samples. The alterations of MCC/CTNNBIP1 showed significant correlation with increased nuclear β-catenin/p-β-catenin(Y654) expression. Also, a significant correlation was seen between nuclear β-catenin expression and overexpression of its target genes like EGFR, MYC and CCND1 in the BC samples (P0.0001). An upregulation of MCC and CTNNBIP1 expression by 5-Aza-2'-deoxycytidine treatment of MCF7 and MDA-MB-231 cell lines lead to downregulation of β-catenin and its target genes. The expression of nuclear p-β-catenin(Y654), EGFR, MYC and CCND1 were significantly high in TNBC (Triple negative BC) and Her2+ compared to Luminal A/B+ subtypes. The TNBC patients in stage III/IV having reduced expression of MCC in the tumors showed poor prognosis. Thus, our data suggests that inactivation of MCC/CTNNBIP1 could be an important event in activation of β-catenin mediated transcription of target genes in BC.

Epidermal growth factor receptor (EGFR) pathway is overexpressed in head and neck cancer (HNC). Lupeol, a natural triterpene (phytosterol found in fruits, vegetables, etc.), has been reported to be effective against multiple cancer indications. Here we investigate the antitumor effects of Lupeol and underlying mechanism in oral cancer. Lupeol-induced antitumor response was evaluated in two oral squamous cell carcinoma (OSCC) cell lines (UPCI:SCC131 and UPCI:SCC084) by viability (MTT), proliferation, and colony formation assays. Lupeol-mediated induction of apoptosis was examined by caspase 3/7 assay and flow cytometry. Effect of Lupeol on EGFR in the presence or absence of EGF was delineated by Western blot. The mRNA stability assay was performed to check the role of Lupeol on COX-2 mRNA regulation. Lupeol inhibited proliferation of OSCC cells in vitro by inducing apoptosis 48 h post treatment. Ligand-induced phosphorylation of EGFR and subsequent activation of its downstream molecules such as protein kinase B (PKB or AKT), I kappa B (IκB), and nuclear factor kappa B (NF-κB) was also found to be, in part, suppressed. Interestingly, Lupeol suppressed expression of COX-2 at mRNA and protein level in a time-dependent manner. Primary explants from oral squamous cell carcinoma tissues further confirmed significant inhibition of proliferation (Ki67) in Lupeol-treated explants as compared to untreated control at 48 h. Together these data suggest that Lupeol may act as a potent inhibitor of the EGFR signaling in OSCC and therefore imply its role in triggering antitumor efficacy.

Head and neck squamous cell carcinoma (HNSCC) is a global disease and mortality burden, necessitating the elucidation of its molecular progression for effective disease management. The study aims to understand the molecular profile of three candidate cell cycle regulatory genes, RBSP3, LIMD1 and CDC25A in the basal/ parabasal versus spinous layer of normal oral epithelium and during head and neck tumorigenesis. Immunohistochemical expression and promoter methylation was used to determine the molecular signature in normal oral epithelium. The mechanism of alteration transmission of this profile during tumorigenesis was then explored through additional deletion and mutation in HPV/ tobacco etiological groups, followed byclinico-pathological correlation. In basal/parabasal layer, the molecular signature of the genes was low protein expression/ high promoter methylation of RBSP3, high expression/ low methylation of LIMD1 and high expression of CDC25A. Dysplastic epithelium maintained the signature of RBSP3 through high methylation/ additional deletion with loss of the signatures of LIMD1 and CDC25A via deletion/ additional methylation. Similarly, maintenance and / or loss of signature in invasive tumors was by recurrent deletion/ methylation. Thus, differential patterns of alteration of the genes might be pre-requisite for the development of dysplastic and invasive lesions. Etiological factors played a key role in promoting genetic alterations and determining prognosis. Tobacco negative HNSCC patients had significantly lower alterations of LIMD1 and CDC25A, along with better survival among tobacco negative/ HPV positive patients. Our data suggests the necessity for perturbation of normal molecular profile of RBSP3, LIMD1 and CDC25A in conjunction with etiological factors for head and neck tumorigenesis, implying their diagnostic and prognostic significance.

The genetic variations of HPV16 in Breast Cancer (BC) are not well studied unlike HPV16 in Cervical Cancer (CACX). In this study, the genetic variations of HPV16 in BC were compared with HPV16 in CACX. In sequencing analysis of LCR, E6 and E7 regions of HPV16 in BC and CACX the A lineage was seen to be 64.2% and 66.6% respectively. The other lineages showed differential frequency in BC and CACX. The mutation frequency index of the regions in BC and CACX was in the following order: LCR>E6>E7. However, the inter-patient genetic diversity in LCR and E6/E7 regions was high in BC than CACX. The LCR region showed more variations than the E6/E7 region in BC. Apart from some common variations, some unique tissue specific variants in LCR and E6/E7 region were seen in BC and in CACX. Besides the selection of some common variants in both BC and CACX, some unique variants in BC (D98Y; 395 G>T) and CACX (R48W; 245 G>T) were observed. The 7521 G>A variant of LCR showed association with Luminal B subtype of BC and progression of CACX. Whereas, 145 G>T (Q14H) and 335 C>T (H78Y) variants of E6 showed association with either early invasiveness of BC and/or poor outcome of the patients. Thus, this study indicates that there may be a difference in the genetic variation of HPV16 in BC and in CACX.

Background: Outgrowth of new blood vessels (neovascularization) allows tumors to supply themselves with oxygen and nutrients, and to rapidly metastasize throughout the body. Triple negative breast cancer (TNBC) is particularly susceptible to neovascularization. However, success with anti-angiogenics is highly variable and often patient-specific. This is particularly true as anti-angiogenics are being combined with immunotherapies. Thus, there is a huge unmet need for clinicians to test and predict clinical efficacy of anti-angiogenics at the individual patient level, prior to treatment. Methods: Here, we characterize a patient-autologous, ex-vivo tumor model, termed CANscript, as a platform to study the intratumor microvascular density (iMVD) of breast cancer samples (N=15). To profile iMVD we used immunohistochemical (IHC) analysis of CD34, an early biomarker of neovascularization. We then introduced anticancer and anti-angiogenic agents (e.g. Avastin) for 72 hours, and subsequently quantified phenotypic response to drugs by testing viability, cell death, proliferation and morphology. These quantitative data were then fed into a machine learning algorithm that provides a clinical response prediction (M-Score). Results: We determined that ex-vivo culture reliably retains baseline heterogeneity of iMVD based on expression of CD34+ nodes per visual field by IHC. Furthermore, we show that anticancer and anti-angiogenic agents will dynamically alter iMVD, ex-vivo, in a patient-specific manner. Finally, we show that prediction of clinical response using the 'M-Score' algorithm associates with diminished expression of CD34 per visual field of IHC after drug pressure. Summary: Neovascularization and iMVD are features of aggressive cancers, such as TNBC. CANscript provides a rapid assessment of clinical response to anticancer drugs, many of which induce their antitumor effect by targeting the tumor vasculature. We show that pharmacodynamics of antiangiogenics can be captured during acute ex-vivo culture under drug pressure, which associate to clinical response prediction. Therefore, we highlight the ability of CANscript as a platform to predict clinical response to anti-angiogenic drugs, and may therefore be a logical 'testing ground' to predict clinical efficacy of antiangiogenic drugs combined with immunotherapies. Citation Format: Smalley M, Alam N, Murmu N, Somashekhar S, Ulaganathan B, Thayakumar A, Maciejko L, Ganesh J, Lawson M, Gertje H, Shanthappa BU, Goldman A. A live tissue platform allows dynamic measurement of neovascularization and prediction of clinical response in human breast cancer samples, ex vivo [abstract]. In: Proceedings of the 2018 San Antonio Breast Cancer Symposium; 2018 Dec 4-8; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2019;79(4 Suppl):Abstract nr P6-07-03.

Objectives Human papillomavirus (HPV) causes tumors primarily Cervical cancer. Recently, inconsistent reports came up in Breast cancer (BC) too. In India, despite treatment 70,218 BC patients die each year. So, we explored the association of HPV, if any, with BC prognosis in Indian pre-therapeutic (PT) and Neo-adjuvant chemotherapy (NACT) patients with subsequent analysis of HPV profile. Methods HPV prevalence was checked and analysis of physical status, copy number, genome variation, promoter methylation and expression (mRNA and protein) of the prevalent subtype was done. Results High prevalence of HPV was observed in both PT (64.0%) and NACT (71.0%) cases with significant association with younger (20-45 yrs) PT patients. Interestingly, HPV infection was significantly increased from adjacent normal breast (9.5%, 2/21), fibro adenomas (30%, 3/10) to tumors (64.8%, 203/313) samples. In both PT and NACT cases, HPV16 was the most prevalent subtype (69.0%) followed by HPV18 and HPV33. Survival analysis illustrated hrHPV infected PT patients had worst prognosis. So, detailed analysis of HPV16 profile was done which showed Europian-G350 as the most frequent HPV16 variant along with high rate of integration. Moreover, low copy number and hyper-methylation of P97 early promoter were concordant with low HPV16 E6 and E7 mRNA and protein expression. Notably, four novel variations (KT020838, KT020840, KT020841 and KT020839) in the LCR region and two (KT020836 and KT020837) in the E6 region were identified for the first time along with two novel E6^E7*I (KU199314) and E6^E7*II (KU199315) fusion transcript variants. Conclusion Thus, significant association of hrHPV with prognosis of Indian BC patients led to additional investigation of HPV16 profile. Outcomes indicated a plausible role of HPV in Indian BC patients.

Head and neck cancers constitute a multifactorial global disease burden and are associated with human papilloma virus (HPV) as a possible risk factor. The aim of the study is to understand the relationship between HPV and the development of head and neck lesions in Indian patients. To this end, frequency of HPV was assessed in relation to different demographic and etiological features and correlated with patient survival. The prevalence of HPV significantly increased from mild dysplastic lesions (43.6%) to head and neck squamous cell carcinoma (HNSCC) stage IV (68.5%) with HPV 16 being pre-dominant in both dysplasia (43.8%) and HNSCC (61.5%). Similar trend was observed in increasing grades of the tumour. In invasive lesions, patients aged below the median age of onset showed significantly higher occurrence of HPV than those above it. Patients harbouring HPV showed a significantly better survival irrespective of age of onset. Likewise, better survival was observed in tobacco habit negative/HPV-positive patients, and as reflected in both univariate and multivariate analysis. Majority of the HPV 16-positive samples showed moderate/high nuclear expression of HPV E6 and E7 proteins in tumours and respective basal layer of adjacent normal tissues. Thus, our data indicate that frequent HPV infection, along with tobacco habit, is a pre-requisite factor for the development of HNSCC of Indian patients but offers a better survival even during tobacco usage, implicating its diagnostic and prognostic importance.

The aim of the study was to understand the role of SLIT2-ROBO1/2-CDC42 signalling pathways in development of breast cancer (BC). Primary BC samples (n = 150), comprising of almost equal proportion of four subtypes were tested for molecular alterations of SLIT2, ROBO1, ROBO2 and CDC42, the key regulator genes of this pathway. Deletion and methylation frequencies of the candidate genes were seen in the following order: deletion, SLIT2 (38.6%) > ROBO1 (30%) > ROBO2 (7.3%); methylation, SLIT2 (63.3%) > ROBO1 (26.6%) >ROBO2 (9.3%). Majority (80%, 120/150) of the tumours showed alterations (deletion/methylation) in at least one of the candidate genes. Overall, alterations of the candidate genes were as follows: SLIT2, 75.3% (101/150); ROBO1, 45.3% (68/150); ROBO2, 15.3% (23/150). Significantly, higher alteration of SLIT2 locus was observed in triple negative breast cancer (TNBC) over HER2 subtype (P = 0.0014). Similar trend is also seen in overall alterations of SLIT2 and/or ROBO1, in TNBC than HER2 subtype (P = 0.0012); of SLIT2 and/or ROBO2 in TNBC than luminal A (P = 0.014) and HER2 subtype (P = 0.048). Immunohistochemical analysis of SLIT2, ROBO1/2 showed reduced expression, concordant with their molecular alterations. Also, high expression of total CDC42 (49/52; 94.2%) and reduced expression of phospho Serine-71 CDC42 (41/52; 78.8%) was observed. Coalterations of SLIT2 and/or ROBO1, SLIT2 and/or ROBO2 had significant association with reduced expression of phospho Serine-71 CDC42 (P = 0.0012-0.0038). Alterations of SLIT2 and/or ROBO1, reduced expression of phospho Serine-71 CDC42 predicted poor survival of BC patients. Results indicate the importance of SLIT2-ROBO1-CDC42 signalling pathway in predicting tumour progression.

Aim: To understand the importance of homologous recombination repair pathway in development of breast carcinoma (BC), alterations of some key regulatory genes like BRCA1, BRCA2, FANCC and FANCD2 were analyzed in pretherapeutic/neoadjuvant chemotherapy (NACT)-treated BC samples. Materials & methods: Alterations (deletion/methylation/expression) of the genes were analyzed in 118 pretherapeutic and 41 NACT-treated BC samples. Results: High deletion/methylation (29–68%) and 64–78% overall alterations of the genes were found in the samples. Concordance was evident between alteration and protein expression of the genes. Estrogen/progesterone receptor-negative tumors showed significantly high alterations even in NACT-treated samples having low CD44 and proliferating cell nuclear antigen expression. Pretherapeutic patients with alterations showed poor prognosis. Conclusion: Alterations of homologous recombination repair pathway genes are needed for the development of BC.

The Accepted Manuscript version of this article (published on 1 June 2016) was withdrawn on 24 November 2017 at the request of the authors.

MCPH1 is a proximal regulator of DNA damage response pathway that is involved in recruitment of phosphorylated ATM to double-stranded DNA breaks. To understand the importance of MCPH1 and ATM in deregulation of DNA damage response pathway in breast carcinoma, we studied m-RNA expression and genetic/epigenetic alterations of these genes in primary breast carcinoma samples. Our study revealed reduced expression (mRNA/protein) and high alterations (deletion/methylation) (96 %, 121 of 126) of MCPH1 and ATM. Mutation was, however, rare in inactivation of MCPH1. In immunohistochemical analysis, reduced protein expression of MCPH1, ATM and p-ATM were concordant with their molecular alterations (P = 0.03–0.01). Alterations of MCPH1 and deletion of ATM were significantly high in estrogen/progesterone receptor–negative than estrogen/progesterone receptor–positive breast carcinoma samples compared to early or late age of onset tumors, indicating differences in pathogenesis of the molecular subtypes (P = 0.004–0.01). These genes also showed differential association with tumor stage, grade and lymph node status in different subtypes of breast carcinoma (P = 0.00001–0.01). Their coalterations showed significant association with tumor progression and prognosis (P = 0.003–0.05). Interestingly, patients with alterations of these genes or MCPH1 alone had poor outcome after treatment with DNA-interacting drugs and/or radiation (P = 0.01–0.05). Inactivation of MCPH1-ATM-associated DNA damage response pathway might have an important role in the development of breast carcinoma with diagnostic, prognostic and therapeutic implications.

The adenomatous polyposis coli (APC) and mutated in colorectal cancer (MCC) genes are key regulatory genes of the Wnt/β-catenin signaling pathway, which are independently involved in maintaining low levels of β-catenin in the cell. In addition to genetic and epigenetic alterations, some genetic polymorphisms in the genes associated with the Wnt signaling pathway have been reported to be associated with an increased risk of cancer, including breast cancer. In the present study we analyzed the association of genotype and haplotype status of two single nucleotide polymorphisms (SNPs), rs2229992 and rs11283943, in the APC and MCC genes, respectively, with an increased risk of breast carcinogenesis in a breast cancer and control population from eastern India. We observed a significant association of the rs11283943 SNP with increased breast cancer risk. Two specific haplotypes involving the minor allele of rs11283943 were found to be associated with an increased breast cancer risk. Kaplan-Meier curves showed a significant association of the 2–2 genotype (genotype homozygous for the rs11283943 minor allele) with decreased survival (p=0.045) of the breast cancer patients in our study, in particular patients with early-onset BC.

Background: It is now clear that response or resistance to therapy is contingent on the tumor microenvironment, which is comprised of malignant cells, normal stroma, and immune landscape; attributes that are unique to each individual patient. This is particularly true for emerging anticancer drugs, such as immune checkpoint inhibitors, which recalibrate the body's own immune defense largely by modulating exhaustion of cytotoxic lymphocytes including T cells and natural killer (NK) cells. However, clinical response to therapy varies enormously. There is a critical gap in our understanding for the mechanisms that drive response or resistance to conventional drugs and immunotherapies at the individual patient level. Methods: Here, we used in-vitro and in-vivo experiments to study the role of NK cells in models of drug tolerance. Next, we employed (CANscriptTM), a clinically-validated ex-vivo tumor model that recreates and preserves the native, patient tumor microenvironment, which integrates an algorithm-driven method to predict clinical response to therapy (M-Score). Utilizing tissue from patients diagnosed with luminal, HER2 positive, and triple-negative (ER- PR- HER2-) breast cancers (N=10), we studied spatial heterogeneity of the tumor-immune contexture, M-Score, and phenotypic alterations under pressure of conventional standard-of-care regimens and immunotherapies including immune-checkpoint inhibitors. To do this, we used a comprehensive panel of immunological assays to evaluate changes in cytotoxic lymphocytes by flow cytometry, immunohistochemistry and cytokine profiling (i.e. CD56, MHC class 1A/B, NKG2D/C, CD8, CD3, PD-1, CTLA-4, TIM-3, LAG-3, 4-1BB, granzyme A/B). Results: We identified that drug resistance is associated to downregulation of activating proteins on NK cells including NKG2D/C, reducing their cytotoxic capability. Furthermore, tumor response, predicted by M-Score, correlates to a unique pattern of spatially-distributed NK cells, which associated a pro-inflammatory cytokine signature from the tumor microenvironment. Interestingly, we determined that therapy-induced expression of exhaustion biomarkers on NK cell exhaustion inversely correlated to the expression of cytotoxic granzyme B in the tumor microenvironment. Conclusions: Taken together, these data demonstrate an integral role that NK cells contribute to resistance and the antitumor response to therapy including both conventional and immuno-modulatory drugs. We further demonstrate that CANscriptTM can be harnessed to study response and resistance of the innate and adaptive immune system. Such an advance in our preclinical methods to study anticancer drugs at the individual patient level can help guide treatment decisions for clinicians while simultaneously functioning as a platform to study clinical efficacy of novel and emerging agents. Citation Format: Munisha Smalley, Basava U. Shanthappa, Hans Gertje, Mark Lawson, Manjusha Biswas, Saravanan Thiyagarajan, Biswanath Majumder, D. C. Doval, Anurag Mehta, Neyaz Alam, Nabendu Murmu, S. P. Somashekhar, Aaron Goldman. Spatial re-distribution of natural killer cells targets drug resistance and anti-tumor response, ex-vivo [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 4064.

To understand the association of candidate tumour suppressor genes SH3GL2, p16INK4a , p14ARF, and p15INK4b in the pathogenesis of head and neck squamous cell carcinoma (HNSCC), we studied the deletion, mutation, and methylation of these genes in 61 dysplastic lesions and 94 HNSCC samples. In mild dysplasia, SH3GL2, p16INK4a, and p14ARF showed a higher frequency of overall alterations (60–70%) than in p15INK4b (40%). However, in subsequent stages of tumour progression, the alteration frequency of these genes did not change significantly. One novel mutation in common exon 2 of p16INK4a/p14ARF and three in exon 9 of SH3GL2 were seen. Concordance was seen in the expression of these genes with their molecular alterations. Deletions of INK4A-ARF and p15INK4b have a significant poor patient outcome. The alterations of p16INK4a , p14ARF, and p15INK4b were positively correlated with tobacco and inversely with HPV, while SH3GL2 alterations were independent of these factors. Based on aetiological factors, four tumour subtypes were recognized: HPV−tobacco− (I), HPV+tobacco− (II), HPV−tobacco+ (III), and HPV+tobacco+ (IV). Groups III and IV showed a high frequency of p16INK4a/p14ARF /p15INK4b alterations with significant poor patient outcome in comparison to group II. Our findings suggest that deregulation of SH3GL2-associated signalling and p16INK4a/p14ARF/p15INK4b-mediated G1–S/G2–M checkpoints of cell cycle are independent pathways for the development of early dysplastic lesions of the head and neck. Copyright  2008 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.

Genetic and epigenetic alterations in genes associated with distinct cellular pathways were checked in fibroepithelial tumors, including fibroadenomas, benign and malignant phyllode and atypical ductal hyperplasia. A panel of 22 genes associated with different cellular pathways such as stem cell renewal (Wnt and Hedgehog), DNA damage response [homologous recombination (HR), mismatch repair (MMR) and nucleotide excision repair (NER)] and cell proliferation signaling pathway were tested. Alterations (genetic/epigenetic) of the genes associated with Wnt signaling pathway were detected in 100% (20/20) of the breast tumors for at least one out of the six Wnt antagonists tested. Frequent molecular alterations (57-64%) were detected in HR and MMR pathway and low frequency of alterations (8-25%) were seen in cell-proliferation and cell signaling pathways showing a differential pattern of alterations in different tumor types. The patterns of alterations, in particular the epigenetic alterations, differed little from that seen previously in breast carcinoma cells, suggesting epigenetic alterations to be an early event in the development of the tumors. In gene ontology analysis, it was evident that Wnt signaling pathway [GO: 0030111, Kegg: 04310], cell proliferation pathway [GO: 0008285] and pathways in cancer [Kegg: 05200] were significantly enriched by differentially altered genes in fibroadenoma and phyllode tumor types. All these results may provide a new breakthrough in early diagnosis, prognosis and treatment of these tumors.

To understand the importance of frequent deletions at chromosome 11q24.1-24.2 region in breast carcinoma, alterations (deletion/methylation) of the candidate genes LOH11CR2A , ROBO3, ROBO4 , HEPACAM , PIG8 and CHEK1 located in this region were analyzed in 106 breast carcinoma samples. Among these genes, LOH11CR2A showed highest frequency of deletion (56%), followed by PIG8 (35%), CHEK1 (31%) and ROBO3 / ROBO4 / HEPACAM loci (28%). Comparable frequency of promoter methylation (26–35%) was observed for LOH11CR2A , CHEK1 and PIG8 . Overall alterations (deletion/methylation) of these genes were in the following order: LOH11CR2A (60%) > PIG8 (46%) > CHEK1 (41%) and showed significant association with each other. Breast carcinoma samples that were estrogen/progesterone receptor negative showed significantly high deletion and overall alterations than estrogen/progesterone receptor positive samples for LOH11CR2A , CHEK1 and PIG8 . The methylation and overall alteration of LOH11CR2A were significantly associated with tumor stages in breast carcinoma. However, in early/late onset and estrogen/progesterone receptor positive/negative breast carcinoma, the overall alterations of LOH11CR2A , PIG8 and CHEK1 were differentially associated with advanced stages, tumor grade and lymph node metastasis. Alterations of PIG8 and CHEK1 were significantly associated with poor prognosis in patients with early age of onset of the disease indicating significant prognostic importance. Quantitative mRNA expression analysis detected reduced expression of the genes in the order LOH11CR2A > CHEK1 > PIG8 . Immunohistochemical analysis showed reduced protein expression of PIG8 and CHEK1 that was concordant with their molecular alterations. Thus, our study suggests that LOH11CR2A, PIG8 and CHEK1 are candidate tumor suppressor genes associated with breast carcinoma and have significant clinical as well as prognostic importance.