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Comprehensive genomic profiling is increasingly used to facilitate precision oncology based on molecular stratification. In addition to conventional tissue comprehensive genomic profiling, comprehensive genomic profiling of circulating tumor DNA has become widely utilized in cancer care owing on its advantages, including less invasiveness, rapid turnaround time, and capturing heterogeneity. However, circulating tumor DNA comprehensive genomic profiling has some limitations, mainly false negatives due to low levels of plasma circulating tumor deoxyribonucleic acid and false positives caused by clonal hematopoiesis. Nevertheless, no guidelines and recommendations fully address these issues. Here, an expert panel committee involving representatives from 12 Designated Core Hospitals for Cancer Genomic Medicine in Japan was organized to develop expert consensus recommendations for the use of circulating tumor deoxyribonucleic acid-based comprehensive genomic profiling. The aim was to generate guidelines for clinicians and allied healthcare professionals on the optimal use of the circulating tumor DNA assays in advanced solid tumors and to aid the design of future clinical trials that utilize and develop circulating tumor DNA assays to refine precision oncology. Fourteen clinical questions regarding circulating tumor deoxyribonucleic acid comprehensive genomic profiling including the timing of testing and considerations for interpreting results were established by searching and curating associated literatures, and corresponding recommendations were prepared based on the literature for each clinical question. Final consensus recommendations were developed by voting to determine the level of each recommendation by the Committee members.

In Japan, comprehensive genomic profiling (CGP) tests for refractory cancer patients have been approved since June 2019, under the requirement that all cases undergoing CGP tests are annotated by the molecular tumor board (MTB) at each government-designated hospital. To investigate improvement in precision oncology, we evaluated and compared the proportion of cases receiving matched treatments according to CGP results and those recommended to receive genetic counseling at all core hospitals between the first period (11 hospitals, June 2019 to January 2020) and second period (12 hospitals, February 2020 to January 2021). A total of 754 and 2294 cases underwent CGP tests at core hospitals in the first and second periods, respectively; 28 (3.7%) and 176 (7.7%) patients received matched treatments (p 0.001). Additionally, 25 (3.3%) and 237 (10.3%) cases were recommended to receive genetic counseling in the first and second periods, respectively (p 0.001). The proportion was associated with the type of CGP test: tumor-only (N = 2391) vs. tumor-normal paired (N = 657) analysis (10.0% vs. 3.5%). These results suggest that recommendations regarding available clinical trials in networked MTBs might contribute to increasing the numbers of matched treatments, and that tumor-normal paired rather than tumor-only tests can increase the efficiency of patient referrals for genetic counseling.

ImportanceQuality assurance of molecular tumor boards (MTBs) is crucial in cancer genome medicine.ObjectiveTo evaluate the concordance of recommendations by MTBs and centrally developed consensus treatment recommendations at all 12 leading institutions for cancer genomic medicine in Japan using 50 simulated cases.Design, Setting, and ParticipantsThis was a prospective quality improvement study of 50 simulated cancer cases. Molecular tumor boards from 12 core hospitals independently recommended treatment for 50 cases blinded to the centrally developed consensus treatment recommendations. The study’s central committee consisted of representatives from all 12 core hospitals in Japan who selected the 50 simulated cases from The Cancer Genome Atlas database, including frequently observed genomic alterations. The central committee recommended centrally developed consensus treatment. The concordance rate for genomically matched treatments between MTBs and centrally developed consensus treatment recommendations was evaluated. Data analysis was conducted from January 22 to March 3, 2021.ExposuresSimulated cases of cancer.Main Outcomes and MeasuresThe primary outcome was concordance, defined as the proportion of recommendations by MTBs concordant with centrally developed consensus treatment recommendations. A mixed-effects logistic regression model, adjusted for institutes as a random intercept, was applied. High evidence levels were defined as established biomarkers for which the treatment was ready for routine use in clinical practice, and low evidence levels were defined as biomarkers for genomically matched treatment that were under investigation.ResultsThe Clinical Practice Guidance for Next-Generation Sequencing in Cancer Diagnosis and Treatment (edition 2.1) was used for evidence-level definition. The mean concordance between MTBs and centrally developed consensus treatment recommendations was 62% (95% CI, 57%-65%). Each MTB concordance varied from 48% to 86%. The concordance rate was higher in the subset of patients with colorectal cancer (100%; 95% CI, 94.0%-100%), ROS1 fusion (100%; 95% CI, 85.5%-100%), and high evidence level A/R (A: 88%; 95% CI, 81.8%-93.0%; R:100%; 95% CI, 92.6%-100%). Conversely, the concordance rate was lower in cases of cervical cancer (11%; 95% CI, 3.1%-26.1%), TP53 mutation (16%; 95% CI, 12.5%-19.9%), and low evidence level C/D/E (C: 30%; 95% CI, 24.7%-35.9%; D: 25%; 95% CI, 5.5%-57.2%; and E: 18%; 95% CI, 13.8%-23.0%). Multivariate analysis showed that evidence level (high [A/R] vs low [C/D/E]: odds ratio, 4.4; 95% CI, 1.8-10.8) and TP53 alteration (yes vs no: odds ratio, 0.06; 95% CI, 0.03-0.10) were significantly associated with concordance.Conclusions and RelevanceThe findings of this study suggest that genomically matched treatment recommendations differ among MTBs, particularly in genomic alterations with low evidence levels wherein treatment is being investigated. Sharing information on matched therapy for low evidence levels may be needed to improve the quality of MTBs.

11032 Background: Treatment recommendations (TRs) by molecular tumor boards (MTBs) based on comprehensive genomic profiling tests are crucial for enrolling cancer patients in genotype-matched trials. Our previous study of Japanese designated ‘Core’ hospitals using simulated cases showed that TRs for biomarkers with high evidence levels (ELs) were highly concordant between central consensus and MTBs, whereas those with low ELs showed low concordance, indicating that a learning program (LP) about obtaining information on matched trials, particularly for those with low ELs, is needed to improve the quality of TRs by MTBs (Kage H. ESMO 2021). Therefore, we conducted a nationwide prospective study to investigate the clinical utility of an LP for MTBs in designated ‘Hub’ hospitals and explore the efficacy of an artificial intelligence (AI)-based annotation system. Methods: Fifty simulated cases were randomly divided into 2 groups (pre- and post-tests) by the central statistician, stratified by high vs. low ELs. Each MTB at ‘Hub’ hospitals (group), treating physicians at ‘Core’ hospitals (individual), and an AI company were eligible. Each participant made TRs for the first 25 cases, then participated in the LP, and made TRs for the second 25 cases. The online LP shared the methodology of making the optimal TRs and showed central consensus TRs for simulated cases. The primary endpoint was the proportion of MTBs that met prespecified ‘accreditation’ criteria for the second 25 cases: > 90% concordance for high ELs and > 40% for low ELs. Expected and threshold proportions of ‘accreditation’ MTBs were 50% and 20%, respectively, and the planned sample size was 24 MTBs. The improvements in concordance rates in TRs on post-tests were key secondary endpoints. The concordance rate of TRs with the AI system was an exploratory endpoint. Results: From May to December 2021, 47 participants applied, and 42 (27 MTBs, 14 individuals, and an AI company) were eligible. The primary ‘accreditation’ rate of MTBs was 55.6% (95% CI, 35.3-74.5%, p < 0.001), and the ‘accreditation’ rate of individuals was 35.7% (95% CI, 12.8-64.9%, p = 0.17). TR concordance rates improved significantly, from 57.5% (95% CI, 51.7-63.1%) to 65.7% (95% CI, 59.2-71.6%) [odds ratio (OR): 1.23, p = 0.04]. A prespecified subgroup analysis showed an improved concordance rate for biomarkers with low ELs for MTBs (OR, 1.32: 95% CI, 1.00-1.73), but not for individuals (OR, 1.11: 95% CI, 0.75-1.63). TR concordance rates with the AI system were 80% (95% CI, 60.0-91.4%) and 84% (95% CI, 64.3-93.9%) for pre- and post-tests, respectively. Conclusions: This LP significantly improved the quality of TRs by MTBs, potentially leading to providing more matched trials to cancer patients. TRs by the AI system showed higher concordance with central consensus TRs than MTBs, suggesting the clinical utility of AI-based TRs over those by MTBs.

Background Bartholin gland carcinomas (BGCs) are rare tumor types, for which no molecular analyses including genomic sequencing have been reported to date. Adenoid cystic carcinomas (ACCs) of the Bartholin’s glands are an atypical histological type of BGC, and currently nothing is known regarding their genetic profiles or similarity to ACC carcinogenesis in other organs including the salivary glands, thereby limiting possible therapeutic options using precision medicine. Case presentation We used targeted gene sequencing to analyze the occurrence of 160 cancer-related genes in two patients with BG-ACC. KRAS and KDM6A mutations were detected in tumor samples collected from each patient. No KRAS mutations have been previously reported in salivary gland ACCs, indicating that the carcinogenesis of BG-ACC differs from that of the salivary gland ACCs. KDM6A mutations are often reported in salivary gland ACCs and facilitate novel gene-targeted therapy, including the use of BET and HDAC inhibitors. Conclusions A better understanding of the underlying genetic mechanisms will help to clarify the carcinogenesis of BG-ACC. In turn, this will enable treatment with novel targeting agents, as well as the initial exploration of gene-based precision oncological therapies, which aim to improve treatment outcomes for patients with this disease.

We established an outpatient service in November 2017 to provide cancer gene profiling test services to cancer patients. To date, we have seen approximately 100 patients. Our staff includes genetic counselors and nurses specialized in genetic medicine. Our experience highlights the importance of healthcare professionals having in-depth knowledge of cancer therapeutic drugs and/or investigational drugs based on cancer genome medicine. Recently, poly(ADP-ribose) polymerase (PARP) inhibitors have been approved for treating breast cancer patients with germline BRCA mutation; thus, in-depth knowledge of genetics and skills for genetic counseling are often considered indispensable in working with cancer patients. However, because the prompt treatment of clear and present cancer is the top priority in clinical settings, providing genetic information at that time, including that of unaffected family members, is of low priority for most patients who are dealing with the severe side effects of anti-cancer therapies. Pharmacists have an essential role to play in cancer therapeutics, talking with patients in order to assess their condition and to clarify the status of their treatment with anticancer agents. Genetic pharmacists should therefore work closely with genetics nurses and genetic counselors in the clinical practice of cancer genomic medicine. In this symposium, I would like to describe our experience caring for patients through our outpatient service, and to discuss the ideal framework for multidisciplinary cooperation to promote cancer genomic medicine.

Background/aims Genistein, a soy isoflavone, has been shown to have anti-cancer effects in various cancers including renal cancer. Long non-coding RNA, HOX transcript antisense RNA (HOTAIR), is involved in cancer progression and metastasis, such as renal cancer. Our aim was to investigate the effects of genistein on HOTAIR chromatin remodeling functions. Methods We used MTS assays and Transwell migration assays to study the effects of genistein on cell proliferation and migration respectively in human renal cell carcinoma (RCC) cell lines. We used Western blots to analyze SNAIL and ZO-1 expression. We performed chromatin immunoprecipitation (ChIP) assays to study recruitment of the polycomb repressive complex 2 (PRC2) to the ZO-1 promoter. We performed RNA immunoprecipitation (RIP) assays to study interaction between HOTAIR and PRC2, SMARCB1 or ARID1A. We also performed transfection experiments to overexpress EED, HOTAIR and knockdown SMARCB1. Results Genistein reduced cell proliferation and migration of human renal cell carcinoma cell lines. ChIP assays indicated that genistein reduces recruitment of the PRC2 to the ZO-1 promoter and increased its expression. RIP assays showed that genistein inhibits HOTAIR interaction with PRC2, leading to tumor suppression. Immunoprecipitation also revealed that genistein reduced EED levels in PRC2, suggesting that decreased EED levels suppress HOTAIR interaction with PRC2. EED overexpression in the presence of genistein restored PRC2 interaction with HOTAIR and reduced ZO-1 transcription, suggesting genistein activates ZO-1 by inhibiting HOTAIR/PRC2 functions. RIP assays also showed that HOTAIR interacts with SMARCB1 and ARID1A, subunits of the human SWI/SNF chromatin remodeling complex and genistein reduces this interaction. Combination of HOTAIR overexpression and SMARCB1 knockdown in the presence of genistein revealed that genistein inhibits SNAIL transcription via the HOTAIR/SMARCB1 pathway. Conclusion Genistein suppresses EED levels in PRC2 and inhibits HOTAIR/PRC2 interaction. Genistein suppresses HOTAIR/PRC2 recruitment to the ZO-1 promoter and enhances ZO-1 transcription. Genistein also inhibits SNAIL transcription via reducing HOTAIR/SMARCB1 interaction. We demonstrate that the reduction of HOTAIR interaction with chromatin remodeling factors by genistein represses HOTAIR/chromatin remodeling pathways to suppress RCC malignancy.

Additional file 1: Figure S1. Details of the KRAS point mutation. Figure S2. Details of the KDM6A alteration. Figure S3. (Copy-number alteration and variant allele frequency (VAF) in case 1. The horizontal axis corresponds to each examined gene and the vertical axis corresponds to (A) copy number or (B) VAF. Figure S4. Copy-number alteration and variant allele frequency (VAF) in case 2. The horizontal axis corresponds to each examined gene and the vertical axis corresponds to the (A) copy number or (B) VAF.

Cytochrome P450 (CYP) 1A1 is a phase I enzyme that can activate various compounds into reactive forms and thus, may contribute to carcinogenesis. In this study, we investigated the expression, methylation status, and functional role of CYP1A1 on prostate cancer cells. Increased expression of CYP1A1 was observed in all cancer lines (PC-3, LNCaP, and DU145) compared to BPH-1 (P < 0.05); and was enhanced further by 5-aza-2′-deoxycytidine treatment (P < 0.01). Methylation-specific PCR (MSP) and sequencing of bisulfite-modified DNA of the xenobiotic response element (XRE) enhancer site XRE-1383 indicated promoter methylation as a regulator of CYP1A1 expression. In tissue, microarrays showed higher immunostaining of CYP1A1 in prostate cancer than normal and benign prostatic hyperplasia (BPH; P < 0.001), and methylation analyses in clinical specimens revealed significantly lower methylation levels in cancer compared to BPH at all enhancer sites analyzed (XRE-1383, XRE-983, XRE-895; P < 0.01). Interestingly, smoking affected the XRE-1383 site where the methylation level was much lower in cancer tissues from smokers than non-smokers (P < 0.05). CYP1A1 levels are thus increased in prostate cancer and to determine the functional effect of CYP1A1 on cells, we depleted the gene in LNCaP and DU145 by siRNA. We observe that CYP1A1 knockdown decreased cell proliferation (P < 0.05) and increased apoptosis (P < 0.01) in both cell lines. We analyzed genes affected by CYP1A1 silencing and found that apoptosis-related BCL2 was significantly down-regulated. This study supports an oncogenic role for CYP1A1 in prostate cancer via promoter hypomethylation that is influenced by tobacco smoking, indicating CYP1A1 to be a promising target for prostate cancer treatment.

Precision medicine, which includes comprehensive genome sequencing, is a potential therapeutic option for treating high-grade serous carcinoma (HGSC). However, HGSC is a heterogeneous tumor at the architectural, cellular, and molecular levels. Intratumoral molecular heterogeneity currently limits the precision of medical strategies based on the gene mutation status. This study was carried out to analyze the presence of 160 cancer-related genetic alterations in three tissue regions with different pathological features in a patient with HGSC. The patient exhibited histological heterogeneous features with different degrees of large atypical cells and desmoplastic reactions. TP53 mutation, ERBB2 and KRAS amplification, and WT1, CDH1, and KDM6A loss were detected as actionable gene alterations. Interestingly, the ERBB2 and KRAS amplification status gradually changed according to the region examined. The difference was consistent with the differences in pathological features. Our results demonstrate the need for sampling of the appropriate tissue region showing progression of pathological features for molecular analysis to solve issues related to tumor heterogeneity prior to developing precision oncology strategies.

Silibinin is the major active constituent of silymarin, an extract of milk thistle seeds. Silibinin has been shown to have significant anti-cancer effects in a variety of malignancies. However, the molecular mechanisms of silibinin action in bladder cancer have not been studied extensively. In the present study, we found that silibinin (10 μM) significantly suppressed proliferation, migration, invasion and induced apoptosis of T24 and UM-UC-3 human bladder cancer cells. Silibinin down-regulated the actin cytoskeleton and phosphatidylinositide 3-kinase (PI3K)/Akt signaling pathways in these cancer cell lines. These pathways were found to crosstalk through RAS cascades. We found that silibinin suppressed levels of trimethylated histone H3 lysine 4 and acetylated H3 at the KRAS promoter. Furthermore, silibinin targets long non-coding RNA: HOTAIR and ZFAS1, which are known to play roles as oncogenic factors in various cancers. This study shows that silibinin exerts anti-cancer effects through down-regulation of actin cytoskeleton and PI3K/Akt pathways and thus suppresses bladder cancer growth and progression.

Non-coding RNA (ncRNA) has been shown to regulate diverse cellular processes and functions through controlling gene expression. Long non-coding RNAs (lncRNAs) act as a competing endogenous RNAs (ceRNAs) where microRNAs (miRNAs) and lncRNAs regulate each other through their biding sites. Interactions of miRNAs and lncRNAs have been reported to trigger decay of the targeted lncRNAs and have important roles in target gene regulation. These interactions form complicated and intertwined networks. Certain lncRNAs encode miRNAs and small nucleolar RNAs (snoRNAs), and may regulate expression of these small RNAs as precursors. SnoRNAs have also been reported to be precursors for PIWI-interacting RNAs (piRNAs) and thus may regulate the piRNAs as a precursor. These miRNAs and piRNAs target messenger RNAs (mRNAs) and regulate gene expression. In this review, we will present and discuss these interactions, cross-talk, and co-regulation of ncRNAs and gene regulation due to these interactions.

The proteoglycan versican (VCAN) promotes tumor progression and enhances metastasis in several cancers; however, its role in clear cell renal cell carcinoma (ccRCC) remains unknown. Recent evidence suggests that VCAN is an important target of chromosomal 5q gain, one of the most prevalent genetic abnormalities in ccRCC. Thus, we investigated whether VCAN expression is associated with the pathogenesis of ccRCC. VCAN expression was analyzed using three RCC and normal kidney cell lines as well as a clinical cohort of 84 matched ccRCC and normal renal tissues. Functional analyses on growth and progression properties were performed using VCAN-depleted ccRCC cells. Microarray expression profiling was employed to investigate the target genes and biologic pathways involved in VCAN-mediated ccRCC carcinogenesis. ccRCC had elevated VCAN expression in comparison with normal kidney in both cell lines and clinical specimens. The elevated expression of VCAN was significantly correlated with metastasis (P < 0.001) and worse 5-year overall survival after radical nephrectomy (P = 0.014). In vitro, VCAN knockdown significantly decreased cell proliferation and increased apoptosis in Caki-2 and 786-O cells, and this was associated with alteration of several TNF signaling–related genes such as TNFα, BID, and BAK. Furthermore, VCAN depletion markedly decreased cell migration and invasion which correlated with reduction of MMP7 and CXCR4. These results demonstrate that VCAN promotes ccRCC tumorigenesis and metastasis and thus is an attractive target for novel diagnostic, prognostic, and therapeutic strategies. Implications: This study highlights the oncogenic role of VCAN in renal cell carcinogenesis and suggests that this gene has therapeutic and/or biomarker potential for renal cell cancer. Mol Cancer Res; 15(7); 884–95. ©2017 AACR.

Loss of chromosome (chr) 8p21 is a frequent genomic alteration in prostate cancer (PCa). Genomic deletions of this region increase significantly with tumor grade and are associated with tumor progression and poor prognosis. A common region of loss of heterozygosity (LOH) has been mapped to the chr8p21 locus that primarily harbors prostate-specific NKX3.1 homeobox gene. We recently demonstrated that this frequently deleted locus is associated with a cluster of microRNA genes- miR-3622a/b- that are lost in prostate cancer and play an important mechanistic role in PCa progression and metastasis. MicroRNA expression profiling in microdissected human PCa clinical tissues showed that miR-3622a/b expression is widely downregulated and is correlated with poor survival outcome in prostate cancer. Our analyses suggested that miR-3622a has potential as a prognostic and diagnostic marker for prostate cancer. Extending these findings, we explored the prognostic potential of serum miR-3622a in prostate cancer patients. Since exosomes provide an important source of non-invasive, circulating biomarkers and represent an enriched source of microRNAs for biomarker profiling, we profiled the microRNA content of exosomes derived from sera of prostate cancer patients. In a pilot study, we profiled exosomal miRNAs from a training cohort of individuals with benign prostate hyperplasia (BPH), an indolent form of PCa or aggressive metastatic PCa. Exosomes were isolated from sera of prostate cancer patients and integrity of exosomal preparations was confirmed by nanoparticle tracking analysis and Western blot analyses for exosomal markers. Expression analyses of exosomal miR-3622a expression showed that this miRNA is enriched in exosomes. Higher levels of miR-3622a were significantly associated with tumor stage and lymph node metastasis in a cohort of prostate cancer clinical specimens. Further, ROC (Receiver Operating Characteristic) analyses showed that exosomal miR-3622a expression can be a single significant parameter to discriminate between BPH and prostate cancer. Overall, our data suggests that exosomal miR-3622a is a promising prognostic biomarker for prostate cancer. patients. Citation Format: Thao Yang, Divya Bhagirath, Kirandeep Sekhon, Nathan Bucay, Shahana Majid, Varahram Shahryari, Marisa Shiina, Yutaka Hashimoto, Priyanka Kulkarni, Pritha Dasgupta, Mitsuho Imai-Sumida, Soichiro Yamamura, Z Laura Tabatabai, Kirsten Greene, Yuichiro Tanaka, Rajvir Dahiya, Guoren Deng, Sharanjot Saini. Exosomal miR-3622a as prognostic marker in prostate cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 4435. doi:10.1158/1538-7445.AM2017-4435

Prostate tumors are highly heterogeneous and molecular characterization based on biopsy sampling is often challenging. Hence, it is desirable to have an easily accessible, minimally invasive way to determine the molecular imprints of a patient’s tumor. Exosomes in blood are an important source of non-invasive, circulating biomarkers and provide an enriched source of microRNAs. With an objective of identifying microRNA based markers, we developed a comprehensive procedure for exosomal microRNA profiling analyses from sera of prostate cancer patients employing Nanostring nCounter technology. In a pilot study, we profiled exosomal miRNAs from a training cohort of normal individuals, patients with benign prostate hyperplasia (BPH), indolent form of PCa and aggressive metastatic prostate cancer. Exosomes were extracted from serum and the integrity of exosomal preparations was confirmed by nanoparticle tracking analysis and Western blot analyses for exosomal markers CD63, CD9 and TSG101. Levels of two miRNAs were significantly upregulated and several candidate miRNAs were markedly downregulated in metastatic PCa serum samples compared with controls. The candidate microRNAs were further validated in a larger cohort by real time PCR based microRNA assays. Of significance, we validated miR-1246 as an exosomal marker for prostate cancer. miR-1246 levels were specifically altered in prostate cancer patients and not significantly altered in BPH. Our analyses suggest that miR-1246 is a promising diagnostic biomarker for prostate cancer. Citation Format: Divya Bhagirath, Thao Yang, Kirandeep Sekhon, Nathan Bucay, Shahana Majid, Yutaka Hashimoto, Priyanka Kulkarni, Pritha Dasgupta, Marisa Shiina, Varahram Shahryari, Mitsuho Imai-Sumida, Soichiro Yamamura, Z Laura Tabatabai, Yuichiro Tanaka, Rajvir Dahiya, Guoren Deng, Sharanjot Saini. An exosomal biomarker for prostate cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 5448. doi:10.1158/1538-7445.AM2017-5448

Genistein, a soy isoflavone, has been shown to have anticancer effects on various cancers in vitro and in vivo including renal cancer. Long non-coding RNAs (lncRNAs) are differentially expressed in various tissues and have important functions in cellular processes such as cell proliferation, motility and apoptosis in various malignancies. HOX transcript antisense RNA (HOTAIR) is a lncRNA localized in the Homeobox C gene cluster on chromosome 12. HOTAIR interacts with the polycomb repressive complex 2 (PRC2), which enhances H3K27 trimethylation and represses the expression of tumor suppressors. In various cancers, HOTAIR is highly expressed and involved in their progression and metastasis. In this study, we investigated the molecular mechanisms of genistein action through a novel pathway that represses HOTAIR. We found that HOTAIR expression is higher in renal cancer cell lines compared to normal controls. Genistein treatment was found to significantly decrease HOTAIR expression in renal cancer cells (786-O and ACHN cells). Genistein treatment also reduced expression of epithelial-to-mesenchyme transition (EMT)-related proteins (ZEB1, Vimentin and Snail), causing reduced cell migration, invasion, and increased apoptosis. We performed RNA immunoprecipitation assays, and found that genistein inhibits HOTAIR binding to PRC2. One of the other EMT markers, a tight junction protein ZO-1, is upregulated by genistein. We are currently investigating if genistein represses PRC2 recruitment to the ZO-1 promoter by inhibiting binding of HOTAIR to PRC2. Our results indicate that genistein is a potent therapeutic agent for renal cancer. Citation Format: Mitsuho Imai-Sumida, Shahana Majid, Pritha Dasgupta, Priyanka Kulkarni, Sharanjot Saini, Divya Bhagirath, Taku Kato, Shigekatsu Maekawa, Yutaka Hashimoto, Marisa Shiina, Guoren Deng, Varahram Shahryari, Yuichiro Tanaka, Rajvir Dahiya, Soichiro Yamamura. Genistein inhibits renal cancer progression through long non-coding RNA HOTAIR suppression [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 3449. doi:10.1158/1538-7445.AM2017-3449

Cytochrome P450 1B1 (CYP1B1) converts xenobiotics to carcinogens and polymorphic variants have been shown to increase activity levels. Lifestyle choices such as tobacco smoking and alcohol consumption are known to enhance the carcinogenesis process and in this study, how these factors may interact with CYP1B1 polymorphisms and affect prostate cancer risk was assessed. Blood genomic DNA from a Caucasian population consisting of 405 healthy men and 400 prostate cancer patients were obtained. Of these, 507 were current or former smokers and 407 were alcohol drinkers. Eight polymorphic sites of the promoter region of CYP1B1 (rs2551188 G to A, rs2567206 G to A, rs2567207 A to G, rs162556 A to G, rs10175368 C to T, rs163090 T to A, rs162330 T to G, and rs162331 A to G) were analyzed in samples using Taqman genotyping assays and real-time PCR. Lifestyle factors and its influence on CYP1B1 polymorphisms toward cancer risks were also evaluated. Overall, both alcohol (P=0.006) and smoker (P=0.069) status were associated with prostate cancer. CYP1B1 variants were also risks for cancer at rs2551188 (P=0.043), rs2567206 (P=0.008), and rs10175368 (P=0.001). Evaluation of linkage disequilibrium show rs2551188, rs2567206, rs2567207, and rs10175368 to be linked and interestingly, the G-G-A-C haplotype (wildtype at respective sites) was significantly reduced in cancer (P=0.0282). When classified by lifestyle factors, no associations for CYP1B1 variants were found for cancer among non-smokers with rs10175368 (P=0.051) being a risk among non-drinkers. On the other hand, variants at both rs2567206 and rs10175368 showed increased cancer risk among smokers (P=0.032 and 0.002, respectively) as well as drinkers (P=0.044 and 0.019, respectively). No genotyping differences were observed when analyzing cancers by pathological grades. These results demonstrate smoker and alcoholic drinker status to modify the risks of CYP1B1 polymorphisms for prostate cancer and this is of importance in understanding their role in the pathogenesis of this disease. Citation Format: Taku Kato, Yutaka Hashimoto, Shigekatsu Maekawa, Marisa Shiina, Mitsuho Imai-Sumida, Pritha Dasgupta, Priyanka Kulkarni, Soichiro Yamamura, Shahana Majid, Sharanjot Saini, Varahram Sharryari, Guoren Deng, Rajvir Dahiya, Yuichiro Tanaka. Effects of tobacco smoking and alcohol consumption on risks of CYP1B1 polymorphisms for prostate cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 2288. doi:10.1158/1538-7445.AM2017-2288

Introduction and Objectives: Although versican (VCAN) is known to promote tumor progression and enhance metastasis of several types of cancers, its role in clear cell renal cell carcinoma (ccRCC) remains unknown. Interestingly, a recent report showed that VCAN is an important target of chromosomal 5q gain, one of the most prevalent genetic abnormalities in ccRCC cases. In the present study, we investigated whether VCAN expression is associated with the pathogenesis of ccRCC. Methods: VCAN expression was analyzed in 3 RCC and normal kidney cell lines, as well as 84 matched ccRCC and normal renal tissues. We also performed various functional analyses of growth and progression properties using VCAN-depleted ccRCC cells. Microarray analysis was then employed to investigate the target genes of the pathway involved in ccRCC tumorigenesis and development. Results: There are 4 isoforms of VCAN containing the N-terminal globular (G1 domain) and C-terminal globular (G3 domain) domains, each of which was found to be over-expressed in the ccRCC samples as compared to the controls. Higher VCAN expression was significantly correlated with metastasis (p Conclusion: Our results demonstrate that VCAN promotes ccRCC tumorigenesis and metastasis, showing it to be an attractive novel target for diagnostic, prognostic, and therapeutic strategies for affected patients. Note: This abstract was not presented at the meeting. Citation Format: Yozo Mitsui, Taku Kato, Shigekatsu Maekawa, Yutaka Hashimoto, Marisa Shiina, Mitsuho Imai Sumida, Ryan Kenji Wong, Soichiro Yamamura, Varahram Shahryari, Shahana Majid, Sharanjot Saini, Guoren Deng, Rajvir Dahiya, Koichi Nakajima, Yuichiro Tanaka. VCAN promotes clear cell renal cell carcinoma tumor progression and metastasis, and predicts poor prognosis [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 5749. doi:10.1158/1538-7445.AM2017-5749

Silibinin is the major active constituent of silymarin, an extract of milk thistle seeds. Silibinin has been shown to have significant anti-cancer effects in a variety of malignancies. However, the molecular mechanisms of silibinin action in bladder cancer have not been studied extensively. In the present study, we found that silibinin (10 μM) significantly suppressed proliferation and invasion of T24 and UM-UC-3 human bladder cancer cells. In this study, we investigated the molecular mechanisms underlying these effects of silibinin. Our results showed that silibinin down-regulates actin cytoskeleton-related Ras-Rac-PAK1 pathways and the phosphatidylinositide 3-kinase (PI3K)/Akt signaling pathway in these bladder cancer cell lines. These pathways were found to promote cellular transformation and cancer development and have crosstalk through Ras cascades. We investigated the role of silibinin on Ras, and found that silibinin suppresses levels of trimethylated histone H3 lysine 4 and acethylated H3 at the K-RAS promoter. We also found that silibinin downregulates long non-coding RNAs: HOTAIR and ZFAS1, which are known to play roles as oncogenic lncRNAs in various cancers. This study shows that silibinin exerts anti-cancer effects through down-regulation of the actin cytoskeleton and PI3K/Akt pathways and thus suppresses the development and progression of bladder cancer. Citation Format: Mitsuho Imai-Sumida, Takeshi Chiyomaru, Shahana Majid, Priyanka Kulkarni, Pritha Dasgupta, Sharanjot Saini, Taku Kato, Shigekatsu Maekawa, Yutaka Hashimoto, Marisa Shiina, Guoren Deng, Varahram Shahryari, Hannah Nip, Rajvir Dahiya, Yuichiro Tanaka, Soichiro Yamamura. Silibinin suppresses bladder cancer through down-regulation of actin cytoskeleton and PI3K/Akt signaling pathways [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 199. doi:10.1158/1538-7445.AM2017-199

Objectives: It is of utmost importance that autoimmune pancreatitis (AIP) be differentiated from pancreatic cancer (PC) because some AIP cases undergo unnecessary laparotomy or pancreatic resection on suspicion of PC. This study aimed to develop an appropriate strategy for differentiating between AIP and PC. Methods: Clinical, serological, and radiological features of 17 AIP patients forming a masslike lesion on pancreas head and 70 patients with pancreatic head cancer were compared. Results: Numerous findings can be used to distinguish between AIP and PC, and the following are more likely in AIP: fluctuating jaundice; elevated serum IgG4 levels; delayed enhancement of the enlarged pancreas and a capsule-like low-density rim on computed tomography; long or skipped narrowed portion with side branches of the main pancreatic duct without upstream dilatation on endoscopic retrograde pancreatography, extrapancreatic lesions, such as stenosis of the intrahepatic bile duct, salivary gland swelling, and retroperitoneal mass; and responsiveness to steroid therapy. Conclusions: In elderly male patients presenting with obstructive jaundice and a pancreatic mass, AIP should be considered in the differential diagnosis. Based on a combination of clinical, serological, and radiological findings, AIP can be differentiated from PC. An algorithm for management of patients with a masslike lesion on pancreas head is presented.

Receptor tyrosine kinases (RTKs) are involved in oncogenesis and disease progression for many cancers. Inhibitors targeting them are vigorously developed and some of them are tested in the clinical setting. Amplifications of certain RTKs (c-Met, FGFR2 and ErbB2) have been associated with human gastric cancer progression. According to our genome-wide scans of genetic lesions in 34 gastric cancer cell lines using high-density single-nucleotide polymorphism genotyping microarrays, we confirmed that the c-met locus was amplified in four gastric cancer cell lines (Hs746T, MKN45, NUGC4 and SNU5). It was reported that somatic mutation is occasionally detected in tumor samples of a certain type of cancer with gene amplification. Previous reports showed gastric cancers harbored mutations of FGFR2 and ErbB2, but c-Met oncogenic mutation had not yet been reported. We performed mutational analysis of the cytoplasmic domains of c-Met using the genome DNA of the gastric cancer cell lines, and found that Hs746T cells had a splice site mutation of exon 14. By cDNA sequencing and Western blotting, we showed that the mutation caused juxtamembrane domain deletion. Previously, this mutation had been detected only in lung cancer specimens and this deletion resulted in the loss of Cbl E3-ligase binding causing decreased ubiquitination and delayed down-regulation. In conclusion, four gastric cancer cell lines harbored amplification of c-met locus, and among them, Hs746T had a putative oncogenic mutation with amplification. This information will be useful for screening of inhibitors targeting gastric cancer with c-Met aberration.

Abnormal nuclear accumulation of beta-catenin (CTNNB1) plays one of the key roles in the upregulation of the Wnt signalling pathway that can cause acceleration of cell proliferation. ICAT, inhibitor of beta-catenin and TCF4/beta-catenin-interacting protein, was isolated and mapped to 1p36, a frequent target for LOH in many human cancers. We have previously observed that a number of tumors showing abnormal accumulation of the CTNNB1 protein do not harbor a mutation of the CTNNB1 gene. We studied the precise localization and genomic structure of the ICAT gene and analyzed its mutations in 178 human tumors developed in organs with frequent nuclear accumulation of the CTNNB1 protein and/or frequent LOHs of 1p36, but no genetic alterations were observed. Our results imply that i) genetic alteration of the ICAT gene does not play an important role in abnormal accumulation of CTNNB1 that would cause up-regulation of the Wnt signalling pathway, ii) mechanisms other than genetic alteration may have inactivated ICAT function, or iii) gene(s) on 1p36 other than ICAT may be the responsible tumor suppressor gene for tumors that show frequent 1p36-LOH.

Abnormal nuclear accumulation of beta-catenin (CTNNB1) plays one of the key roles in the upregulation of the Wnt signalling pathway that can cause acceleration of cell proliferation. ICAT, inhibitor of beta-catenin and TCF4/beta-catenin-interacting protein, was isolated and mapped to 1p36, a frequent target for LOH in many human cancers. We have previously observed that a number of tumors showing abnormal accumulation of the CTNNB1 protein do not harbor a mutation of the CTNNB1 gene. We studied the precise localization and genomic structure of the ICAT gene and analyzed its mutations in 178 human tumors developed in organs with frequent nuclear accumulation of the CTNNB1 protein and/or frequent LOHs of 1p36, but no genetic alterations were observed. Our results imply that i) genetic alteration of the ICAT gene does not play an important role in abnormal accumulation of CTNNB1 that would cause up-regulation of the Wnt signalling pathway, ii) mechanisms other than genetic alteration may have inactivated ICAT function, or iii) gene(s) on 1p36 other than ICAT may be the responsible tumor suppressor gene for tumors that show frequent 1p36-LOH.

Pancreatic ductal adenocarcinoma (PDAC), one of the most lethal neoplasms, is characterized histologically by expanded stromal components with marked fibrosis, which suggests that tumor-stromal interaction might play an important role in PDAC progression. We have established pancreas epithelium-specific transforming growth factor-ß receptor type II (Tgfbr2) knockout mice in the context of active Kras (KrasG12D) expression (Kras+Tgfbr2KO) (Ijichi et al., Genes Dev. 2006), which developed differentiated PDAC with 100% penetrance and recapitulated histology of human PDAC described above. In the AACR annual meeting 2009, we reported that tumor-stromal interaction in the PDAC model promoted tumor progression and blockade of CXC chemokines/CXCR2 axis inhibited tumor growth and extended survival of the mice. We examined the underlying mechanisms of anti-tumor effects. Treating the Kras+Tgfbr2KO PDAC mice with the CXCR2 inhibitor decreased CTGF expression and macrophage infiltration in vivo, resulting in decrease of microvessel density and inhibiton of tumor size. The CXC chemokines released from the PDAC cells, CTGF, macrophage, are all considered to promote angiogesis. Taken together, treatment of the mice with the CXCR2 inhibitor achieved anti-tumor effects through anti-angiogenesis via modulating network of an array of components in the tumor microenvironment. On the other hand, treating the mice with gemcitabine, a standard chemoreagent for PDAC, also showed anti-tumor effects, but it was not through anti-angiogenesis. PDAC is well known as a hypovascular tumor, however, angiogenesis might be important for tumor progression and could be a potent therapeutic target. Pursuing optimum combination of anti-angiogenesis and a conventional chemotherapy would provide us a more effective therapeutic strategy for PDAC. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 2259.