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Anti-apoptotic proteins, including Bcl-2 and Bcl-xL, hinder cancer treatment, and several drugs targeting these molecules have been developed. One is ABT-263 (navitoclax), which targets Bcl-2, Bcl-xL, and Bcl-w. On the other hand, hydroxychloroquine (HCQ) has been used as a drug for malaria infection and autoimmune disease. HCQ can exert a similar effect as chloroquine with fewer adverse events. In addition, HCQ exerts antitumor activity. In the present study, the effects of HCQ on ABT-263-induced antitumor activities were examined using three human pancreatic cancer cell lines (PANC-1, MiaPaCa-2, and BxPC-3).In vitro effects of HCQ and ABT-263 were examined by cell viability, colony-forming assays, and flow cytometry. Protein expression was determined by immunoblotting. In vivo effects of HCQ and ABT-263 were examined by a xenograft mice model.Combined treatment with HCQ and ABT-263 synergistically decreased the viability of only BxPC-3 cells. This synergistic effect was not observed when HCQ was combined with ABT-199, an inhibitor specific to Bcl-2. The combination of HCQ and ABT-263 induced caspase-dependent apoptosis. Protein expression of Bcl-xL was more highly expressed in BxPC-3 cells than in the other two cell lines, and the combination of HCQ with a Bcl-xL inhibitor or siRNA-mediated knockdown of Bcl-xL induced apoptosis in BxPC-3 cells. Combination therapy with HCQ and ABT 737, an ABT-263 analogue, suppressed the in vivo growth of BxPC-3 with transient body-weight loss.HCQ effectively promotes Bcl-xL inhibition-induced apoptosis in BxPC-3 human pancreatic cancer cells.

Background HPV-positive oropharyngeal squamous cell carcinomas (OPSCCs) are sensitive to chemo-radiation therapy and have favorable survival outcomes compared with HPV-negative cancers. These tumors are usually not related to tobacco and alcohol exposure. Therefore, diagnosing HPV-positive OPSCCs for the appropriate disease management is crucial, and no suitable markers are available for detecting early malignancies in HPV-infected tissues. In this study, we attempt to find HPV-specific epigenetic biomarkers for OPSCCs. Methods A total of 127 surgical samples were analyzed for HPV positivity and promoter methylation of a panel of genes. HPV detection was performed by PCR detection of HPV E6 and E7 viral oncoproteins. In addition, promoter methylation of a total of 8 genes (DAPK, FHIT, RASSF1A, TIMP3, AGTR1, CSGALNACT2, GULP1 and VGF) was analyzed by quantitative-methylation specific PCR (QMSP), and their associations with HPV positivity or RB/p16 expressions were evaluated. Results AGTR1 and FHIT were frequently methylated in HPV-positive OPSCC samples with a good area under the curve (AUC over 0.70). In addition, these genes' promoter methylation was significantly associated with p16 positive and RB negative cases, which were the characteristics of OPSCC cases with favorable survival outcomes. Either AGTR1 or FHIT methylated cases were significantly associated with HPV-positive cancers with 92.0% sensitivity (P P = 0.047) than both unmethylated cases. Conclusions A combination of AGTR1 and FHIT methylation demonstrated a suitable detection marker of OPSCCs derived from the HPV-infected field, familiar with p16-positive and RB-negative phenotypes.

Mutations in mitochondrial DNA (mtDNA) have been linked to risk, progression, and treatment response of head and neck squamous cell carcinoma (HNSCC). Due to their clonal nature and high copy number, mitochondrial mutations could serve as powerful molecular markers for detection of cancer cells in bodily fluids, surgical margins, biopsies and lymph node (LN) metastasis, especially at sites where tumor involvement is not histologically apparent. Despite a pressing need for high-throughput, cost-effective mtDNA mutation profiling system, current methods for library preparation are still imperfect for detection of low prevalence heteroplasmic mutations. To this end, we have designed an ultra-deep amplicon-based sequencing library preparation approach that covers the entire mitochondrial genome. We sequenced mtDNA in 28 HNSCCs, matched LNs, surgical margins and bodily fluids, and applied multiregional sequencing approach on 14 primary tumors. Our results demonstrate that this quick, sensitive and cost-efficient method allows obtaining a snapshot on the mitochondrial heterogeneity, and can be used for detection of low frequency tumor-associated mtDNA mutations in LNs, sputum and serum specimens. These findings provide the foundation for using mitochondrial sequencing for risk assessment, early detection, and tumor surveillance.

Pericardial cysts are rare benign intrathoracic lesions, and calcified pericardial cysts are even more uncommon. Most pericardial cysts are asymptomatic, but patients may present with chest pain, dyspnea and any complications of pericardial effusion. We present a case of a left-sided calcified pericardial cyst, highlighting its rarity and symptoms in relation to its location.

Simple Summary Effective cancer treatment hinges upon overcoming therapeutic resistance mechanisms that allow for the continued proliferation of cancer cell subpopulations. Exposure to pharmacotherapy invariably leads to resistance as tumor cells with selected advantageous features evade destruction and alter the tumor composition. Cancer stem cells (CSCs) with features of plasticity that allow for regeneration and differentiation are particularly responsible for this phenomenon. Advances in tumor biology and molecular signaling have highlighted their role in neoplastic initiation, invasion, and maintenance. Novel strategies to direct therapy against these tumor cell subpopulations have the potential to dramatically alter tumor response and change the course of cancer care. Abstract Resistance to cancer therapy remains a significant obstacle in treating patients with various solid malignancies. Exposure to current chemotherapeutics and targeted agents invariably leads to therapy resistance, heralding the need for novel agents. Cancer stem cells (CSCs)—a subpopulation of tumor cells with capacities for self-renewal and multi-lineage differentiation—represent a pool of therapeutically resistant cells. CSCs often share physical and molecular characteristics with the stem cell population of the human body. It remains challenging to selectively target CSCs in therapeutically resistant tumors. The generation of CSCs and induction of therapeutic resistance can be attributed to several deregulated critical growth regulatory signaling pathways such as WNT/β-catenin, Notch, Hippo, and Hedgehog. Beyond growth regulatory pathways, CSCs also change the tumor microenvironment and resist endogenous immune attack. Thus, CSCs can interfere with each stage of carcinogenesis from malignant transformation to the onset of metastasis to tumor recurrence. A thorough review of novel targeted agents to act against CSCs is fundamental for advancing cancer treatment in the setting of both intrinsic and acquired resistance.

Objectives In this study we describe the tumor microenvironment, the signaling pathways and genetic alterations associated with the presence or absence of CD8+ T-cell infiltration in primary squamous cell carcinoma of the head and neck (SCCHN) tumors. Materials and Methods Two SCCHN multi-analyte cohorts were utilized, the Cancer Genome Atlas (TCGA) and the Chicago Head and Neck Genomics (CHGC) cohort. A well-established chemokine signature classified SCCHN tumors into high and low CD8+ T-cell inflamed phenotypes (TCIP-H, TCIP-L respectively). Gene set enrichment and iPANDA analyses were conducted to dissect differences in signaling pathways, somatic mutations and copy number aberrations for TCIP-H versus TCIP-L tumors, stratified by HPV status. Results TCIP-H SCCHN tumors were enriched in multiple immune checkpoints irrespective of HPV-status. HPV-positive tumors were enriched in markers of T-regulatory cells (Tregs) and HPV-negative tumors in protumorigenic M2 macrophages. TCIP-L SCCHN tumors were enriched for the β-catenin/WNT and Hedgehog signaling pathways, had frequent mutations in NSD1, amplifications in EGFR and YAP1, as well as CDKN2A deletions. TCIP-H SCCHN tumors were associated with the MAPK/ERK, JAK/STAT and mTOR/AKT signaling pathways, and were enriched in CASP8, EP300, EPHA2, HRAS mutations, CD274, PDCD1LG2, JAK2 amplifications. Conclusions Our findings support that combinatorial immune checkpoint blockade and depletion strategies targeting Tregs in HPV-positive and M2 macrophages in HPV-negative tumors may lead to improved antitumor immune responses in patients with TCIP-H SCCHN. We highlight novel pathways and genetic events that may serve as candidate biomarkers and novel targeted therapies to enhance the efficacy of immunotherapy in SCCHN patients.

Mammary gland cancer is the most common cancer occurring in women globally. Incidences of this cancer in Japan are on the increase. Annually, more than 70,000 new cases are recorded in Japan and about 1.7 million in the world. Many cases are still difficult to cure completely, and animal models are required for the characterization of the biology, therapeutic strategy, and preventive measures for spontaneous mammary tumor. The mouse model used currently has some limitations owing to structural differences between mouse and human mammary glands. Tupaia belangeri (tree shrew), which belongs to the Tupaiidae family, shows relatively high genetic homology and structural similarity to human mammary glands. Here, we characterized the spontaneous mammary tumors in 61 female tree shrews of different ages. The incidence rate was 24.6% (15/61), and the rate of simultaneous or metachronous multiplex tumors was 60% (9/15). From the incidence pattern, some cases seemed to be of familial mammary gland tumor, as the offspring of female tree shrews No. 3 and 9 and male tree shrew No. 11 showed a high incidence rate, of 73.3% (11/15). Average incidence age for tumor development was 2 years and 3 months, and the earliest was 10 months. Histochemical analysis indicated that spontaneous mammary gland tumors in the tree shrew show the features of intraductal papillary adenomas (22 cases), except 2 tubulopapillary carcinoma cases (No. 75 and 131). All the cases were positive for the progesterone receptor, whereas 91.3% were positive for the estrogen receptor, and 4.3% were HER-2 positive. We have also confirmed the expression of nectin-4 in some mammary tumor cells. Additionally, we subjected tree shrews to cytodiagnosis or X-ray CT. Thus, the findings of this study highlight the potential of the tree shrew as a valuable new animal model for mammary gland tumor study.

Hepatocellular carcinoma (HCC) is the sixth most common cancer and the third most common cause of cancer mortality worldwide. Infection with hepatitis B virus (HBV) and/or hepatitis C virus (HCV) is the most predominant cause of HCC. Concerns arise for the presence of additional risk factors, as there is still a large proportion of patients without HBV or HCV infection. Previous studies have reported that higher intake of fruits and vegetables and reduced consumption of red/processed meat might play a protective role in HCC etiology, though the nationwide proof is limited. Hence, we studied multiple risk factors including food habit, lifestyle, and clinical implications of HCC patients in Bangladeshi. Demographic, clinical, and biochemical data, as well as data on food habits, were collected in this study. Our results indicated that a high intake of rice (AOR 4.28, 95% CI 1.48 to 14.07, p = 0.011), low intake of fruits (AOR = 4.41 95% CI 1.48-15.46; p = 0.012), leafy vegetables (AOR = 2.80, 95% CI 1.32-6.08; p = 0.008), and fish (AOR = 4.64 95% CI 2.18-10.23; p

Many epigenetically inactivated genes involved in ovarian cancer (OC) development and progression remain to be identified. In this study we undertook an integrated approach that consisted of identification of genome-wide expression patterns of primary OC samples and normal ovarian surface epithelium along with a pharmacologic unmasking strategy using 3 OC and 3 immortalized normal ovarian epithelial cell lines. Our filtering scheme identified 43 OC specific methylated genes and among the 5 top candidates (GULP1, CLIP4, BAMBI, NT5E, TGFβ2), we performed extended studies of GULP1. In a training set, we identified GULP1 methylation in 21/61 (34%) of cases with 100% specificity. In an independent cohort, the observed methylation was 40% (146/365) in OC, 12.5% (2/16) in borderline tumors, 11% (2/18) in cystadenoma and 0% (0/13) in normal ovarian epithelium samples. GULP1 methylation was associated with clinicopathological parameters such as stage III/IV (p = 0.001), poorly differentiated grade (p = 0.033), residual disease (p

Most cancers are genetically heterogeneous diseases, and many genes or gene products have altered their expressions or activities in cancer cells due to a variety of genetic and epigenetic mechanisms in DNA. Copy number aberrations (CNAs), common genomic events during carcinogenesis, are known to affect a large fraction of genome and extensively studied for last several decades.

YAP1 is one of the most important effectors of the Hippo pathway and has crosstalk with other cancer promoting pathways. YAP1 contributes to cancer development in various ways that include promoting malignant phenotypes, expansion of cancer stem cells and drug resistance of cancer cells. Because pharmacologic or genetic inhibition of YAP1 suppresses tumor progression and increases the drug sensitivity, targeting YAP1 may open a fertile avenue for a novel therapeutic approach in relevant cancers. Recent enormous studies have established the efficacy of immunotherapy, and several immune checkpoint blockades are in clinical use or in the phase of development to treat various cancer types. Immunosuppression in the tumor microenvironment (TME) induced by cancer cells, immune cells and associated stromal cells promotes tumor progression and causes drug resistance. Accumulated evidences of scientific efforts from the last few years suggest that YAP1 influences macrophages, myeloid-derived suppressor cells and regulatory T-cells to facilitate immunosuppressive TME. Although the underlying mechanisms is not clearly discerned, it is evident that YAP1 activating pathways in different cellular components induce immunosuppressive TME. In this review, we summarize the evidences involved in the dual roles of YAP1 in cancer development and immunosuppression in the TME. We also discuss the possibility of YAP1 as a novel therapeutic target.

Chronic arsenic exposure is associated with the development of urothelial carcinoma of the bladder (UCB). To elucidate the contribution of arsenic exposure to urothelial cancer stem cell (CSC) generation, we established an in vitro stepwise malignant model transformed by chronically exposing human urothelial cells to arsenic. Using this model, we found that chronic arsenic exposure endows urothelial cells with malignant stemness properties including increased expression of stemness-related factors such as SOX2, sphere formation, self-renewal, invasion and chemoresistance. SOX2 was gradually and irreversibly overexpressed in line with acquired sphere-forming and self-renewal abilities. Following gene set enrichment analyses of arsenic-exposed and arsenic-unexposed cells, we found COX2 as an enriched gene for oncogenic signature. Mechanistically, arsenic-induced COX2/PGE2 increases SOX2 expression that eventually promotes malignant stem cell generation and repopulation. In urine samples from 90 subjects exposed to arsenic and 91 control subjects, we found a significant linear correlation between SOX2 and COX2 expression and the potential of SOX2 and COX2 expression as urinary markers to detect subjects exposed to arsenic. Furthermore, the combination marker yielded a high sensitivity for UCB detection in a separate cohort. Finally, our in vitro model exhibits basal-type molecular features and dual inhibition of EGFR and COX2 attenuated stem cell enrichment more efficiently than an EGFR inhibitor alone. In conclusion, the COX2/PGE2-SOX2 axis promotes arsenic-induced malignant stem cell transformation. In addition, our findings indicate the possible use of SOX2 and COX2 expression as urinary markers for the risk stratification and detection of UCB.

Urothelial Carcinoma of Bladder (UCB) is a disease of multiple origins of the same bladder with varying in morphology and genetic changes. The origin of spatially different tumors in the same bladder has been proposed to occur through clonal changes with intraluminal migration or field effect with synchronous transformation of multiple cells by carcinogens. A clear molecular understanding of the origin of multifocal UCB will have important implications for the proper tailoring of personalized targeted treatment and diagnostics. Despite efforts to identify intertumoral heterogeneity, the clonal origin of UCB is still debated. To further understand the genetic mechanism behind the multifocality of UCB, we performed targeted next-generation sequencing (tNGS) on 39 UCB lesions obtained from 15 cystectomized bladders. Germline distant muscle DNA was used as a control. We technically validated selected mutational events by using ultra-sensitive droplet digital PCR (ddPCR). Our data showed that multifocal UCB is of clonal origin, with lesions from individual patients displaying nearly identical mutational landscapes, with few heterogeneous events that may have been acquired at a later point. tNGS demonstrated similar genetic drivers within the same patient and reveal that clonal expansion is an early event of tumorigenesis. In addition, we found distinct gene mutations in different UCB patients suggesting prominent interpatient heterogeneity of clonal events in the UCB. Among others, our result showed that TP53, FGFR3, and NOTCH4 are the most frequently mutated genes in UCB samples. ddPCR assay of mutational events on TP53, FGFR3, AKT, PIK3CA genes confirmed TNGS data and solidified that mutation in TP53, FGFR3, AKT, PIK3CA genes are associated with tumorigenesis of uroepithelium. In summary, this study conveys insight into the clonal origin of multifocal UCB and demonstrates that TNGS is a sensitive and reliable method for interrogating the molecular landscape of UCB. These findings also may inform clinical practice as sequencing of a single focus from a patient presenting with the multifocal disease may be sufficient to understand the mutational profile and guide personalized treatment and risk stratification. Citation Format: M. Talha Ugurlu, Rachel Goldberg, Pritam Sadhukhan, Mohammad O. Hoque. Targeted sequencing revealed clonal genetic changes in spatially different foci in urothelial carcinoma of bladder [abstract]. In: Proceedings of the AACR-NCI-EORTC Virtual International Conference on Molecular Targets and Cancer Therapeutics; 2021 Oct 7-10. Philadelphia (PA): AACR; Mol Cancer Ther 2021;20(12 Suppl):Abstract nr P094.

Overcoming acquired drug resistance remains a core challenge in the clinical management of human cancer, including in urothelial carcinoma of the bladder (UCB). Cancer stem-like cells (CSC) have been implicated in the emergence of drug resistance but mechanisms and intervention points are not completely understood. Here, we report that the proinflammatory COX2/PGE2 pathway and the YAP1 growth-regulatory pathway cooperate to recruit the stem cell factor SOX2 in expanding and sustaining the accumulation of urothelial CSCs. Mechanistically, COX2/PGE2 signaling induced promoter methylation of let-7, resulting in its downregulation and subsequent SOX2 upregulation. YAP1 induced SOX2 expression more directly by binding its enhancer region. In UCB clinical specimens, positive correlations in the expression of SOX2, COX2, and YAP1 were observed, with coexpression of COX2 and YAP1 particularly commonly observed. Additional investigations suggested that activation of the COX2/PGE2 and YAP1 pathways also promoted acquired resistance to EGFR inhibitors in basal-type UCB. In a mouse xenograft model of UCB, dual inhibition of COX2 and YAP1 elicited a long-lasting therapeutic response by limiting CSC expansion after chemotherapy and EGFR inhibition. Our findings provide a preclinical rationale to target these pathways concurrently with systemic chemotherapy as a strategy to improve the clinical management of UCB. Significance: These findings offer a preclinical rationale to target the COX2 and YAP1 pathways concurrently with systemic chemotherapy to improve the clinical management of UCB, based on evidence that these two pathways expand cancer stem-like cell populations that mediate resistance to chemotherapy. Cancer Res; 78(1); 168–81. ©2017 AACR.

Background: While patient-derived xenografts (PDXs) offer a powerful modality for translational cancer research, a precise evaluation of how accurately patient responses correlate with matching PDXs in a large, heterogeneous population is needed for assessing the utility of this platform for preclinical drug-testing and personalized patient cancer treatment. Patients and methods: Tumors obtained from surgical or biopsy procedures from 237 cancer patients with a variety of solid tumors were implanted into immunodeficient mice and whole-exome sequencing was carried out. For 92 patients, responses to anticancer therapies were compared with that of their corresponding PDX models. Results: We compared whole-exome sequencing of 237 PDX models with equivalent information in The Cancer Genome Atlas database, demonstrating that tumorgrafts faithfully conserve genetic patterns of the primary tumors. We next screened PDXs established for 92 patients with various solid cancers against the same 129 treatments that were administered clinically and correlated patient outcomes with the responses in corresponding models. Our analysis demonstrates that PDXs accurately replicate patients' clinical outcomes, even as patients undergo several additional cycles of therapy over time, indicating the capacity of these models to correctly guide an oncologist to treatments that are most likely to be of clinical benefit. Conclusions: Integration of PDX models as a preclinical platform for assessment of drug efficacy may allow a higher success-rate in critical end points of clinical benefit.

Cancer stem cells (CSC) are highly resistant to conventional chemotherapeutic drugs. YAP1 and STAT3 are the two transcription factors that facilitate the therapeutic resistance and expansion of CSCs. The objective of this study was to understand the cross-talk between YAP1 and STAT3 activities and to determine the therapeutic efficacy of targeting dual CSC-regulating pathways (YAP1 and STAT3) combined with chemotherapy in lung adenocarcinoma. Here, we showed that YAP1 contributes to CSC regulation and enhances tumor formation while suppressing apoptosis. Mechanistically, YAP1 promotes phosphorylation of STAT3 by upregulating IL6. In lung adenocarcinoma clinical specimens, YAP1 expression correlated with that of IL6 (P < 0.01). More importantly, YAP1 and phosphorylated STAT3 (pSTAT3) protein expressions were significantly correlated (P < 0.0001) in primary lung adenocarcinoma as determined by IHC. Immunoblotting of 13 lung adenocarcinoma patient-derived xenografts (PDX) showed that all YAP1-expressing PDXs also exhibited pSTAT3. Additional investigations revealed that chemotherapy resistance and malignant stemness were influenced by upregulating NANOG, OCT4, and SOX2, and the expression of these targets significantly attenuated by genetically and pharmacologically hindering the activities of YAP1 and STAT3 in vivo and in vitro. Therapeutically, the dual inhibition of YAP1 and STAT3 elicits a long-lasting therapeutic response by limiting CSC expansion following chemotherapy in cell line xenograft and PDX models of lung adenocarcinoma. Collectively, these findings provide a conceptual framework to target the YAP1 and STAT3 pathways concurrently with systemic chemotherapy to improve the clinical management of lung adenocarcinoma, based on evidence that these two pathways expand CSC populations that mediate resistance to chemotherapy.

Cancer stem cells (CSCs) are subpopulations of tumor cells with the ability to self-renew, differentiate, and initiate and maintain tumor growth, and they are considered to be the main drivers of intra- and inter-tumoral heterogeneity. While conventional chemotherapy can eradicate the majority of non-CSC tumor cells, CSCs are often drug-resistant, leading to tumor recurrence and metastasis. The heterogeneity of CSCs is the main challenge in developing CSC-targeting therapy; therefore, we and other investigators have focused on developing novel therapeutic strategies that combine conventional chemotherapy with inhibitors of CSC-regulating pathways. Encouraging preclinical findings have suggested that CSC pathway blockade can indeed enhance cellular sensitivity to non-targeted conventional therapy, and this work has led to several ongoing clinical trials of CSC pathway inhibitors. Our studies in bladder cancer and lung adenocarcinoma have demonstrated a crucial role of YAP1, a transcriptional regulator of genes that promote cell survival and proliferation, in regulating CSC phenotypes. Moreover, using cell lines and patient-derived xenograft models, we showed that inhibition of YAP1 enhances the efficacy of conventional therapies by attenuating CSC stemness features. In this review, we summarize the therapeutic strategies for targeting CSCs in several cancers and discuss the potential and challenges of the approach.

// Mariana Brait 1, * , Mithu Banerjee 1, * , Leonel Maldonado 1, 4 , Akira Ooki 1 , Myriam Loyo 1 , Elisa Guida 1 , Evgeny Izumchenko 1 , Leslie Mangold 2 , Elizabeth Humphreys 2 , Eli Rosenbaum 5 , Alan Partin 2 , David Sidransky 1, 3 , Mohammad Obaidul Hoque 1, 2, 3 1 Department of Otolaryngology and Head and Neck Surgery, The Johns Hopkins University School of Medicine, Baltimore, Maryland, USA 2 Department of Urology, The Johns Hopkins University School of Medicine, Baltimore, Maryland, USA 3 Department of Oncology, The Johns Hopkins University School of Medicine, Baltimore, Maryland, USA 4 Department of Pathology, University of South Alabama Medical Center, Mobile, Alabama, USA 5 Department of Urological Oncology, Davidoff Center, Beilinson Hospital, Eliahu Hakim, Ramat Aviv, Israel * These authors contributed equally to this work Correspondence to: Mohammad Obaidul Hoque, email: mhoque1@jhmi.edu Keywords: prostate cancer, methylation, early detection Received: June 08, 2016 Accepted: December 31, 2016 Published: January 28, 2017 ABSTRACT Background: Prostate cancer (PC) is the second most common cancer among men worldwide. Currently, the most common non-invasive approach for screening and risk assessment of PC is measuring the level of serum prostate-specific antigen (PSA). However, the sensitivity of PSA is 42.8 % and specificity is 41.1%. As a result, the serum PSA test leads to numerous unneeded biopsies. Therefore, a rigorous search for biomarkers for early detection of PC is ongoing. In this study, we aim to assess a panel of epigenetic markers in an intend to develop an early detection test for PC. Results: The sensitivity and specificity of hypermethylation of MCAM was 66% and 73% respectively which is an improvement from the sensitivity and specificity of PSA. Considering a combination marker panel of MCAM, ERα and ERβ increased the sensitivity to 75% and the specificity became 70% for the minimally invasive early detection test of PC. Materials and Methods: Sixteen primary matched tumor and serum were analyzed by quantitative methylation specific PCR (QMSP) to determine analytical and clinical sensitivity of the genes tested ( SSBP2, MCAM, ERα, ERβ, APC, CCND2, MGMT, GSTP1, p16 and RARβ2 ). Additionally, serum samples from eighty four cases of PC, thirty controls and seven cases diagnosed as high grade Prostatic Intraepithelial Neoplasia (HGPIN) were analyzed. Conclusions: Promoter methylation of MCAM, ERα and ERβ have a potential to be utilized as biomarker for the early detection of prostate PC as their sensitivity and specificity seem to be better than serum PSA in our cohort of samples. After robust validation in a larger prospective cohort, our findings may reduce the numbers of unwarranted prostate biopsies.

Chemoresistance and poor immune sensitivity are one of the major challenges to treat different cancer types including lung, bladder and head & neck. Different epigenetic regulators and cancer stem cells (CSCs) regulatory pathways play a critical role in the development of therapeutic resistance. Prolonged chemotherapy treatment enriches CSCs in the tumor microenvironment and in a previous study we have shown that inhibition of the CSCs regulatory pathway/s combined with chemotherapy decreased CSC properties and efficiently inhibit tumor growth in patient-derived xenografts (PDXs) models. The purpose of this study is to determine the chemo and immunotherapeutic efficacy in combination with several CSC pathway/s modulatory agent/agents to treat different solid tumors. To identify relevant CSC regulating pathways, we tested the therapeutic efficacy of different agents such as 5-Azacytidine (DNA methyltransferase 1 inhibitor), Vismodegib (SMO inhibitor), Romidepsin (HDAC1 inhibitor), Olaparib (PARP1 inhibitor), and GSK343 (EZH2 inhibitor) using different bladder and head & neck cancer cell lines and also determined the numerous CSCs properties. Our data indicate that in vitro treatment with these potential therapeutic agents decreases cell viability, sphere formation and wound healing that suggest that, these drugs may be effective in reducing CSCs population when combined with chemo and/or immunotherapy. Our immunoblotting experiments indicate reduced expression of some CSCs markers and factors such as such as NANOG, OCT4, CD133 and CD24. Interestingly, after treating the cells with some of these agents and array analysis, we have found differential expression of some of the key genes regulating the immunogenic response. Further, to confirm the array-based data, selected candidate immune-associated genes were analyzed by qRT-PCR. Moreover, our preliminary data suggest that combination treatment with epigenetic drugs and an immune agent may effectively reduce tumor growth in vivo compared to either of these drugs. In conclusion, although comprehensive mechanistic studies are ongoing, our initial results highlight the importance of considering combination of epigenetic and immune therapy for patients with diverse cancer types that respond poorly to immune and chemotherapy. Citation Format: Pritam Sadhukhan, Masahiro Shibata, M. Talha Ugurlu, Rachel Goldberg, Mohammad O. Hoque. Modulating cancer stem cell pathways to enhance systemic therapeutic efficacy [abstract]. In: Abstracts: AACR Special Virtual Conference on Epigenetics and Metabolism; October 15-16, 2020; 2020 Oct 15-16. Philadelphia (PA): AACR; Cancer Res 2020;80(23 Suppl):Abstract nr PO-016.

BACKGROUND Evaluation of programmed cell death ligand 1 (PD-L1) expression can be made on both resection specimens and diagnostic biopsies; however, more than 30% of patients with advanced non-small cell lung cancer (NSCLC) do not have adequate histologic material to perform PD-L1 assays and require additional biopsies. In addition, in our practice, more than 16% of cases have cytological smears as the only available material. Our aim was to validate the PD-L1 immunocytochemistry assay on cytological smears and compare its accuracy with the results obtained from tissue cores and whole tumor sections using the clinically relevant cutoff of 50%. METHOD We compared the PD-L1 staining results of cytological smears to those from tissue cores or whole sections in 50 and 53 NSCLC cases, respectively, using the SP263 assay after scanning hematoxylin and eosin slides. RESULTS We found an overall agreement of 90.6% between cytological smears and whole sections; specifically, we found absolute concordance between smears with PD-L1 expressed in

Pembrolizumab is the only programmed cell death 1/programmed death-ligand 1 inhibitor for treatment of patients with non-small cell lung cancer, with a companion diagnostic assay, the 22C3 PharmDx. Although in many studies 22C3 and Ventana's SP263 appear to yield overlapping results, they show discrepancies at clinically relevant cutoffs (1% and 50%). We provide a solid comparison between 22C3 and SP263 assays in a large cohort of non-small cell lung cancer cases taking into account interobserver variability between trained pathologists who are used to either clone in their clinical practice. Serial sections of tissue microarrays, built from 198 cases of resected lung cancer, were stained for 22C3 on the Dako Link-48 platform and for SP263 on the Ventana Benchmark Ultra, following manufacturer's instructions. A protocol was also developed to run the 22C3 antibody on the Ventana platform. The pathologist used to 22C3 scored consistently higher than the pathologist used to SP263 at both 1% and 50% cutoff for all assays. For 22C3 and SP263 on respective platforms, we found statistically significant differences in terms of proportion of positive cases at both cutoffs; at 50% cutoff, around half of the cases positive with SP263 would have been defined negative with 22C3 by both pathologists. Important differences were also observed, when comparing clone 22C3 and SP263, both run on the Ventana platform. The lowest differences were seen with 22C3 run on both platforms. Assays 22C3 and SP263 show important discrepancies in identifying programmed death-ligand 1-positive cases at clinically relevant cutoffs, with possible underestimation of patients suitable for pembrolizumab therapy.

Background CD24 is a cornerstone of tumour progression in urothelial carcinoma of the bladder (UCB). However, its contribution to cancer stem cell (CSC)-like traits and the clinical utility of CD24 as a urinary biomarker for cancer detection have not been determined. Methods The functional relevance of CD24 was evaluated using in vitro and in vivo approaches. The clinical utility of CSC-related molecules was assessed in urine samples by quantitative RT-PCR. Results The knockdown of CD24 attenuated cancer stemness properties. The high-CD24-expressing cells, isolated from patient-derived UCB xenograft tumours, exhibited their enhanced stemness properties. CD24 was overexpressed not only in primary tumours but also in urine from UCB subjects. By assessment of 15 candidate CSC-related molecules in urine samples of a training cohort, a panel of three molecules (CD24, CD49f, and NANOG) was selected. The combination of these three molecules yielded a sensitivity and specificity of 81.7% and 74.3%, respectively, in an independent cohort. A combined set of 84 cases and 207 controls provided a sensitivity and specificity of 82% and 76%, respectively. Conclusion CD24 has a crucial role in maintaining the urothelial cancer stem-like traits and a panel of CSC-related molecules has potential as a urinary biomarker for non-invasive UCB detection.

S100A2, a member of the S100 protein family, is known to be downregulated in a number of human cancers, leading to its designation as a potential tumor suppressor gene. Here, we investigated the expression and methylation status of S100A2 in head&neck and bladder cancer. Reduced mRNA and protein expression was observed in 8 head&neck and bladder cancer cell lines. To explore the mechanism responsible for the downregulation of S100A2, we treated six cell lines with 5-aza-2′-deoxycytidine. We found S100A2 is silenced in association with aberrant promoter-region methylation and its expression is restored with 5-aza-2′-deoxycytidine treatment. Of 31 primary head&neck cancer cases and 31 bladder cancer cases, promoter methylation was detected in 90% and 80% of cases, respectively. Interestingly, only 1/9 of normal head&neck tissues and 2/6 of normal bladder tissues showed promoter methylation. S100A2 promoter methylation can be detected in urine and is more frequent in bladder cancer patients than in healthy subjects (96% vs 48% respectively). Moreover, increased methylation of S100A2 is linked to the progression of the tumor in bladder cancer (p

// Enrico Munari 1, 2 , Giuseppe Zamboni 1, 2 , Gianluigi Lunardi 3 , Marcella Marconi 1 , Marco Sommaggio 1 , Matteo Brunelli 2 , Guido Martignoni 2, 4 , George J. Netto 5 , Mohammad O. Hoque 6 , Francesca Moretta 7 , Maria Cristina Mingari 8 , Maria Cristina Pegoraro 9 , Francesca Romana Mariotti 10 , Paola Vacca 10 , Lorenzo Moretta 10 and Giuseppe Bogina 1 1 Department of Pathology, Sacro Cuore Don Calabria Hospital, 37024 Negrar VR, Italy 2 Department of Diagnostics and Public Health, University of Verona, 37134 Verona VR, Italy 3 Department of Oncology, Sacro Cuore Don Calabria Hospital, 37024 Negrar VR, Italy 4 Department of Pathology, Pederzoli Hospital, 37019 Peschiera del Garda VR, Italy 5 Department of Pathology, The University of Alabama at Birmingham, Birmingham, AL 35233, USA 6 Department of Otolaringology, Johns Hopkins University School of Medicine, Baltimore, MD 21287, USA 7 Department of Laboratory Medicine, Sacro Cuore Don Calabria Hospital, 37024 Negrar VR, Italy 8 Department of Experimental Medicine (DIMES), University of Genova, 16132 Genova GE, Italy 9 Department of Oncology, Pederzoli Hospital, 37019 Peschiera del Garda VR, Italy 10 Immunology Research Area, IRCCS Bambino Gesu Pediatric Hospital, 00146 Rome RM, Italy Correspondence to: Enrico Munari, email: enrico_munari@yahoo.it Keywords: PD-L1; lung; cancer; heterogeneity; metastases Received: March 01, 2018 Accepted: June 23, 2018 Published: July 13, 2018 ABSTRACT We assessed the concordance, in terms of PD-L1 expression, between primary and metastatic non-small cell lung carcinoma (NSCLC) of different histotypes using validated SP263 clone. A few samples of local recurrences have also been analyzed. Whole sections of consecutive cases of primary NSCLC and paired relapses undergone surgical resection have been stained with PD-L1 clone SP263; for scoring purposes, a three-tiered system was applied using the following thresholds

Introduction Determination of programmed death ligand 1 (PD-L1) expression defines eligibility for treatment with pembrolizumab in patients with advanced NSCLC. This study was designed to better define which value across core biopsy specimens from the same case more closely reflects the PD-L1 expression status on whole sections and how many core biopsy specimens are needed for confident classification of tumors in terms of PD-L1 expression. Methods We built tissue microarrays as surrogates of biopsies collecting five cores per case from 268 cases and compared PD-L1 staining results obtained by using the validated clone SP263 with the results obtained by using whole tumor sections. Results We found an overall positivity in 39% of cases at a cutoff of 1% and in 10% of cases at a cutoff of 50%. The maximum value across cores was associated with high concordance between cores and whole sections and the lowest number of false-negative cases overall. To reach high concordance with whole sections, four and three cores are necessary at cutoffs of 1% and 50%, respectively. Importantly, with 20% as the cutoff for core biopsy specimens, fewer than three cores showed high sensitivity and specificity in identifying cases with 50% or more of tumor cells positive for PD-L1 on whole sections. Specifically, for PD-L1 expression values of 20% to 49% on cores, the probabilities of a tumor specimen expressing PD-L1 in at least 50% of cells on a whole section were 46% and 24% with one and two biopsy specimens, respectively. Conclusions An accurate definition of the criteria to determine the PD-L1 status of a given tumor may greatly help in selecting those patients who could benefit from anti–programmed cell death 1/PD-L1 treatment.

Purpose: To establish a novel panel of cancer-specific methylated genes for cancer detection and prognostic stratification of early-stage non–small cell lung cancer (NSCLC). Experimental Design: Identification of differentially methylated regions (DMR) was performed with bumphunter on “The Cancer Genome Atlas (TCGA)” dataset, and clinical utility was assessed using quantitative methylation-specific PCR assay in multiple sets of primary NSCLC and body fluids that included serum, pleural effusion, and ascites samples. Results: A methylation panel of 6 genes (CDO1, HOXA9, AJAP1, PTGDR, UNCX, and MARCH11) was selected from TCGA dataset. Promoter methylation of the gene panel was detected in 92.2% (83/90) of the training cohort with a specificity of 72.0% (18/25) and in 93.0% (40/43) of an independent cohort of stage IA primary NSCLC. In serum samples from the later 43 stage IA subjects and population-matched 42 control subjects, the gene panel yielded a sensitivity of 72.1% (31/41) and specificity of 71.4% (30/42). Similar diagnostic accuracy was observed in pleural effusion and ascites samples. A prognostic risk category based on the methylation status of CDO1, HOXA9, PTGDR, and AJAP1 refined the risk stratification for outcomes as an independent prognostic factor for an early-stage disease. Moreover, the paralog group for HOXA9, predominantly overexpressed in subjects with HOXA9 methylation, showed poor outcomes. Conclusions: Promoter methylation of a panel of 6 genes has potential for use as a biomarker for early cancer detection and to predict prognosis at the time of diagnosis. Clin Cancer Res; 23(22); 7141–52. ©2017 AACR.

Purpose: Recurrent prostate cancer remains a major problem. Staging, grading and prostate specific antigen level at surgery are helpful but still imperfect predictors of recurrence. For this reason there is an imperative need for additional biomarkers that add to the prediction of currently used prognostic factors.Materials and Methods: We evaluated the extent of promoter methylation of genes previously reported as aberrantly methylated in prostate cancer (AIM1, APC, CCND2, GPX3, GSTP1, MCAM, RARβ2, SSBP2 and TIMP3) by quantitative fluorogenic methylation-specific polymerase chain reaction. We used cancer tissue from a nested case-control study of 452 patients surgically treated for prostate cancer. Recurrence cases and controls were compared and the association between methylation extent and recurrence risk was estimated by logistic regression adjusting for patient age at prostatectomy, prostatectomy year, stage, grade, surgical margins and preprostatectomy prostate specific antigen. All statistical test...

Tumors are hierarchically organized by a rare population of cancer stem cells (CSCs) that contributes to cancer initiation, progression, and treatment failure. A better understanding of the molecular mechanisms underlying urothelial CSC regulation and the identification of key molecules associated with CSC generation and maintenance are pivotal for the molecular-targeting therapy. CD24 is overexpressed and acts as a lynchpin of tumorigenesis and metastatic progression in urothelial carcinoma of the bladder (UCB). However, its contribution to cancer stem-like traits of CD24 have not been determined. The functional relevance of CD24 was evaluated using in vitro and in vivo approaches. The knockdown of CD24 attenuated cancer stemness properties including sphere formation, self-renewal, invasion, chemo-resistance, and tumor initiation. The high-CD24-expressing cells, isolated from patient-derived UCB xenograft tumors, exhibited their enhanced stemness properties. Biologically, CD24 was associated with the expression of several key CSC-related molecules, including CD133, ABCG2, and YAP1. In human UCB samples, CD24 was overexpressed not only in primary tumors, but also in urine from UCB subjects. Conclusion: CD24 plays a crucial role in maintaining the urothelial cancer stem-like traits. Note: This abstract was not presented at the meeting. Citation Format: Akira Oki, Mohammad Obaidul Hoque. CD24 regulates cancer stem cell like traits in bladder cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 1162.

Recent studies have identified transcriptions factors YAP1 and STAT3, as therapeutic targets in cancer, but efforts targeting either YAP1 or STAT3 pathway have met with limited success. Our study is focused on understanding the cross-talk between YAP1 and STAT3 signaling, impact of this cross-talk on lung tumorigenesis, drug resistance and blocking this pathway as a potential therapeutic strategy in lung adenocarcinoma (LUAD). Mechanistically, we demonstrate that YAP1 promotes the phosphorylation of STAT3 through IL-6 upregulation. In clinical LUAD samples, a positive correlation between YAP1 and phosphorylated STAT3 protein as well as an association between YAP1 and IL-6 mRNA was observed. Pre-clinically, genetic and pharmacologic dual inhibitions of YAP1 and STAT3 showed decreased cell growth and increased sensitivity to chemotherapy through the attenuation of cancer-stemness like features. Although verteporfin and S3I-201 were employed as YAP1 and STAT3 inhibitors respectively, verteporfin was found to suppress not only YAP1 but also STAT3, and their combination induced synergistic cytotoxic effects. Furthermore, addition of verteporfin and S3I-201 to standard-of-care chemotherapy regimen significantly inhibited tumor growth in patient-derived xenograft mouse models and attenuated in vivo expansion of malignant stemness. These findings provide the conceptual framework for combined targeting of YAP1 and STAT3 in LUAD. Citation Format: Masahiro Shibata, Akira Ooki, Yoshikuni Inokawa, Evgeny Izumchenko, Enrico Munari, Giuseppe Bogina, Edward Gabrielson, Anju Singh, Mohammad Obaidul Hoque. YAP1 induces STAT3 phosphorylation & combined blockade enhances chemotherapeutic efficacy [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 3060.

Colorectal cancer (CRC) represents one of the highest incidences of cancers worldwide. Phosphatidylinositol glycan, class K (PIGK), is a crucial member of the glycosyl-phosphatidylinositol transamidase (GPIT) protein complex that attaches a diverse group of macromolecules to the plasma membrane of eukaryotes. However, the precise role of PIGK in tumorigenesis remains largely unknown. Recently, we reported low expression of PIGK protein in primary tumors compared to paired normal tissues of colorectal cancer (CRC) patients. To understand the mechanism underlying this phenomenon, we performed sequencing of all 10 exons of the PIGK gene in 45 CRC patients. Corresponding PIGK protein expression was also evaluated in these patients by immunohistochemistry. No mutation was detected in the coding regions, however, we found a single nucleotide polymorphism (C/C→C/G or G/G; rs1048575) in the 3'UTR of the PIGK gene in 67% (30/45) of the patients. Most of the patients (22/26, 85%) with the altered alleles were of Jewish origin. In comparison, 47% (8/17) of the Arabian patients exhibited the altered C/G alleles. We observed a significantly low (p

Purpose: Hypermethylation of tumor suppressor gene promoters has been found in head and neck squamous carcinoma (HNSCC) and other solid tumors. We evaluated these alterations in pretreatment salivary rinses from HNSCC patients by using real-time quantitative methylation-specific PCR (Q-MSP). Experimental Design: Pretreatment saliva DNA samples from HNSCC patients were evaluated for patterns of hypermethylation by using Q-MSP. Target tumor suppressor gene promoter regions were selected based on a previous study describing a screening panel for HNSCC in a high-risk population subjects. The selected genes were: DAPK, DCC, MINT-31, TIMP-3, p16, MGMT, CCNA1. Results: We analyzed the panel in a cohort of 61 HNSCC patients. Thirty-three of the analyzed patients (54.1%) showed methylation of at least one of the selected genes in the saliva DNA. Pretreatment methylated saliva DNA was not significantly associated with tumor site (P = 0.209) nor clinical stage (P = 0.299). However, local disease control and overall survival were significantly lower in patients presenting hypermethylation in saliva rinses (P = 0.010 and P = 0.015, respectively). Multivariate analysis confirmed that this hypermethylation pattern remained as an independent prognostic factor for local recurrence (HR = 12.2; 95% CI = 1.8–80.6; P = 0.010) and overall survival (HR = 2.8; 95% CI = 1.2–6.5; P = 0.016). Conclusions: We were able to confirm an elevated rate of promoter hypermethylation in HNSCC saliva of patients by using a panel of gene promoters previously described as methylated specifically in HNSCC. Detection of hypermethylation in pretreatment saliva DNA seems to be predictive of local recurrence and overall survival. This finding has potential to influence treatment and surveillance of HNSCC patients. Clin Cancer Res; 17(14); 4782–9. ©2011 AACR.

Strategies to address resistance to platin drugs are greatly needed in human epithelial cancers (e.g., ovarian, head/neck, and lung) where platins are used widely and resistance occurs commonly. We found that upon ΔNp63α overexpression, AKT1 and phospho-AKT1 levels are upregulated in cancer cells. Investigations using gel-shift, chromatin immunoprecipitation and functional reporter assays implicated ΔNp63α in positive regulation of AKT1 transcription. Importantly, we found that ΔNp63α, AKT1, and phospho-AKT levels are greater in 2008CI3 CDDP-resistant ovarian cancer cells than in 2008 CDDP-sensitive cells. siRNA-mediated knockdown of ΔNp63α expression dramatically decreased AKT1 expression, whereas knockdown of either ΔNp63α or AKT1 decreased cell proliferation and increased death of ovarian and head/neck cancer cells. Conversely, enforced expression of ΔNp63α increased cancer cell proliferation and reduced apoptosis. Together, our findings define a novel ΔNp63α-dependent regulatory mechanism for AKT1 expression and its role in chemotherapeutic resistance of ovarian and head/neck cancer cells. Cancer Res; 71(3); 1167–76. ©2011 AACR.

Head and Neck Squamous Cell Carcinoma (HNSCC) is the sixth most prevalent cancer worldwide, and despite advances in treatment options, the five-year survival rate for patients with HNSCC is dismal. Monotherapy (such as surgery or radiation) can be effective if detected early, however more advanced cases require treatment with various combinations of surgery, radiation and chemotherapy. Recently, interest in the utilization of immunotherapy for advanced HNSCC has increased, including the FDA approval of pembrolizumb and nivolumab (checkpoint inhibitors) for patients with recurrent or metastatic HNSCC. However, about 80% of patients remain unresponsive to checkpoint inhibitors. Numerous pre-clinical and clinical attempts aim to increase the sensitivity of immuno-agents. One such attempt is combination of immune-agents with epigenetic therapy. Epigenetic alterations in tumor cells modulate gene expression patterns that may facilitate evasion of immune recognition. The efficacy of epigenetic modulating agents to reverse these alterations is documented in various cancer types including HNSCC. Here, we sought to explore whether the utilization of combination therapy of epigenetic drugs (DNA methyltransferase inhibitor, 5azacytidine and the histone deacetylase inhibitor, romidepsin) with checkpoint inhibitors to increase the efficacy of immune-agents in a pre-clinical model of HNSCC. To explore the gene expression alterations induced by epigenetic drugs in HNSCC, we treated six HNSCC cell lines in vitro with 5azacytidine and romidepsin, and analyzed gene expression patterns by microarray and quantitative RT-PCR (QRT-PCR). We found significant differences in immune-related gene expression patterns in epigenetic drug-treated cell lines compared to untreated controls. Further, to explore the immune-associated gene expression changes, we performed QRT-PCR using a Taqman low density (TLDA) human immune array. By this analysis, we found changes in expression patterns of several immune related genes including HMOX1, NFKB2, HLA-DRA, STAT3, IL12a, CSF1, CSF2, FAS, and IL-18 in epigenetic drug-treated cells. Additionally, by QRT-PCR analysis of several candidates of the viral defense pathway, we found significant upregulation of Interferon type 1 (IFN 1) related genes STAT1, OASL, IFI6 and IRF7 in epigenetic drug-treated cell lines. These results indicate that HNSCC cell lines acquire a new immune signature following treatment with epigenetic agents. Next, we used an in vivo syngeneic model of HNSCC, to compare tumor growth following treatment with mono or combination therapy. Our preliminary data suggests that combination treatment with epigenetic drugs and an immune-agent more effectively reduces tumor growth than either of these drugs alone. In conclusion, our results highlight the importance of considering combination of epigenetic and immune therapy for HNSCC patients that respond poorly to immune therapy alone. Citation Format: Rachel Goldberg, Masahiro Shibata, Evgeny Izumchenko, Luigi Marchionni, David Sidransky, Mohammad O. Hoque. An epigenetic and immunologic approach to optimize therapy for HNSCC [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 2750.

PAX6 is an essential transcription factor for embryonic stem cell maintenance, but its contribution to lung cancer stem cells (CSCs) remains unexplored. We demonstrate that PAX6 acts as an oncogene responsible for induction of cancer stemness properties and plays a biological role in tumor initiation and CSC expansion in lung adenocarcinoma (LUAD). Mechanistically, PAX6 activates Hedgehog-GLI signaling in a SMO-independent noncanonical manner, resulting in SOX2 upregulation directly by the binding of GLI to the proximal promoter region of the SOX2 gene. The overexpressed SOX2 enhances the expression of key pluripotent factors (OCT4 and NANOG) and suppresses differentiation lineage factors (HOPX and NKX2-1). Thus, PAX6 drives cancer cells toward a stem-like state via the GLI-SOX2 signaling axis in LUAD. In contrast, in the differentiated non-CSCs that represent most of the tumor cell population, PAX6 is transcriptionally silenced by its promoter methylation. In human lung cancer tissues, the positive linear correlations of PAX6 expression with GLI and SOX2 expression and its negative correlations with HOPX and NKX2-1 expression were observed. Therapeutically, the blockade of the PAX6-GLI-SOX2 signaling axis elicits a long-lasting therapeutic efficacy by limiting CSC expansion following chemotherapy. Our findings provide a rationale for targeting the PAX6-GLI-SOX2 signaling axis with chemotherapy as an effective therapeutic strategy. Citation Format: Akira Oki, Mohammad O. Hoque. PAX6 drives cancer cells toward a stem like state via GLI-SOX2 signaling axis in lung adenocarcinoma [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 1132.

Mitochondria encoded Cytochrome B (CYTB) gene mutations were reported in tumors of different anatomic origin but the functional significance of these mutations are not well studied. Earlier, we found a 7-amino acid deletion mutation in the CYTB gene in a primary bladder cancer patient. In the present study, we overexpressed this 7-amino acid deletion mutation of CYTB gene in SV-40 transformed human uroepithelial HUC-1 cells. The nuclear transcribed mitochondrial CYTB (mtCYTB) was targeted into the mitochondria and generated increased copies of mitochondria and mitochondrial COX-I protein in the transfected HUC-1 cells. The pro-apoptotic protein Bax largely remained confined to the cytoplasm of the mtCYTB transfected HUC-1 cells without release of Cytochrome C. The downstream apoptotic proteins PARP also remained uncleaved along with increased Lamin B1 in the mtCYTB transfected cells. Our results demonstrate that forced overexpression of mtCYTB in transformed human uroepithelial HUC-1 cells triggered mitochondrial proliferation and induction of an anti-apoptotic signaling cascade favoring sustained cellular growth. Coding mitochondrial DNA mutations appear to have significant functional contribution in tumor progression.

Aberrant promoter methylation of several known or putative tumor suppressor genes occurs frequently during carcinogenesis, and this epigenetic change has been considered as a potential molecular marker for cancer. We examined the methylation status of nine genes (APC, CDH1, CTNNB1, TIMP3, ESR1, GSTP1, MGMT, THBS1, and TMS1), by quantitative methylation specific PCR. Synchronous preinvasive lesions (atypical ductal hyperplasia and/or ductal carcinoma in situ) and invasive ductal breast carcinoma from 52 patients, together with pure lesions from 24 patients and 12 normal tissues paired to tumor and 20 normal breast distant from tumor were analyzed. Aberrant promoter methylation was detected in both preinvasive and invasive lesions for genes APC, CDH1, CTNNB1, TIMP3, ESR1, and GSTP1. However, hierarchical mixed model and Generalized Estimating Equations model analyses showed that only APC, CDH1, and CTNNB1 promoter regions showed a higher frequency and methylation levels in pathologic samples when compared with normal breast. Whereas APC and CTNNB1 did not show differences in methylation levels or frequencies, CDH1 showed higher methylation levels in invasive tumors as compared with preinvasive lesions (P < 0.04, Mann-Whitney test with permutation correction). The analysis of APC, CDH1, and CTNNB1 methylation status was able to distinguish between normal and pathologic samples with a sensitivity of 67% (95% confidence interval, 60-71%) and a specificity of 75% (95% confidence interval, 69-81%). Our data point to the direct involvement of APC, CDH1, and CTNNB1 promoter methylation in the early stages of breast cancer progression and suggest that they may represent a useful tool for the detection of tumor cells in clinical specimens. (Cancer Epidemiol Biomarkers Prev 2009;18(10):2694–700)

Promoter hypermethylation is associated with the loss of expression of tumor-suppressor genes in cancer. Currently, several genome-wide technologies are available and have been utilized to examine the extent of DNA methylation in discovery-based studies involving several physiological and disease states. Although early in the process, aberrant DNA methylation is gaining strength in the fields of cancer risk assessment, diagnosis and therapy monitoring in different cancer types. There is a need to improve existing methods for early diagnosis of prostate cancer and to identify men at risk for developing aggressive disease. Because of the ubiquity of DNA methylation changes and the ability to detect methylated DNA in several body fluids (e.g., blood and urine), this specifically altered DNA may serve, on one hand, as a possible new screening marker for prostate cancer and, on the other hand, as a tool for therapy monitoring in patients having had neoplastic disease of the prostate. Since many prostate cancer patients present with advanced disease and some present with nonspecific elevation of prostate-specific antigen without prostate cancer, early detection with high specificity and sensitivity is considered to be one of the most important approaches to reduce mortality and unwanted tension of the men with high prostate-specific antigen. Therefore, an effective screening test would have substantial clinical benefits. Furthermore, methylation markers of risk of progression of disease in patients having prostate cancer permits immediate commencement of specific treatment regimens and probably longer survival and better quality of life. This review illustrates the current benefits and limitations of potentially useful prostate cancer methylation markers that have considerable existing data and touches upon other future markers as well as the field of methylation in prostate cancer.

DNA methylation has a role in mediating epigenetic silencing of CpG island genes in cancer and other diseases. Identification of all gene promoters methylated in cancer cells “the cancer methylome” would greatly advance our understanding of gene regulatory networks in tumorigenesis. We previously described a new method of identifying methylated tumor suppressor genes based on pharmacologic unmasking of the promoter region and detection of re-expression on microarray analysis. In this study, we modified and greatly improved the selection of candidates based on new promoter structure algorithm and microarray data generated from 20 cancer cell lines of 5 major cancer types. We identified a set of 200 candidate genes that cluster throughout the genome of which 25 were previously reported as harboring cancer-specific promoter methylation. The remaining 175 genes were tested for promoter methylation by bisulfite sequencing or methylation-specific PCR (MSP). Eighty-two of 175 (47%) genes were found to be methylated in cell lines, and 53 of these 82 genes (65%) were methylated in primary tumor tissues. From these 53 genes, cancer-specific methylation was identified in 28 genes (28 of 53; 53%). Furthermore, we tested 8 of the 28 newly identified cancer-specific methylated genes with quantitative MSP in a panel of 300 primary tumors representing 13 types of cancer. We found cancer-specific methylation of at least one gene with high frequency in all cancer types. Identification of a large number of genes with cancer-specific methylation provides new targets for diagnostic and therapeutic intervention, and opens fertile avenues for basic research in tumor biology. [Cancer Res 2008;68(8):2661–70]

Purpose: Prostate cancer is a highly prevalent malignancy and constitutes a major cause of cancer-related morbidity and mortality. Owing to the limitations of current clinical, serologic, and pathologic parameters in predicting disease progression, we sought to investigate the prognostic value of promoter methylation of a small panel of genes by quantitative methylation-specific PCR (QMSP) in prostate biopsies. Experimental Design: Promoter methylation levels of APC, CCND2, GSTP1, RARB2, and RASSF1A were determined by QMSP in a prospective series of 83 prostate cancer patients submitted to sextant biopsy. Clinicopathologic data [age, serum prostate-specific antigen (PSA), stage, and Gleason score] and time to progression and/or death from prostate cancer were correlated with methylation findings. Log-rank test and Cox regression model were used to identify which epigenetic markers were independent predictors of prognosis. Results: At a median follow-up time of 45 months, 15 (18%) patients died from prostate cancer, and 37 (45%) patients had recurrent disease. In univariate analysis, stage and hypermethylation of APC were significantly associated with worse disease–specific survival, whereas stage, Gleason score, high diagnostic serum PSA levels, and hypermethylation of APC, GSTP1, and RASSF1A were significantly associated with poor disease-free survival. However, in the final multivariate analysis, only clinical stage and high methylation of APC were significantly and independently associated with unfavorable prognosis, i.e., decreased disease-free and disease-specific survival. Conclusions: High-level APC promoter methylation is an independent predictor of poor prognosis in prostate biopsy samples and might provide relevant prognostic information for patient management.

We hypothesized that comprehensive breast cancer methylation profiling might provide biomarkers for diagnostic assessment of suspicious breast lesions using fine needle aspiration biopsy (FNA).Twenty-three gene promoters were surveyed by quantitative methylation-specific PCR in bisulfite-modified DNA from 66 breast carcinomas (BCa), 31 fibroadenomas (FB) and 12 normal breast (NT) samples to define a set of genes differentially methylated in malignant and non-malignant tissues. This set was tested in 78 FNA washings obtained pre-operatively (66 malignant, 12 benign), with histopathological diagnosis. Receiver operator characteristic (ROC) curve analysis identified a gene panel which might distinguish cancer from non-cancerous lesions. Finally, this panel was validated in an independent series of FNA washings (45 cases) in which cytomorphology did not reach definitive diagnosis.In tissue samples, 14-3-3-sigma, DAPK, CCND2, RASSF1A, CALCA, APC, HIN1, RARbeta2, TIG1, and GSTP1 methylation levels differed significantly among BCa, FB, and NT. ROC curve analysis identified a panel of four gene loci (CCND2, RASSF1A, APC, and HIN1) that discriminated BCa from benign lesions in a set of 78 FNA washings from histologically characterized breast lesions. When this panel was tested in the validation dataset of 45 FNA washings, breast cancer was identified with perfect specificity (100%) when 3 of 4 gene loci tested positive, providing estimated added information of 91% over cytomorphologic evaluation alone.Our data provide evidence that multigene methylation analysis augments diagnostic accuracy of cytological assessment of suspicious breast lesions, and might be a valuable ancillary tool for breast cancer diagnosis.

To evaluate gemcitabine-cisplatin (GC) neoadjuvant cisplatin-based chemotherapy (NAC) for pathologic response (pR) and cancer-specific outcomes following radical cystectomy (RC) for muscle-invasive bladder cancer and identify clinical parameters associated with pR.We studied 150 consecutive cases of muscle-invasive bladder cancer that received GC NAC followed by open RC (2000-2013). A cohort of 121 patients treated by RC alone was used for comparison. Pathologic response and cancer-specific survival (CSS) were compared. We created the Johns Hopkins Hospital Dose Index to characterize chemotherapeutic dosing regimens and accurately assess sufficient neoadjuvant dosing regarding patient tolerance.No significant difference was noted in 5-year CSS between GC NAC (58%) and non-NAC cohorts (61%). The median follow-up was 19.6 months (GC NAC) and 106.5 months (non-NAC). Patients with residual non-muscle-invasive disease after GC NAC exhibit similar 5-year CSS relative to patients with no residual carcinoma (P = 0.99). NAC pR (≤ pT1) demonstrated improved 5-year CSS rates (90.6% vs. 27.1%, P0.01) and decreased nodal positivity rates (0% vs. 41.3%, P0.01) when compared with nonresponders (≥ pT2). Clinicopathologic outcomes were inferior in NAC pathologic nonresponders when compared with the entire RC-only-treated cohort. A lower pathologic nonresponder rate was seen in patients tolerating sufficient dosing of NAC as stratified by the Johns Hopkins Hospital Dose Index (P = 0.049), congruent with the National Comprehensive Cancer Network guidelines. A multivariate classification tree model demonstrated 60 years of age or younger and clinical stage cT2 as significant of NAC response (P0.05).Pathologic nonresponders fare worse than patients proceeding directly to RC alone do. Multiple predictive models incorporating clinical, histopathologic, and molecular features are currently being developed to identify patients who are most likely to benefit from GC NAC.

Current cervical cancer screening is based on morphological assessment of Pap smears and associated with significant false negative and false positive results. Previously, we have shown that detection of hypermethylated genes in cervical scrapings using quantitative methylation-specific PCR (QMSP) is a promising tool for identification of squamous cell cervical cancer. Aim of the present pilot-study was to evaluate presence of hypermethylated genes in cervical carcinogenesis, both in squamous cell as well as adenocarcinomas. Cervical scrapings were obtained from 30 patients diagnosed with cervical cancer (20 squamous cell carcinomas and 10 adenocarcinomas) and 19 women with histologically normal cervices. The scraped cells were used for, determination of promoter hypermethylation by QMSP for 12 genes and for morphological assessment. Overall, CALCA, DAPK, ESR1, TIMP3, APC and RAR-beta(2) promoters were significantly more often hypermethylated in cancers than in controls, while adenocarcinomas were more often hypermethylated above the highest control ratio for APC, TIMP3 and RASSF1A promoters. Combining 4 genes (CALCA, DAPK, ESR1 and APC) yielded a sensitivity of 89% (with all adenocarcinomas identified), equal to cytomorphology (89%) and high-risk human papilloma virus (Hr-HPV; 90%). The 4-gene QMSP proved theoretically superior to cytomorphology as well as Hr-HPV in specificity (100% vs. 83 and 68%, respectively), because cytology identified 3 controls as moderate or severe dyskaryosis and 6 controls were positive for Hr-HPV. In conclusions, QMSP of 4 gene promoters combined appears to have comparable sensitivity and potentially better specificity in comparison to "classic" cytomorphological assessment and Hr-HPV detection. QMSP holds promise as a new diagnostic tool for both squamous cell carcinoma and adenocarcinoma of the cervix. (c) 2006 Wiley-Liss, Inc.

Background: The noninvasive identifi cation of bladder tu mors may improve disease control and prevent disease progression. Aberrant promoter methylation (i.e., hypermethylation) is a major mechanism for silencing tumor suppressor genes and other cancer-associated genes in many human cancers, including bladder cancer. Methods: A quantitative fl uorogenic real-time polymerase chain reaction (PCR) assay was used to examine primary tumor DNA and urine sediment DNA from 15 patients with bladder cancer and 25 control subjects for promoter hypermethylation of nine genes (APC, ARF, CDH1, GSTP1, MGMT, CDKN2A, RAR β 2, RASSF1A, and TIMP3) to identify potential biomarkers for bladder cancer. We then used these markers to examine urine sediment DNA samples from an additional 160 patients with bladder cancers of various stages and grades and from an additional 69 age-matched control subjects. Data were analyzed on the basis of a prediction model and were internally validated using a jacknife procedure . All statistical tests were two-sided. Results: For all 15 patients with paired DNA samples, the promoter methylation pattern in urine matched that in the primary tumors. Four genes displayed 100% specifi city. Of the 175 bladder cancer patients, 121 (69%, 95% confi dence interval [CI] = 62% to 76%) displayed promoter methylation in at least one of these genes (CDKN2A, ARF, MGMT, and GSTP1), whereas all control subjects were negative for such methylation (100% specifi city, 95% CI = 96% to 100%). A logistic prediction model using the methylation levels of all remaining fi ve genes was developed and internally validated for subjects who were negative on the four-gene panel . This combined, two-stage predictor produced an internally validated ROC curve with an overall sensitivity of 82% (95% CI = 75 % to 87%) and specifi city of 96% (95% CI = 90% to 99%). Conclusion: Testing a small panel of genes with the quantitative methylation – specifi c PCR assay in urine sediment DNA is a powerful noninvasive approach for the detection of bladder cancer. Larger independent confi rmatory cohorts with longitudinal follow-up will be required in future studies to defi ne the impact of this technology on early detection, prognosis, and disease monitoring before clinical application. [J Natl Cancer Inst 2006;98: 996 – 1004 ]

Purpose Aberrant promoter hypermethylation of several known or putative tumor suppressor genes occurs frequently during the pathogenesis of prostate cancers and is a promising marker for cancer detection. We sought to develop a test for prostate cancer based on a quantitative methylation-specific polymerase chain reaction (QMSP) of multiple genes in urine sediment DNA. Patients and Methods We tested urine sediment DNA for aberrant methylation of nine gene promoters (p16INK4a, p14ARF, MGMT, GSTP1, RARβ2, CDH1 [E-cadherin], TIMP3, Rassf1A, and APC) from 52 patients with prostate cancer and 21 matched primary tumors by quantitative fluorogenic real-time polymerase chain reaction. We also analyzed urine sediments from 91 age-matched individuals without any history of genitourinary malignancy as controls. Results Promoter hypermethylation of at least one of the genes studied was detected in urine samples from all 52 prostate cancer patients. Urine samples from the 91 controls without evidence of genitourinary cancer revealed no methylation of the p16, ARF, MGMT, and GSTP1 gene promoters, whereas methylation of RARβ2, TIMP3, CDH1, Rassf1A, and APC was detected at low levels. Conclusion Overall, methylation found in urine samples matched the methylation status in the primary tumor. A combination of only four genes (p16, ARF, MGMT, and GSTP1) would theoretically allow us to detect 87% of prostate cancers with 100% specificity. Our data support further development of the noninvasive QMSP assay in urine DNA for early detection and surveillance of prostate cancer.

Purpose: Promoter hypermethylation is an alternative pathway for gene silencing in neoplastic cells and a promising cancer detection marker. Although quantitative methylation-specific PCR (QMSP) of the GSTP1 promoter has demonstrated near perfect specificity for cancer detection in prostate biopsies, we postulated that identification and characterization of additional methylation markers might further improve its high (80–90%) sensitivity. Experimental Design: We surveyed nine gene promoters (GSTP1, MGMT, p14/ARF, p16/CDKN2A, RASSF1A, APC, TIMP3, S100A2, and CRBP1) by QMSP in tissue DNA from 118 prostate carcinomas, 38 paired high-grade prostatic intraepithelial neoplasias (HGPIN), and 30 benign prostatic hyperplasias (BPH). The methylation levels were calculated and were correlated with clinical and pathologic indicators. Results: Only the methylation frequencies of GSTP1 and APC were significantly higher in prostate carcinoma compared with BPH (P < 0.001). Methylation levels of GSTP1, APC, RASSF1A, and CRBP1, differed significantly between prostate carcinoma and HGPIN, and/or HGPIN or BPH (P < 0.0001).With QMSP and empirically defined cutoff values, the combined use of GSTP1 and APC demonstrated a theoretical sensitivity of 98.3% for prostate carcinoma, with 100% specificity. Methylation levels were found to correlate with tumor grade (GSTP1 and APC) and stage (GSTP1, RASSF1A, and APC). Conclusions: Our data demonstrate the existence of a progressive increase of promoter methylation levels of several cancer-related genes in prostate carcinogenesis, providing additional markers to augment molecular detection of prostate carcinoma. Because methylation levels of GSTP1, APC, and RASSF1A are associated with advanced grade and stage, QMSP might augment the pathologic indicators currently used to predict tumor aggressiveness.

Purpose: Aberrant promoter hypermethylation of several known or putative tumor suppressor genes occurs frequently during the pathogenesis of lung cancers and is a promising marker for cancer detection. We investigated the feasibility of detecting aberrant DNA methylation in the bronchoalveolar lavage (BAL) samples of lung cancer patients. Experimental Design: We examined the tumor and the matched BAL DNA for aberrant methylation of eight gene promoters (CDH1, APC, MGMT, RASSF1A, GSTP1, p16, RAR-β2, and ARF) from 31 patients with primary lung tumors by quantitative fluorogenic real-time PCR. BAL from 10 age-matched noncancer patients was used as a control. Results: Promoter hypermethylation of at least one of the genes studied was detected in all 31 lung primary tumors; 27 (87%) CDH1, 17 (55%) APC, 14 (45%) RASSF1A, 12 (39%) MGMT, 7 (23%) p16, 3 (10%) GSTP1, 3 (10%) RAR-β2, and 0 (0%) ARF. Methylation was detected in CDH1 (48%), APC (29%), RASSF1A (29%), MGMT (58%), p16 (14%), GSTP1 (33%), RAR-β2 (0%), and ARF (0%) of BAL samples from matched methylation-positive primary tumors, and in every case, aberrant methylation in BAL DNA was accompanied by methylation in the matched tumor samples. BAL samples from 10 controls without evidence of cancer revealed no methylation of the MGMT, GSTP1, p16, ARF, or RAR-β2 genes whereas methylation of RASSF1, CDH1, and APC was detected at low levels. Overall, 21 (68%) of 31 BAL samples from cancer patients were positive for aberrant methylation. Conclusion: Our findings suggest that promoter hypermethylation in BAL can be detected in the majority of lung cancer patients. This approach needs to be evaluated in large early detection and surveillance studies of lung cancer.

Exposure to toxicants leads to cumulative molecular changes that overtime increase a subject's risk of developing urothelial carcinoma. To assess the impact of arsenic exposure at a time progressive manner, we developed and characterized a cell culture model and tested a panel of miRNAs in urine samples from arsenic-exposed subjects, urothelial carcinoma patients, and controls. To prepare an in vitro model, we chronically exposed an immortalized normal human bladder cell line (HUC1) to arsenic. Growth of the HUC1 cells was increased in a time-dependent manner after arsenic treatment and cellular morphology was changed. In a soft agar assay, colonies were observed only in arsenic-treated cells, and the number of colonies gradually increased with longer periods of treatment. Similarly, invaded cells in an invasion assay were observed only in arsenic-treated cells. Withdrawal of arsenic treatment for 2.5 months did not reverse the tumorigenic properties of arsenic-treated cells. Western blot analysis demonstrated decreased PTEN and increased AKT and mTOR in arsenic-treated HUC1 cells. Levels of miR-200a, miR-200b, and miR-200c were downregulated in arsenic-exposed HUC1 cells by quantitative RT-PCR. Furthermore, in human urine, miR-200c and miR-205 were inversely associated with arsenic exposure (P = 0.005 and 0.009, respectively). Expression of miR-205 discriminated cancer cases from controls with high sensitivity and specificity (AUC = 0.845). Our study suggests that exposure to arsenic rapidly induces a multifaceted dedifferentiation program and miR-205 has potential to be used as a marker of arsenic exposure as well as a maker of early urothelial carcinoma detection. Cancer Prev Res; 8(3); 208–21. ©2015 AACR.

The 5-year cancer specific survival (CSS) for patients with muscle invasive urothelial carcinoma of the bladder (MIBC) treated with cystectomy alone is approximately 50%. Platinum based neoadjuvant chemotherapy (NAC) plus cystectomy results in a marginal 5-10% increase in 5-year CSS in MIBC. Interestingly, responders to NAC (ypT2) have a 5-year CSS of 90% which is in stark contrast to the 30-40% CSS for those whose MIBC is resistance to NAC. While the implementation of NAC for MIBC is increasing, it is still not widely utilized due to concerns related to delay of cystectomy, potential side-effects, and inability to predict effectiveness. Recently suggested molecular signatures of chemoresponsiveness, which could prove useful in this setting, would be of considerable utility but are yet to be translated into clinical practice.mRNA expression data from a prior report on a NAC-treated MIBC cohort were re-analyzed in conjunction with the antibody database of the Human Protein Atlas (HPA) to identify candidate protein based biomarkers detectable by immunohistochemistry (IHC). These candidate biomarkers were subsequently tested in tissue microarrays derived from an independent cohort of NAC naive MIBC biopsy specimens from whom the patients were treated with neoadjuvant gemcitabine cisplatin NAC and subsequent cystectomy. The clinical parameters that have been previously associated with NAC response were also examined in our cohort.Our analyses of the available mRNA gene expression data in a discovery cohort (n = 33) and the HPA resulted in 8 candidate protein biomarkers. The combination of GDPD3 and SPRED1 resulted in a multivariate classification tree that was significantly associated with NAC response status (Goodman-Kruskal γ = 0.85 p0.0001) in our independent NAC treated MIBC cohort. This model was independent of the clinical factors of age and clinical tumor stage, which have been previously associated with NAC response by our group. The combination of both these protein biomarkers detected by IHC in biopsy specimens along with the relevant clinical parameters resulted in a prediction model able to significantly stratify the likelihood of NAC resistance in our cohort (n = 37) into two well separated halves: low-26% n = 19 and high-89% n = 18, Fisher's exact p = 0.0002).We illustrate the feasibility of translating a gene expression signature of NAC response from a discovery cohort into immunohistochemical markers readily applicable to MIBC biopsy specimens in our independent cohort. The results from this study are being characterized in additional validation cohorts. Additionally, we anticipate that emerging somatic mutations in MIBC will also be important for NAC response prediction. The relationship of the findings in this study to the current understanding of variant histologic subtypes of MIBC along with the evolving molecular subtypes of MIBC as it relates to NAC response remains to be fully characterized.