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Background: Chronic kidney disease is frequently associated with persistent inflammation, which results in fibrosis in the stroma, a reduced number of renal tubules, the formation of multiple cystic lesions, ultimately terminating in renal failure and hemodialysis. Given the high incidence of renal cell carcinoma (RCC) in hemodialysis patients, suggesting a relationship between tissue remodeling by chronic inflammation and carcinogenesis. However, little is known about the genetic background of cancer development from remaining tubules and cystic lesions in hemodialysis patients. Method: We enrolled 5 patients under hemodialysis who were accompanied by acquired cystic kidney disease and underwent radical nephrectomy for RCC. Surgical specimens were fixed with alcohol-based solution and paraffin-embedded, and after H&E staining, subjected to laser capture microdissection (LCM) to collect remaining tubules and cysts containing approximately 200 cells. DNA was extracted and analyzed for somatic mutations and copy number alteration by whole-exome sequencing. Result: In total, we collected 161 LCM samples, including 118 from proximal tubules, 17 from collecting ducts, and 26 from cysts. Median variant allele frequencies of detected mutations were 0.237 in proximal tubules, 0.133 in collecting duct, and 0.381 in cysts, indicating larger clonal expansion in proximal tubules and cysts than in collecting ducts. Proximal tubules and cysts contained recurrent mutations in FAT1 (11% and 8%, respectively), STAG2 (5% and 8%), and PTEN (3% and 4%), whereas no recurrent driver mutations were identified in collecting ducts. In copy number analysis, 26% of proximal tubules, 35% of collecting ducts, and 80% of cysts had copy number alterations. Of note, samples from cysts had more copy number alterations compared to remaining tubules (on average, 2.3 vs. 1.1). Proximal tubules had recurrent copy number gains of chromosomes 3, 7, 10, 18, and 20, and loss of chromosome 22 and samples from collecting ducts showed gains of chromosome 7 and loss of chromosome 18. In cysts, gains of chromosomes 2, 3, 7, 10, 12, and 16 and loss of chromosomes 15, 16, 21, and 22 were frequently observed. Copy number profile in cysts was similar to that of papillary RCC and acquired cystic disease-associated RCC. Conclusion: In the end-stage cystic kidney, proximal tubules and cysts showed an enrichment of driver mutations commonly found in papillary RCC and RCC with acquired cystic disease, reflecting the fact that the incidence of these two types of RCC increases as the duration of dialysis becomes longer. In addition, clear cell RCC drivers, such as VHL mutation and loss of chromosome 3, were not observed, possibly explaining the rare occurrence of clear cell RCC in hemodialysis patients. Since cysts had more frequent copy number alterations than remaining tubules, cysts in the end-stage kidney might be precursor lesions of RCC. Citation Format: Kosuke Ieiri, Nobuyuki Kakiuchi, Tomonori Hirano, Tomomi Nishimura, Koichi Watanabe, Hiroko Tanaka, Satoru Miyano, Dai Takamatsu, Keisuke Monji, Eiji Kashiwagi, Masaki Shiota, Junichi Inokuchi, Masatoshi Eto, Seishi Ogawa. Genomic analysis of end-stage renal disease [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 6066.

Introduction: The majority of patients with pancreatic cancer are inoperable at the time of diagnosis and even those cases with successful tumor resection for early-stage tumors frequently had second tumors within the remaining pancreas shortly after the surgery, contributing to a poor clinical outcome. Moreover, synchronous multiple pancreatic cancers were often reported. One of the long-standing issues about such synchronous and/or metachronous multiple pancreatic cancers is their clonal origins. In this study, we aimed to reveal the origin of multiple pancreatic cancers using an unbiased detection of somatic mutations in primary and metachronous cancers as well as adjacent precursor lesions. Methods: Serially obtained formalin-fixed paraffin-embedded surgical specimens from 18 patients who had undergone curative surgery for an early-stage pancreatic cancer were subjected to laser microdissection for the enrichment of tumor and precancerous lesions, from which DNA was extracted and analyzed for somatic mutations using whole-exome sequencing (WES) with matched normal DNA. Based on shared and private mutations across different samples, we interrogated history of clonal evolution of these lesions. Results: We analyzed 18 patients with multiple pancreatic cancers of 2 metachronous and synchronous, 14 metachronous, and 2 synchronous tumors. In metachronous cases, pathology for primary cancer were margin-negative in all patients, and the median interval between the initial and second surgery was 38.9 months (7 - 85 months). In addition, a total of 20 pancreatic intraepithelial neoplasia (PanIN) from 5 of these cases were analyzed. We identified a median of 66 (range: 31-232) and 20 (14-42) somatic mutations in cancer and PanIN lesions, respectively. None of the patients have known pathogenic germline variants. All samples had one or more driver mutations. In most cases (N=17), sharing many somatic mutations, multiple tumors had a common clonal origin. In another synchronous case, the synchronous tumors shared only one mutation, suggesting that they were clonally independent tumors. By contrast, none of the mutations other than hotspot KRAS mutations were shared between precursor and cancer lesions. While multiple independent clones were observed in precancerous lesions, most of multiple cancers originated from a common ancestor. Moreover, negative pathology of the margins at the initial surgery suggested that those multiple lesions arose from distant dissemination or metastasis, rather than contiguous, intraductal invasion. Conclusions: In conclusion, our study suggests that even early pancreatic cancer might be disseminated within the pancreas and contribute to synchronous and metachronous cancers. Citation Format: Tomonori Hirano, Nobuyuki Kakiuchi, Yasuhide Takeuchi, Tomomi Nishimura, Toshihiko Masui, Kazuyuki Nagai, Takayuki Anazawa, Sachiko Minamigushi, Hironori Haga, Norimitsu Uza, Hiroshi Seno, Yuzo Kodama, Atsuhiro Masuda, Takeshi Tanaka, Seishi Ogawa, Hiroko Tanaka, Satoru Miyano. Origin of synchronous or metachronous multiple pancreatic cancers [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 1511.

Introduction: The epithelial cells of the endometrium undergo rapid proliferation and shedding each month under the influence of estrogen and progesterone. Recent studies have demonstrated pervasive driver mutations in the endometrium with ageing. However, previous observations have been limited to the microscopic scale, and it is still unclear how clonal expansion occurs over the entire endometrium and what driver mutations are responsible therein. To address these issues, we performed multisampling of the endometrium with extensive spatial mapping. Methods: We collected multiple endometrial glands from normal endometrium in a 5mm lattice in 19 patients who underwent hysterectomy (3-31 samples per case), followed by DNA extraction, detected somatic mutations by whole exome sequencing, to identify large clones that spread across samples based on shared mutations. Results: In total, 216 bulk endometrial glands were analyzed. The number of detected somatic mutations per sample ranged from 6 to 98 (mean±SD: 37.8±16.0), and most of the samples had at least one known driver gene mutation (mean±SD: 4.38±2.27). The mutational signature analysis revealed the predominance of the age-related SBS1 and SBS5 signatures. dN/dS analysis identified 21 genes that were positively selected in normal endometrium, including PIK3CA, PIK3R1, KRAS, PPP2R1A, ARHGAP35, and FBXW7, most of which are also known as driver genes for endometrial cancer. The number of mutations and their frequency for each driver gene did not differ by background diseases: endometriosis, endometriosis-associated ovarian cancer and other non-endometrial related diseases. We found clones showing a large expansion involving multiple samples in 8 cases, most of which harbored one or more driver mutations, such as PIK3CA and KRAS. The size of the largest clone carrying a KRAS mutation, which was found in a 32-year-old woman with cervical cancer, spanned as long as 10 mm in diameter. This suggests that clonal expansion in the endometrium could be very rapid even in healthy women, which contrasts to the observations that clonal expansion in other organs is usually seen in aged individuals. Conclusion: We confirmed the high frequency of driver mutations in the normal endometrium, most of which overlapped to those found in endometrial cancers, suggesting that these driver genes are involved in the early phase of the development in the endometrial cancer. Some of the driver-mutated clones expanded in macroscopic size. Our findings provide an interesting insight into the clonal structure and underlying genetic variation in the normal endometrium. Citation Format: Koichi Watanabe, Nobuyuki Kakiuchi, Shiro Takamatsu, Sachiko Kitamura, Mana Taki, Koji Yamanoi, Ryusuke Murakami, Ken Yamaguchi, Junzo Hamanishi, Hiroko Tanaka, Satoru Miyano, Masaki Mandai, Seishi Ogawa. Clonal expansion with driver mutations in normal human endometrium [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 128.

Introduction: Adjuvant chemotherapy (ACT) has a key role in the treatment of high-risk colorectal cancer (CRC) patients to prevent post surgical recurrence. However, the efficacy of ACT is offset by the development of complication such as peripheral neuropathy, which leads to compromised quality of life, while ACT is expected to prevent tumor recurrence in only 5% and 20% in Stage2 and 3 patients, respectively. Thus, there is an urgent need to develop a biomarker that accurately predicts the effect of ACT and therefore helps guide ACT eligibility. Methods: We enrolled a total of 3,023 cases with CRC, for whom targeted-capture sequencing was performed that covered 169 known or putative driver genes and 1,660 polymorphoic sites to detect somatic mutations and copy number alterations (CNAs), respectively. RNA sequencing was also performed to detect driver gene fusions and to profile gene expression. We identified, through univariate analysis, gene mutations/CNAs as well as clinical parameters using those cases who did not received ACT to establish a model that predict time to recurrence (TTR) using the Cox proportional hazard modelling. Thereafter, the model was applied to those who did receive ACT to assess the impact of the model in the prediction of TTR. Results: We identified a total of 32145 mutations and/or 44511 CNAs in 2970 (98%) out of 3,023 cases. Mutations were found in 55 driver genes, including 15 previously unknown drivers, such as MAP2K1, AKT1, and TCF7. According to the number of mutations, 278 casese were classified as hypermutated cases. The remaining ‘non-hypermutated cases’ (n=2692) were classified into APC-mutated, RNF43/ZNRF3-mutated, CTNNB1-mutated, RSPO-fusion(+) and other Wnt-mutation(−) cases. The TTR prediction model was constructed by repeating Cox regression 1000 times using randomly selected 80% of the non-hypermutated cases and variables identified in univariate analysis. Adopting those variables selected in >70% models, we established the final model including both genetic (mutations/CNAs) and clinical variables, based on which we classified patients into 4 risk groups, low/intermediate/high/very-high. Finally, by applying the classification to those who received ACT, we demonstrated that 18% of Stage2 and the majority of Stage3 cases with high/very-high risks were predicted to benefit from ACT, whereas most of the Stage2 and as many as 19% of Stage3 cases received ACT with no significant improvement of TTR. Approximately 5% of Stage1 cases belonged to the high-risk category and potentially benefit from ACT. Conclusions: On the basis of large-scale genotyping of clinically well-annotated CRC cases, we proposed molecular classification of CRC and established a model that enabled prediction of TTR and risk stratification, which was successfully used to identify cases who benefited (within Stage 2/3 cases) or will benefit (in Stage1 cases) from ACT to prolong TTR. Citation Format: Yoshikage Inoue, Nobuyuki Kakiuchi, Kenichi Yoshida, Yasuhito Nanya, Yasuhide Takeuchi, Yuichi Shiraishi, Kenichi Chiba, Tetusichi Yoshizato, Hiroko Tanaka, Satoshi Nagayama, Kazutaka Obama, Satoru Miyano, Seishi Ogawa. Genomic characterization and risk stratification of colorectal cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 978.

KRAS is one of the most frequently mutated genes in cancer with alterations occurring in > 14% of all tumors. Recent advances have led to the discovery and development of inhibitors that bind the inactive (GDP-bound) form of KRASG12C. The most advanced of these first-generation molecules demonstrated clinical response rates of 30-45% and approximately 6-month progression-free survival in lung cancer patients. While significant, a majority of patients failed to achieve a clinical response and acquired resistance can be rapid. One hypothesis to explain tumor resistance is the failure of existing inhibitors to recognize the activated (GTP-bound) form of KRASG12C that can be upregulated in response to these first-generation inhibitors. Here we report the discovery of a series of novel inhibitors that effectively inhibit both the GTP- and GDP-bound forms of KRASG12C. These “dual-acting” inhibitors bind in the switch II pocket of both GTP-bound and GDP-bound KRASG12C and rapidly form a covalent bond with cysteine 12. This results in significantly increased inhibition of RAF1 and PI3Kα effector interactions (IC50 < 5 nM at 2 hrs.) in comparison to inactive state inhibitors. Dual targeting of both GTP- and GDP-bound KRASG12C results in potent cellular activity in models that are both sensitive (NCI-H358 and MIA PaCa-2) and resistant (NCI-H2122) to adagrasib and sotorasib. In contrast to adagrasib and sotorasib which are less effective in the NCI-H2122 cell line model, dual-acting inhibitors of GTP- and GDP-bound KRASG12C elicit rapid inhibition of pERK in < 1 hour with sustained inhibition of MAPK signaling through 48 hours. To model resistance to first generation inhibitors, an A59G mutation was introduced into KRASG12C, abrogating GTPase activity. This decreases the activity of both adagrasib and sotorasib in tumor cell viability assays by more than an order of magnitude whereas dual-acting inhibitors of GTP- and GDP-bound KRASG12C are equally effective in the G12C/A59G and parental G12C cell lines. Evaluation of dual-acting inhibitors of KRASG12C in vivo demonstrated rapid and > 90% KRASG12C target occupancy, resulting in regression of MIA PaCa-2 tumors. Dual-acting inhibitors of both the active and inactive states of KRASG12C may provide the potential for broader and more durable responses in the clinic. Citation Format: Philamer Calses, Sam Clark, Jacob Corpuz, Susan Fong, Phil Gerkin, Mohammad Hekmatnejad, Evan McMahon, Megan Murray, Truc Nguyen, Tony Phan, Allison Roberts, Phillip Schwartz, Mikayla Shanafelt, Hiroko Tanaka, Jennifer Tomczyk, John Widen, Monika Williams, John Eksterowicz, Daniel Erlanson, Marie Evangelista, Johannes Hermann, Richard M. Neve, Snahel Patel, Kevin R. Webster. Discovery of novel dual-acting KRASG12C inhibitors that target both the active and inactive forms of the protein [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 3601.

[Introduction] Early detection of pancreatic cancer is a key to curable surgery. However, many patients are diagnosed with an advanced disease and even in those who have an early stage tumor successfully resected, the development of metachronous cancer in the residual pancreas prevents a long-term survival. Thus, it is important to control metachronous tumors to improve clinical outcomes, whose pathogenesis, however, is poorly understood. In this study, we aimed to reveal the origin of metachronous tumors using an unbiased detection of somatic mutations in primary and metachronous tumors as well as adjacent precursor lesions. [Methods] Serially obtained formalin-fixed paraffin-embedded surgical specimens from 12 patients who had undergone curative surgery for an early-stage pancreatic cancer were subjected to laser microdissection for enrichment of tumor and precancerous components, from which DNA was extracted and analyzed for somatic mutations using whole-exome sequencing (WES) with matched normal DNA. Based on shared and private mutations across different samples, we interrogated history of clonal evolution of these lesions. [Results] Pathology for resected primary cancer was margin-negative in all patients. The median interval between the initial and second surgery was 34.6 months (12 - 65.1 months). Paired primary and metachronous cancers were analyzed in all 12 patients. An additional 5 pancreatic intraepithelial neoplasia (PanIN) samples were also analyzed in one case. We identified a median of 86 (range: 40-145) and 20 (14-42) somatic mutations using WES in cancers and PanIN lesions, respectively. None of the patients have known pathogenic germline variants. All samples had one or more driver mutations. In each patient, all driver mutations and many passenger mutations were shared between primary and metachronous cancer samples, suggesting that those metachronous tumors are evolutionally closely related to the primary cancer, despite long years before recurrence. Negative pathology of the margins at the initial surgery suggested that those metachronous lesions originated from distant dissemination or metastasis, rather than contiguous, intraductal invasion. By contrast, none of the mutations other than hotspot KRAS mutations were shared between precursor lesions and cancer samples. Despite multiple independent clones in the precancerous lesion, all metachronous tumors originated from the primary lesions in this study. [Conclusions] Our study suggests that even early pancreatic cancer might be disseminated within the pancreas and give rise to metachronous cancers, suggesting the importance of close monitoring of recurrence in the residual pancreas. Citation Format: Tomonori Hirano, Nobuyuki Kakiuchi, Yasuhide Takeuchi, Tomomi Nishimura, Toshihiko Masui, Sachiko Minamiguhi, Hironori Haga, Kenichi Chiba, Hiroko Tanaka, Yuichi Shiraishi, Satoru Miyano, Uza Norimitsu, Yuzo Kodama, Hiroshi Seno, Seishi Ogawa. Genetic analysis of metachronous pancreatic cancers [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 2185.

Skin is the largest organ system and is exposed to ultraviolet (UV) or sun light, a well-known mutagenic insult in various skin cancers, depending on the body site. A previous study showed that keratinocytes in sun-exposed eyelids harbor a high number of somatic mutations affecting a number of common driver mutations found in skin cancers, including NOTCH1, NOTCH2, NOTCH3, FAT1, TP53, and RBM10, suggesting pervasive expansion of clones before the development of skin cancer. However, clonal expansion in other skin sites in association not only with UV but also other insults, such as chronic inflammation is yet to be evaluated. Thus, to fully characterize the clonal expansion in the skin, we performed sequencing analysis of apparently normal skin, as well as that affected by chronic inflammatory conditions such as psoriasis and atopic dermatitis. We analyzed small fragments (0.2mm2) of epidermal samples accrued from surplus biopsy materials using exome sequencing. Analysis of bulk epidermal samples revealed a median of ~30 mutations/exome. Sun-exposed skin was notable for a high mutation burden (>200 mutations/exome). On the basis of the analysis of their variant allele frequencies, the median clone size was estimated to be ~0.1mm2. Several cases harbored large of clones that spanned multiple samples, which were invariably found in sun-exposed skin. In one of such cases, a clone spanned a region having more than 2mm in diameter. Skin affected by chronic inflammation showed a higher number of mutations. dN/dS analysis revealed positively selected driver genes that were similar to the previous report. Diseased skin showed a similar mutation profile that is seen in apparently normal sun-exposed skin. Analysis of mutational signature disclosed signatures that are related ageing (SBS1) and UV light exposure (SBS7). The contribution of SBS7 was prominent in the sun-exposed area but also detected in areas not exposed to sun light, such as the back, buttock, or thighs. By contrast, inflammation-affected skin samples are characterized by a large contribution of the SBS1 signature, likely reflecting increased cell cycles. In conclusion, our analysis shows UV light is a relevant driver of clonal expansion of skin keratinocytes. Chronic inflammation accelerates an accumulation of somatic mutations in and/or clonal expansion of keratinocytes. Citation Format: Yoshihiro Ishida, Nobuyuki Kakiuchi, Yoichi Fujii, Tomonori Hirano, Yoshikage Inoue, Tomomi Nishimura, Tatsuki Ogasawara, Hirona Maeda, Yuichi Shiraishi, Kenichi Chiba, Hiroko Tanaka, Satoru Miyano, Atsushi Otsuka, Kenji Kabashima, Seishi Ogawa. Clonal expansion of skin keratinocyte [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 2675.

Introduction POLE-mutated tumor is a rare subtype of colorectal cancer (POLE-CRC), whose pathogenesis has been poorly understood because of its rarity. In this study, we have investigated the clinical and genetic features of POLE-CRC in the largest cohort of this unique subtype of CRC ever analyzed. Methods We included a total of 3,265 patients who had been treated at the Cancer Institute Hospital of Japanese Foundation for Cancer Research between 2004 and 2017, which were screened for POLE mutations by targeted-panel sequencing. The POLE-mutated samples were further analyzed by whole exome sequencing. All samples were well-annotated for clinical information. Results In total, 44 samples showed prominent hypermutation with a median of 5,346 (range: 837-16,990) with the predominance of COSMIC signature 10 associated with defective POLE functions. By contrast, copy number variations (CNV) in POLE-CRC was much less common compared to non-hypermutated CRC. In accordance with previous reports, mutational hotspots in the POLE gene were found at codons 411, 286, 459, and 456. Patients with POLE-CRC were significantly younger (median of 50 years), compared with non-hypermutated CRC (median of 65 years, p Conclusions POLE-CRC is a CRC subtype with a favorable prognosis compared with other CRC subtypes and is characterized by high frequency of genetic alterations affecting APM, which is implicated in immune evasion of this unique subtype of CRC with an extremely high mutational burden. Citation Format: Yoshikage Inoue, Nobuyuki Kakiuchi, Kenichi Yoshida, Yasuhide Takeuchi, Yusuke Shiozawa, Yuichi Shiraishi, Kenichi Chiba, Tetsuichi Yoshizato, Hiroko Tanaka, Ai Okada, Satoshi Nagayama, Satoru Miyano, Yoshiharu Sakai, Seishi Ogawa. Frequent genomic alterations to evade the immune system in colorectal cancer with POLE gene mutation [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 3547.

Myxofibrosarcoma (MFS) is a rare subtype of soft tissue sarcomas (STSs) preferentially affecting the elderly. Histologically, MFS is distinct from other STSs, in that it is characterized by the proliferation of pleomorphic spindle cells with varying degrees of myxoid components. However, the molecular pathogenesis of MSF is poorly understood. We conducted an integrated molecular study involving 70 samples from primary MFS patients, in which samples were analyzed by whole-genome sequencing (WGS, n=5), whole-exome sequencing (WES, n=44), targeted-capture sequencing (TS, n=65), RNA sequencing (n=3), and immunohistochemistry (IHC, n=50). Copy number (CN) alterations were detected by sequencing-based CN analysis. We combined our STS data with those from from the Cancer Genome Atlas and European Genome-phenome Archive cohort, including 116 MSF samples, and compared to the data from other STS samples (n=189). To further investigate the genetic basis of mixed histological components and chronological changes in MFS, we performed WES of multi-regional and/or multi-time-point samples from 8 MSF cases. A total of 9,246 mutations were identified by WES in 130 primary MFS samples with a median of 55.5 mutations/sample. Mutations were dominated by age-related C to T transitions at CpG sites. When combining the results from TS, among 188 primary MFS samples, most frequently mutated and/or CN-altered genes included TP53 (n=82, 43.6%), RB1 (n=31, 16.5%), ATRX (n=29, 15.4%), and CDKN2A (n=19, 10.2%). A fusion gene involving TRIO was newly identified by RNA sequencing (n=1). A similar mutational profile was observed in undifferentiated pleomorphic sarcoma (UPS), in which TP53 (59.1%), ATRX (34.0%), RB1 (22.7%), and CDKN2A (20.5%) were the major mutational targets, suggesting the common molecular pathogenesis between these two subtypes. Combined with frequent positive staining in IHC (n=22, 44.0%), TP53 was affected in 55.3% of the MFS cases (n=104). Five MFS cases evaluated by WGS showed complex structural abnormalities suggestive of increased genetic instability, where a median of 179 structural variations were detected per sample. WES with multi-regional sampling (n=5) disclosed a high level of intratumor heterogeneity, in which less than 29.0% of mutations were shared by different samples taken from the same tumor. An analysis of longitudinal samples (n=6) revealed significantly higher numbers of mutations in relapse samples (1.6 times on average, p = 0.03). In all cases, TP53 lesions were present from at the time of the initial diagnosis, while most of other lesions were subclonal and acquired during the clinical course. In summary, the genetic profile of MFS is characterized by frequent abnormalities in TP53, RB1, CDKN2A and ATRX, and closely related to other STSs, particularly to UPS. Clonal TP53 abnormalities resulted in complex chromosomal structure and a high degree of intratumor heterogeneity. Citation Format: Yasuhide Takeuchi, Annegret Kunitz, Adriane Halik, Hiromichi Suzuki, Kenichi Yoshida, Yuichi Shiraishi, Kenichi Chiba, Hiroko Tanaka, Nobuyuki Kakiuchi, Yusuke Shiozawa, Akira Yokoyama, Yoshikage Inoue, Tetsuichi Yoshizato, Kosuke Aoki, Yoichi Fujii, Yasuhito Nannya, Hideki Makishima, Satoru Miyano, Hironori Haga, Frederik Damm, Seishi Ogawa. Frequent abnormalities in TP53 and increased genetic instability in myxofibrosarcoma [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 225.

[Introduction] Early detection of pancreatic cancer is a key to curable surgery. However, many patients are diagnosed in an advanced stage and even in those cases who had a surgical resection of early stage tumors, metachronous pancreatic cancer in residual pancreas prevents cure. Thus, it is important to control metachronous tumors to improve clinical outcomes, whose pathogenesis is poorly understood. In this study, we aimed to reveal the origin of metachronous pancreatic cancers using an unbiased detection of somatic mutations in primary and metachronous cancers as well as adjacent precursor lesions. [Methods] Serially obtained formalin-fixed paraffin-embedded surgical specimens from 8 patient who had undergone curative surgery for an early stage pancreatic cancer were subjected to laser microdissection for the enrichment of tumor and PanIN lesions, from which DNA was extracted and analyzed for somatic mutations of each lesion by whole-exome sequencing (WES) with matched normal DNA. Based on shared and private mutations across different samples, we interrogated history of clonal evolution of these lesions. [Results] Pathology for primary cancer were margin-negative in all patients. The median interval between the initial and second surgery was 29.8 months (23 - 64.4 months). We analyzed a median of two cancer and one PanIN samples (0-5), from which we identified a median number of 45(range: 26-69) and 20 (14-42) somatic mutations using WES, respectively. None of the patients have known pathogenic germline variants. All samples had one or more driver mutations. In each patient, all driver mutations and more than half of passenger mutations were shared between primary and metachronous cancer samples, suggesting that those metachronous tumors are evolutionally closely related to the primary cancer. Negative pathology of the margins at the initial surgery suggested that those metachronous lesions originated from distant dissemination or metastasis, rather than contiguous, intraductal invasion. By contrast, none of the mutations other than hotspot KRAS mutations were shared between precursor lesions and cancer samples. Despite multiple independent clones in the precancerous lesion, all metachronous tumors originated from the primary lesions in this study. [Conclusions] Our study suggests that even early pancreatic cancer might be disseminated within the pancreas and contribute to metachronous cancers, suggesting the importance of close monitoring of the recurrence in the residual pancreas. Citation Format: Hirano Tomonori, Nobuyuki Kakiuchi, Yasuhide Takeuchi, Yoshikage Inoue, Tomomi Nishimura, Yoichi Fujii, Akira Yokoyama, Hideki Makishima, Toshihiko Masui, Shinji Uemoto, Sachiko Minamiguchi, Hironori Haga, Kenichi Chiba, Hiroko Tanaka, Yuichi Shiraishi, Satoru Miyano, Norimitsu Uza, Yuzo Kodama, Hiroshi Seno, Seishi Ogawa. Genetic analysis of metachronous pancreatic cancers [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 5877.

Pancreatoblastoma is a rare pediatric pancreatic malignancy for which the molecular pathogenesis is not understood. In this study, we report the findings of an integrated multiomics study of whole-exome and RNA sequencing as well as genome-wide copy number and methylation analyses of ten pancreatoblastoma cases. The pancreatoblastoma genome was characterized by a high frequency of aberrant activation of the Wnt signaling pathway, either via somatic mutations of CTNNB1 (90%) and copy-neutral loss of heterozygosity (CN-LOH) of APC (10%). In addition, imprinting dysregulation of IGF2 as a consequence of CN-LOH (80%), gain of paternal allele (10%), and gain of methylation (10%) was universally detected. At the transcriptome level, pancreatoblastoma exhibited an expression profile characteristic of early pancreas progenitor-like cells along with upregulation of the R-spondin/LGR5/RNF43 module. Our results offer a comprehensive description of the molecular basis for pancreatoblastoma and highlight rational therapeutic targets for its treatment. Significance: Molecular genetic analysis of a rare untreatable pediatric tumor reveals Wnt/IGF2 aberrations and features of early pancreas progenitor-like cells, suggesting cellular origins and rational strategies for therapeutic targeting. Cancer Res; 78(4); 865–76. ©2017 AACR.

E3 ubiquitin ligases play critical roles in directing cellular protein fate by controlling the specificity of ubiquitin conjugation to substrate proteins and targeting them for cellular relocalization or degradation by the ubiquitin proteasome system. The E3 ubiquitin ligase CBL-B is expressed in immune cell lineages and negatively regulates activity of the T-cell receptor (TCR) by imposing a requirement for a costimulatory signal to mount a productive immune response upon TCR engagement. Mice deficient in Cbl-b, and more specifically in the RING Zn-finger ligase domain of Cbl-b, demonstrate a tumor rejection phenotype mediated by CD8+ T cells (Paolino et al., JI, 2011). We have reproduced these results and demonstrate that Cbl-b deficient mice show enhanced anti-tumor activity. In addition, we show that CD4+ and CD8+ T cells from mice deficient in Cbl-b have 5 to 10-fold enhanced secretion of IL-2 and IFN γ when stimulated ex vivo. These data provide a genetic rationale for the development of a small molecule inhibitor of CBL-B ligase activity for use in patients with tumor-mediated immune suppression of effector T cells. We have identified a series of small molecule inhibitors of CBL-B activity with biochemical potency at low nanomolar concentrations. CBL-B inhibitors increased cytokine secretion in vitro at low nanomolar concentrations, as measured by IL-2 and IFN γ secretion, in primary human and mouse T cells stimulated with CD3/CD28 or CD3 alone. The compounds also stimulated proliferation and elevated levels of the T cell surface activation markers CD25 and CD69. CBL-B inhibitors enhanced an antigen recall response in human PBMCs ex vivo, as measured by approximately 5-fold higher secretion of GM-CSF, TNF-α and RANTES, and demonstrated effects in an ex vivo model of exhausted T cell function. Oral dosing of an optimized CBL-B inhibitor enhanced anti-CD3 stimulated T cell activation in mouse CD4+ and CD8+ T cells, demonstrating a dose proportional pharmacodynamic effect. Oral administration over 28 days in the syngeneic CT-26 tumor model was well tolerated and resulted in single agent tumor growth inhibition. These data support the continued advancement of small molecule oral CBL-B inhibitors for future development in immuno-oncology. Citation Format: Jennifa Gosling, Ryan Rountree, Asad Taherbhoy, Chenbo Wang, Thomas Cummins, Frederick Cohen, Hiroko Tanaka, Dahlia Weiss, Mario Cardozo, Christopher Karim, May Tan, Joseph Juan, Austin Tenn-McClellan, Szerenke Kiss von Soly, Julie Sheung, Kathleen Boyle, Ketki Dhamnaskar, Katherine Kurylo, Jilliane Bruffey, Jennifer McKinnell, Dane Karr, Andria Christianson, Anne-Renee Van Der Vuurst de Vries, Pallavur Sivakumar, Mark Gallop, Paul A. Barsanti, Anjanabha Saha, Neil F. Bence, Christoph W. Zapf. Genetic and pharmacologic evaluation of the ubiquitin ligase CBL-B as a small-molecule, tumor immunotherapy target [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 2696.

[Introduction] Early detection of pancreatic cancer is a key to curable surgery, although many are diagnosed in advanced stage. However, even in those cases in which cancer was detected early enough for curative resection, metachronous pancreatic cancer may occur in residual pancreas, whose pathogenesis, including its relationship with primary cancer, has been poorly understood. In this study, we aimed to reveal the origin of metachronous pancreatic cancers using an unbiased detection of somatic mutations in primary and metachronous cancers as well as adjacent precursor lesions. [Methods] Samples were obtained from longitudinal sampling from above lesions using laser microdissection from formalin-fixed paraffin-embedded surgical specimens. DNA was extracted and analyzed for somatic mutations of each lesion by whole-exome sequencing with matched normal DNA. On the basis of shared and private mutations across different samples, we interrogated history of clonal evolution of these lesions. [Results] Four patients were enrolled who underwent curative surgery for early pancreatic cancer and subsequently for metachronous tumors in the residual pancreas. Pathology from surgery for primary cancer were margin-negative in all patients. The median interval between the initial and second surgery was 29.8 months (22.8 - 38.3 months). The number of precursor lesions analyzed was from 0 to 5 per patient. The median number of somatic mutations per sample was 78 (range: 41-92) in cancers and 20 (14-42) in precursor lesions. None of the patients have known pathogenic germline mutations. With a median of 78 and 20 mutations per sample, all samples had one or more driver mutations. In each case, all driver mutations that were detected in cancers were shared between primary and metachronous cancer. It indicated that metachronous cancer branched off from primary cancers at later stage of carcinogenesis. And negative margin at initial surgery suggested that metachronous cancer was formed by dissemination or metastasizing rather than Intraductal progression. By contrast, none of the mutations other than a hotspot KRAS mutations were shared between precursor lesions and cancers, suggesting multiple independent clonal growth of precancerous cells in the cancer bearing pancreas. [Conclusions] Our study shows that metachronous pancreatic cancers are derived from the same origin of initial cancer. Therefore, even in the case of early pancreatic cancer careful checkup for recurrence in the residual pancreas is important. Citation Format: Hirano Tomonori, Nobuyuki Kakiuchi, Yasuhide Takeuchi, Yoshikage Inoue, Tomomi Nishimura, Yoichi Fujii, Akira Yokoyama, Hideki Makishima, Toshihiko Masui, Shinji Uemoto, Sachiko Minamiguchi, Hironori Haga, Kenichi Chiba, Hiroko Tanaka, Yuichi Shiraishi, Satoru Miyano, Norimitsu Uza, Yuzo Kodama, Hiroshi Seno, Tsutomu Chiba, Seishi Ogawa. Genetic analysis of metachronous pancreatic cancers [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 5126.

Myxofibrosarcoma (MFS) is a rare subtype of soft tissue sarcomas (STSs) preferentially affecting the elderly. Histologically, MFS is distinct from other STSs, in that it is characterized by the proliferation of pleomorphic spindle cells with varying degrees of myxoid components. However, the molecular pathogenesis of MSF is poorly understood. We conducted an integrated molecular study involving 70 samples from primary MFS patients, in which samples were analyzed by whole-genome sequencing (WGS) (n=5), whole-exome sequencing (WES) (n=44), targeted-capture sequencing (n=65), RNA sequencing (n=3), and immunohistochemistry (IHC, n=50). Copy number (CN) alterations were detected by sequence-based CN analysis. The obtained genomic data were combined with those from STS cases from the Cancer Genome Atlas (TCGA) cohort, including 17 MSF samples and compared to the data from other STS samples (n=189). To further investigate the genetic basis of mixed histological components and chronological changes in MFS, we performed WES of multi-regional and/or multi-time-point samples from 8 MSF cases. A total of 4,613 mutations were identified by WES in 61 primary MFS samples with a median of 44.0 mutations/sample. Mutations were dominated by age-related C to T transitions at CpG sites. Among 84 primary MFS samples, most frequently mutated and/or CN-altered genes included TP53 (n=64, 76.2%), RB1 (n=29, 34.5%), CDKN2A (n=25, 30.0%), and ATRX (n=18, 21.4%). A fusion gene involving TRIO was newly identified by RNA sequencing in a single case. A similar mutational profile was observed in undifferentiated pleomorphic sarcoma (UPS), in which TP53 (59.1%), ATRX (34.0%), RB1 (22.7%), and CDKN2A (20.5%)) were the major mutational targets, suggesting the common molecular pathogenesis between these two subtypes. Combined with frequent positive staining in IHC (n=22, 44.0%), TP53 was affected in as many as 83.3% of the MFS cases (n=70). Five MFS cases evaluated by WGS showed complex structural abnormalities suggestive of increased genetic instability, where a median of 179 structural variations were detected per sample. WES with multi-regional sampling (n=5) disclosed a high level of intratumor heterogeneity, in which less than 29.0% of mutations were shared by different samples taken from the same tumor. Finally, an analysis of longitudinal samples (n=6) revealed significantly higher numbers of mutations in relapse samples (1.6 times on average, p = 0.03). In all cases, TP53 lesions were present from at the time of initial diagnosis, while most of the other lesions were subclonal and acquired during the clinical course. In summary, the genetic profile of MFS is characterized by frequent abnormalities in TP53, RB1, CDKN2A and ATRX, and closely related to other STSs, particularly to UPS. Clonal TP53 abnormalities resulted in complex chromosomal structure and a high degree of intratumor heterogeneity. Citation Format: Yasuhide Takeuchi, Annegret Kunitz, Hiromichi Suzuki, Kenichi Yoshida, Yuichi Shiraishi, Kenichi Chiba, Hiroko Tanaka, Teppei Shimamura, Nobuyuki Kakiuchi, Yusuke Shiozawa, Akira Yokoyama, Tetsuichi Yoshizato, Kosuke Aoki, Yoichi Fujii, Yasuhito Nannya, Hideki Makishima, Satoru Miyano, Hironori Haga, Frederik Damm, Seishi Ogawa. Myxofibrosarcoma is characterized by frequent abnormalities in TP53 and increased genetic instability [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 738.

Introduction POLE-mutated tumor is a rare subtype of colorectal cancer (POLE-CRC) and has been poorly understood because of its rarity. In this study, we have investigated the clinical and genetic features of POLE-CRC in the largest cohort of this unique subtype of CRC. Methods We included a total of 3,240 patients who had been treated at the Cancer Institute Hospital of Japanese Foundation for Cancer Research between 2004 and 2017, which were screened for POLE mutations, either by targeted-panel sequencing (n=544) or amplicon-based deep sequencing (n=2,696). POLE mutation-positive samples were further analyzed by whole exome sequencing. Results In total, 74 POLE variants were detected in 69 samples, of which 40 showed prominent hypermutation (median; 5,346, range; 837-16,990) with the predominance of COSMIC signature 10 associated with defective POLE functions. Patients with POLE-CRC were significantly younger (median; 50, range; 33-84), compared with those with non-hypermutated CRC (median; 65, p-value Conclusions Similar to MSI-CRC, POLE-CRC shows a favorable prognosis, where the mutations affecting APM are positively selected to evade immune surveillance, suggesting a possible role of checkpoint blockade in its therapeutics. Citation Format: Yoshikage Inoue, Nobuyuki Kakiuchi, Kenichi Yoshida, Yusuke Shiozawa, Yuichi Shiraishi, Kenichi Chiba, Tetsuichi Yoshizato, HIroko Tanaka, Satoshi Nagayama, Satoru Miyano, Yoshiharu Sakai, Seishi Ogawa. Mutational landscape of colorectal cancer with POLE gene mutation [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 1683.

Clonal expansion in aged normal tissues has been implicated in cancer development. However, its chronology and risk-dependence are poorly understood. Esophageal squamous cell carcinoma (ESCC) is a predominant esophageal cancer among Asian populations and substantially affected by heavy smoking and drinking, likely through a ‘field effect’. To elucidate the role of these lifestyle risks on ESCC development, we investigated clonal expansion in physiologically normal esophageal epithelia (PNE) using multiple microscale sampling, as small as 0.2 mm2 in size, followed by an unbiased detection of somatic mutations with whole exome sequencing. Mutations were detected in most of PNE samples (151/157), where none of the mutations were shared between samples collected >10 mm apart. The number of mutations and their allele frequency increased with age, suggesting age-related clonal expansion in PNE (ARCE), which was significantly promoted by heavy smoking and drinking. Mutations were dominated by age-related patterns and a still poorly-defined, ‘esophagus-specific signature, as well as a COSMIC 16-like signature. The latter has recently been related to alcohol drinking and was enriched in high-risk samples, which was confirmed by whole genome sequencing of single cell-derived colonies. As many as 10 genes were significantly mutated or positively selected in ARCE. Among most commonly affected genes were NOTCH1, TP53, FAT1, PPM1D, NOTCH2, and NOTCH3, which substantially differed from those in ESCC, showing prominent over-representation of NOTCH1, PPM1D, FAT1 and NOTCH2, and significant underrepresentation of TP53, NFE2L2, and CDKN2A were significantly underrepresented, suggesting different mechanisms of positive selection between ARCE and ESCC. Driver mutations were detected more frequently and in higher numbers in high-risk PNE samples than low-risk ones, with accentuated NOTCH1, TP53 and PPM1D mutations. Analyses of densely collected micro-scale samples (0.2 mm2) disclosed fine structure of ARCE with its chronological history. Driver-mutated clones emerge multifocally from early childhood as early as Citation Format: Akira Yokoyama, Nobuyuki Kakiuchi, Tetsuichi Yoshizato, Yasuhito Nannya, Hiromichi Suzuki, Yasuhide Takeuchi, Yusuke Shiozawa, Yusuke Sato, Kosuke Aoki, Soo kim, Yoichi Fujii, Kenichi Yoshida, Keisuke Kataoka, Masahiro M. Nakagawa, Yoshikage Inoue, Tomonori Hirano, Yuichi Shiraishi, Kenichi Chiba, Hiroko Tanaka, Masashi Sanada, Shinya Ohashi, Shin’ichi Miyamoto, Shigeru Tsunoda, Koshi Mimori, Sachiko Minamiguchi, Satoru Miyano, Hideki Makishima, Manabu Muto, Seishi Ogawa. Chronology and risk-dependence of age-related remodelling of oesophageal epithelia [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 3322.

Pancreatic neuroendocrine neoplasms (PanNENs) are pancreatic tumors with neuroendocrine differentiation. PanNENs are classified into three grades according to the proliferating cell fraction as measured by Ki-67 or mitotic index. Among them, PanNEN grade 3 (PanNEN G3) shows the highest Ki-67 index > 20%, which is morphologically divided into two categories, well-differentiated (PanNET G3) and poorly differentiated (PanNEC) tumors. Although sharing some overlapping histological features, both tumors also differ from each other with respect of clinical characteristics. To date, the molecular basis of these difference and similarity are poorly understood. To understand their molecular pathogenesis, we performed comprehensive genetic analysis on PanNEN G3. In total, we enrolled 25 PanNEN G3 cases, including 13 PanNET G3, 10 PanNEC, and 2 PanNEN G3 NOS, which were analyzed for somatic mutations. All tumors showed neuroendocrine markers such as synaptophysin or chromogranin A. The median Ki67 index of PanNET G3 was 30.5% (15-78%), which was lower than that of PanNEC (median 70.9%, range 44-100 %). Genomic DNA was extracted from microdissected tumors and paired normal tissues from formalin-fixed paraffin-embedded PanNEN G3 specimens guided by H&E stained sections and subjected to whole-exome sequencing (WES). Copy number alterations were also analyzed on the basis of sequencing data. All tumor specimens were centrally reviewed by the two expert pathologists and assigned to PanNET G3, PanNEC, or PanNEN G3 not otherwise specified (PanNEN G3 NOS). WES analysis detected a total of 1,688 somatic mutations with a median of 45 (range 21-450)/sample. There was no clear difference in mutational burden between PanNET G3 and PanNEC. Combined with copy number alterations, mutations most frequently affected TP53 (40%), followed by MEN1(28%), SMAD4 (28%), KRAS(24%), and CDKN2A (20%). Frequently mutated in PanNET G1/2, MEN1, DAXX, and ATRX mutations were associated with well-differentiated morphology (n=7/7), while tumors with KRAS or RB1 mutations exhibited poorly differentiated histology (n=5/5). MEN1, DAXX, and ATRX mutations frequently co-occurred with mutations in mTOR pathway genes, such as NF1, TSC2, or DEPDC5 (q Our results help understand a molecular basis of PanNEN G3, contributing to a better understanding and classification of PanNET G3 and PanNEC. Citation Format: Nobuyuki Kakiuchi, Kenichi Yoshida, Yusuke Shiozawa, Akira Yokoyama, Keisuke Kataoka, Yoshikage Inoue, Yasuhide Takeuchi, Tomonori Hirano, Yoichi Fujii, Hiroo Ueno, Susumu Hijioka, Nobumasa Mizuno, Waki Hosoda, Yasushi Yatabe, Kenichi Chiba, Hiroko Tanaka, Yuichi Shiraishi, Satoru Miyano, Toshihiko Masui, Shinji Uemoto, Akihiko Yoshizawa, Hironori Haga, Norimitsu Uza, Hiroshi Seno, Yuzo Kodama, Seishi Ogawa. Genetic analysis of pancreatic neuroendocrine neoplasms grade 3 [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 3429.

Myxofibrosarcoma (MFS) is a rare subtype of soft tissue sarcomas (STSs) preferentially affecting elderly. Histologically, MFS is distinct from other STSs, in that it is characterized by the proliferation of pleomorphic spindle cells with varying degrees of myxoid components. However, the molecular pathogenesis of MSF is poorly understood. In this study, we conducted an integrated molecular analysis of 44 samples from primary MFS patients, in which samples were analyzed by whole-genome sequencing (WGS) (n=2), whole-exome sequencing (WES) (n=44), RNA sequencing (n=3), DNA methylation array (n=16), and immunohistochemistry (IHC, n=27). Copy number alterations (CNAs) were identified by sequence based copy number analysis. The obtained genomic data were combined with those from STS cases from the Cancer Genome Atlas (TCGA) cohort, including 17 MSF samples and compared to the data from other STS samples (n=189). To further investigate the genetic basis of mixed histological components and chronological changes in MFS, we performed analysis from multi-regional and/or multi-time-point samples from 8 MSF cases. A total of 4,613 mutations were identified by WES in 61 primary MFS samples with a median of 44.0 mutations/sample. Mutations were dominated by age-related C to T transitions at CpG sites. Most frequently mutated genes included TP53 (n=21, 34.4%), ATRX (n=9, 14.8%), and RB1 (n=3, 4.9%). A fusion gene associated with TRIO was detected by RNA sequencing in a single case. Among other STSs, undifferentiated pleomorphic sarcoma (UPS) harbored the most similar genetic abnormalities (most frequently mutated genes included TP53 (40.9%), ATRX (29.5%), and RB1 (11.3%)), suggesting that these two subtypes are genetically closely related. Also combined cases with CNAs (n=35) and strong staining in IHC (n=13), TP53 abnormality was found in most cases (n=56, 91.8%). Two MSF cases evaluated by WGS showed complex structural abnormalities, where 491 and 198 somatic structural variations were detected suggestive of increased genetic instability. Multi-regional sampling (n=5) disclosed a high level of intratumor heterogeneity with less than 29.0% of mutations being shared by different samples. Multi-time points sampling (n=6) revealed that the number of mutations was significantly higher in relapse samples (odds ratio 1.6, p = 0.03). While in all cases, TP53 lesions were observed at the initial time-point, others were subclonal and acquired during the clinical course. Finally, in methylation analysis, 3,817 differentially methylated regions were detected (Stouffer's p < 0.05), based on which MFS were clustered into two distinct subtypes. In summary, the genetic profile of MFS is characterized by frequent abnormalities in TP53, ATRX, and RB1 and closely related to other STSs, especially to UPS. Clonal TP53 abnormalities resulted in complex chromosomal structure and a high degree of intratumor heterogeneity. Citation Format: Yasuhide Takeuchi, Annegret Kunitz, Hiromichi Suzuki, Kenichi Yoshida, Nobuyuki Kakiuchi, Yusuke Shiozawa, Akira Yokoyama, Yoichi Fujii, Tetsuichi Yoshizato, Kosuke Aoki, Keisuke Kataoka, Yasuhito Nannya, Yuichi Shiraishi, Teppei Shimamura, Kenichi Chiba, Hiroko Tanaka, Hideki Makishima, Satoru Miyano, Hironori Haga, Frederik Damm, Seishi Ogawa. Comprehensive analysis of genetic alterations and intratumor heterogeneity in myxofibrosarcoma [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 3401.

Colitis-associated cancer (CAC) is a common complication among patients with a long history of ulcerative colitis (UC). Previous studies revealed that dysplasia precedes CAC development, and more than 20% of CACs are multicentric, suggesting the presence of a field effect on colorectal carcinogenesis in UC patients. However, the genetic basis of the field effect in UC patients is poorly understood.To address this issue, we isolated single crypts from non-tumorous colon mucosa from 6 UC patients, as well as those from apparently normal mucosa from non-UC patients, and “single crypt-derived DNA” was analyzed for somatic mutations using whole-exome sequencing (WES). In total, 19 crypts from histologically non-dysplastic, but inflamed mucosa, and 2 from apparently normal mucosa were isolated from 6 UC patients, including 4 with pancolitis, 1 with left-sided colitis and 1 with proctitis, while 32 crypts were obtained from normal mucosa from 20 non-UC patients, including 10 with colorectal cancer, 6 with adenoma and 4 healthy volunteers. Regardless of the disease status, mutations were detected in all crypts analyzed with the median of 43/crypt (3 − 121), showing high variant allele frequencies (VAFs) (~0.5), suggesting that these mutations were shared by all cells within single crypts and therefore by crypt-maintaining stem cells. The number of mutations increased with age, in crypts from both UC and non-UC patients (P < 10-9), showing clear age-related C to T transition in both normal and UC-inflamed crypts. Strikingly, however, the age-adjusted number of mutations was significantly higher in UC-derived crypts than in those from non-UC individuals (P < 10-3). KRAS and TP53 mutations were reported to be the most prevalent alterations in UC-associated CAC, but only rarely seen in UC-derived normal crypts. Next, to evaluate clonal expansion of mutated clones, we performed WES for the bulk specimens composed of 20 - 30 mutually adjacent crypts. However, this time, mutations and copy number alterations (CNAs) with high VAFs were detected only in UC-inflamed mucosa, suggesting that clonal crypt expansion occurred only in UC-derived mucosa. WES was also performed for 14 CACs. The number of non-synonymous mutations (101/sample) in CAC were significantly higher than that in UC-derived normal crypts (38/sample) and recurrently involved known driver genes, such as TP53 (71%) and KRAS (36%). CNAs were also more frequent in CAC than UC-derived, normal crypts. In conclusion, our study revealed that somatic mutations accumulated in normal mucosa of both UC and non-UC cases, but the mutational burden increased in UC-inflamed mucosa. Chronic inflammation caused clonal expansion of crypts even in histologically non-dysplastic mucosa in UC case, accompanied by acquisition of driver mutations and CNAs, which could trigger the development of CAC. Our findings provide novel insights into the carcinogenic sequence in UC-derived CAC. Citation Format: Nobuyuki Kakiuchi, Kenichi Yoshida, Yusuke Shiozawa, Akira Yokoyama, Keisuke Kataoka, Yoshikage Inoue, Yasuhide Takeuchi, Yasunori Kogure, Ayana Kon, Masahiro Nakagawa, Kenichi Chiba, Hiroko Tanaka, Yuichi Shiraishi, Satoru Miyano, Kenji Kawada, Hideaki Okajima, Yoshiharu Sakai, Takaki Sakurai, Hironori Haga, Hiroshi Nakase, Motoi Uchino, Hiroki Ikeuchi, Takako Kihara, Seiichi Hirota, Takahiro Horimatsu, Minoru Matsuura, Hiroyuki Marusawa, Hiroshi Seno, Seishi Ogawa. The genetic aberrations in carcinogenic sequence of colitis-associated 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 5754. doi:10.1158/1538-7445.AM2017-5754

Introduction Colorectal cancer (CRC) is the second leading cause of cancer death in Japan. The overall survival rate is approximately 85% and 70% for stage II and stage III CRCs, respectively, where over 45,000 deaths were ascribed to recurrence or metastasis. Survival benefit of adjuvant therapy is well defined only among stage III CRC patients, whereas that for stage II CRC patients remains modest, or according to recent reports, might be even deleterious among those with microsatellite instability (MSI). In an attempt to reveal the landscape of driver mutations in CRC and their association with clinical outcomes, we conducted targeted-capture sequencing of known driver mutations in stage II and III CRCs. Methods A total of 248 patients with stage II (n = 145) and stage III (n = 103) CRC, were enrolled. All patients were treated at a single institute between 2004 and 2016 and clinically well-annotated. Targeted-capture sequencing was performed using RNA baits designed for detection of oncogenic variants in 128 known driver genes in CRC. Additional baits for 1,605 SNPs, 7 introns and 18 microsatellites were also included to assess genome-wide copy numbers, fusion genes and MSI, respectively. Results The median age at diagnosis was 64 (32-95) with a median observation period of 1,460 days. Of the 101 patients with recurrence, 59 and 42 patients had stage II and III diseases, respectively. Sixty percent of stage III patients received adjuvant chemotherapy, whereas only 10% of stage II patients received adjuvant chemotherapy. The average depth of the targeted-capture sequencing was 909×. In total, 2,243 somatic mutations (median 5 per patient, range 0-106) were detected in 243 patients (97.9%). VTI1A-TCF7L2 translocation was identified in one sample. Except for a modest overrepresentation of TP53 mutations, frequency distribution of mutations was similar to those reported in previous reports, where APC (83%), TP53 (79%), KRAS (45%), and PIK3CA (18%) represented the most frequent mutational targets. Although none of these mutations were significantly associated with tumor recurrence or overall survival, we found that FBXW7 mutations had a positive correlation with tumor free survival. Twenty-one patients had more than 20 mutations, suggesting the presence of defects in DNA repair and/or replication. In fact, analysis of 18 microsatellites suggested the presence of MSI in 12 of the 21 samples (4%). The remaining patients with hypermutated cancer were explained by somatic mutations in POLE gene. Conclusions We revealed the landscape of driver mutations in Japanese patients with stage II and III CRC with their implication in clinical outcomes. Hypermutated samples accounted for 8% of the whole cohort, which were explained by MSI or replication errors due to somatic POLE mutations. Our findings help to understand the relationships between driver mutations and recurrence for CRCs after curative resection. Citation Format: Yoshikage Inoue, Nobuyuki Kakiuchi, Kenichi Yoshida, Yusuke Shiozawa, Yuichi Shiraishi, Kenichi Chiba, Keisuke Kataoka, Hiroo Ueno, Hiroko Tanaka, Satoshi Nagayama, Satoru Miyano, Yoshiharu Sakai, Seishi Ogawa. Landscape of driver gene mutations in stage II and stage III colorectal 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 4385. doi:10.1158/1538-7445.AM2017-4385

Backgrounds: Upper urinary tract urothelial carcinoma (UTUC) is relatively rare, accounting for 5-10% of urothelial malignancies with frequent multifocal development. To clarify distinct characteristics of UTUC, we comprehensively investigated the genetic alterations of this disease. Materials & methods: Surgical specimens of UTUC and matched normal samples were obtained from 99 patients with various stages who underwent nephroureterectomy, and subjected to whole exome and RNA sequencing. We compared our results in UTUC with datasets previously reported in bladder urothelial carcinoma (BUC). Mutations in apparently normal urothelial epithelia in 5 cases were also interrogated. Results: Genetic alterations were most frequently observed in TERT promoter (51% of cases), followed by KMT2D (48%), FGFR3 (44%), CDKN2A (42%), TP53 (31%), and RAS pathway (HRAS/KRAS/NRAS, 21%). More than 95% of cases harbored either TP53/MDM2, FGFR3, or RAS pathway mutations in an almost mutually exclusive manner, based on which UTUCs are classified into 3 distinct subgroups with unique molecular and clinical features; FGFR3-mutated tumors showed a significantly better postoperative overall survival than those with TP53/MDM2 (p Mutation patterns were compared between different urothelial cancers with regard to their location. Despite common genes affected, their mutation frequencies were substantially different; KMT2D mutations were more frequent in UTUC, while RB1 alterations were more prevalent in BUC. In addition, KMT2D mutations were significantly more common in UTUCs in the ureter than those in renal pelvis (85% vs. 35%, p In the analysis of normal ureter tissues (N=25 from 5 patients), driver mutations were identified in 6 samples from 2 patients. In one case, tumor and normal samples shared 10-42 mutations, indicating that the cancer evolved within a background of clonal precancerous proliferation in apparently normal epithelia. By contrast, in the other case, none of the mutations were shared between tumors and normal epithelia, suggesting the presence of a field effect on urothelial carcinogenesis. Conclusions: UTUC tumors are classified into 3 molecularly and clinically distinct subtypes based on the status of mutations in TP53/MDM2, FGFR3, and RAS pathway. Depending on their location, urothlial cancers have different genetic backgrounds, where a field effect unique to urothelial epithelia might contribute to multifocal occurrence of UTUC. Citation Format: Yoichi Fujii, Yusuke Sato, Hiromichi Suzuki, Tetsuichi Yoshizato, Yusuke Shiozawa, Kenichi Yoshida, Yuichi Shiraishi, Kenichi Chiba, Hiroko Tanaka, Tohru Nakagawa, Haruki Kume, Hiroaki Nishimatsu, Toshikazu Okaneya, Masashi Sanada, Hideki Makishima, Satoru Miyano, Yukio Homma, Seishi Ogawa. Distinct genomic landscape of upper urinary tract urothelial carcinoma [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 2456. doi:10.1158/1538-7445.AM2017-2456

Pleuropulmonary blastoma (PPB) is a rare pediatric malignancy whose pathogens are poorly understood. Recent reports suggest that germline mutations in the microRNA-processing enzyme DICER1 may contribute to PPB development. To investigate the genetic basis of this cancer, we performed whole-exome sequencing or targeted deep sequencing of multiple cases of PPB. We found biallelic DICER1 mutations to be very common, more common than TP53 mutations also found in many tumors. Somatic ribonuclease III (RNase IIIb) domain mutations were identified in all evaluable cases, either in the presence or absence of nonsense/frameshift mutations. Most cases had mutated DICER1 alleles in the germline with or without an additional somatic mutation in the remaining allele, whereas other cases displayed somatic mutations exclusively where the RNase IIIb domain was invariably affected. Our results highlight the role of RNase IIIb domain mutations in DICER1 along with TP53 inactivation in PPB pathogenesis. Cancer Res; 74(10); 2742–9. ©2014 AACR.

Background Esophageal squamous cell carcinoma (ESCC) represents the most common form of esophageal cancer worldwide, especially in East Asia, where alcohol drinking and smoking have been implicated in the field carcinogenesis of ESCC. However, the oncogenic process therein has been poorly understood in terms of gene mutations. Patients & Methods A total of 100 samples, including cancer, dysplastic, and non-dysplastic esophageal tissues, were obtained from 24 individual with (N = 14) or without (N = 10) ESCC (a median of 2.5 samples per case: 1−29) either by endoscopy or surgery and were subjected to whole exome sequencing (WES). An additional paired cancer/non-caner samples from 32 patients was analyzed by targeted sequencing (TS). All samples were analyzed for copy number alterations (CNAs) using SNP array- and/or digital sequencing-based karyotyping. Results In WES, clonal evolution in esophageal epithelia, as determined by the presence of somatic mutations, was detected in 21 of 21 cancer, 12 of 12 dysplastic, and 63 of 67 non-dysplastic samples, where the mean number of mutation per sample showed a significant trend to increase in cancer (65) and dysplastic samples (50) compared to non-dysplastic samples (13) (P = 2.1×10-11). CNAs, especially those involving CDKN2A, CCND1, YAP1, and EGFR, were frequently affected in cancer samples, but rarely so in non-dysplastic samples. Non-dysplastic samples tended to have smaller allelic burden and therefore, clone size, compared to dysplastic and cancer samples (P = 2.2×10-16). Mutations had a predominant age-related signature in non-dysplastic samples but increasing APOBEC3A/3B patterns was observed in cancer and dysplastic samples. Shared mutations were found only within cancer tissues but never among dysplastic or non-dysplastic samples, suggesting the latter lesions are clonally independent from each other. In accordance with previous reports, TP53 mutations were found in 21/21 cancer samples and also found in dysplastic (11/12) and non-dysplastic samples at a lower frequency (26/67). Strikingly, non-dysplastic samples harbored a very high frequency of NOTCH1 mutations (51/67), which were also found in cancer (3/21) and non-dysplastic (8/12) samples but at much lower frequencies (P = 6.6×10-7). TS of validation samples confirmed the trend of higher NOTCH1 (84% vs. 25%) and lower TP53 mutation rates (38% vs. 100%) in non-dysplastic samples compared to cancer samples. The number of mutations in non-dysplastic samples was higher in drinkers than non-drinkers. Multiple NOTCH1 mutations were more common in cancer patients and drinkers than non-drinkers. Conclusion Clonal proliferation in non-cancer esophageal epithelia is common even in non-ESCC cases and extensive in ESCC cases. NOTCH1 and TP53 mutations play major roles in clonal evolution in common but may have differential impacts on esophageal carcinogenesis, which is likely to be shaped by APOBEC-induced mutations and CNAs. Citation Format: Akira Yokoyama, Hiromichi Suzuki, Tetsuichi Yoshizato, Kosuke Aoki, Yusuke Shiozawa, Youichi Fujii, Yusuke Sato, Nobuyuki Kakiuchi, Sugi Kin, Keisuke Kataoka, Kenichi Yoshida, Hideki Makishima, Yusuke Amanuma, Shinya Oohashi, Yuichi Shiraishi, Kenichi Chiba, Hiroko Tanaka, Brown J.B., Masashi Sanada, Shigeru Tsunoda, Sachiko Minamiguchi, Yoshiharu Sakai, Hironori Haga, Tsutome Chiba, Satoru Miyano, Manabu Muto, Seishi Ogawa. Clonal evolution in noncancerous esophageal mucosa in normal and cancer-bearing individuals. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 164.

Backgrounds Upper urinary tract urothelial carcinoma (UUTUC) is a relatively rare and poor prognostic cancer which accounts for 5-10% of all urothelial malignancies. Despite the histological similarity, there are epidemiological and clinical differences between UUTUC and bladder urothelial carcinoma (BUC). Compared to BUC, the molecular pathogenesis of UUTUC is poorly understood. Urothelial carcinoma is also characterized by frequent multifocal lesions, suggesting field effects from mutagenic agents in urine and/or a dissemination from the primary tumor. To reveal the origin of the multifocality as well as the molecular pathogenesis of UUTUC, we performed comprehensive molecular analysis of UUTUC with regional multiple sampling. Materials & methods Surgical specimens of UUTUC and matched normal samples were obtained from 57 patients with various stages who underwent nephroureterectomy. Morphologically normal urothelial epithelia were also obtained in 5 cases, all of which were confirmed to be pathologically intact by an expert pathologist. Genomic DNA was extracted from each specimen and was subjected to whole exome sequencing using Hiseq 2500 for the detection of both somatic mutations and copy number lesions. Results In copy number analysis, recurrent deletions in 8p, 9p and 9q were identified, where homo deletions of 9p21.3 (CDKN2A) were most frequent (40.7%) Focal amplifications in 11q13.3 (CCND1) and 12q15 (MDM2) were also found. On average, 230 somatic nonsynonymous mutations per sample were detected. The mutation signature was biased to age-related and APOBEC patterns. Mutations were most frequently observed in KMT2D (42% of cases), followed by TP53 (32%), FGFR3 (24%), KDM6A (24%), EP300 (21%) and CREBBP (13%), which were also reported to be significantly mutated in BUC. However, the frequencies of these genetic lesions were substantially different between UUTUC and BUC. For example, abnormalities in RB1, CDKN1A and PIK3CA were not detected or rare in UUTUC but have been reported to be common targets of genetic lesions in BUC. In the analysis of normal epithelia, an average of 12.7 somatic mutations per sample were found but with low variant allele frequencies between 0.05−0.1. Somatic mutations in adjacent epithelia and preoperative urine were frequently shared by primary tumor, supporting a possibility of tumor disseminations from the original sites through urinary flow. In contrast, in other cases, distant epithelia harbored driver gene mutations that were not shared by the primary tumors, suggesting the presence of a mutagenic field effect on urothelial carcinogenesis. Conclusions Despite the similarity in their mutation spectrum, UUTUC and BUC seemed to have distinct pathogenesis in terms of the difference in mutation frequencies. Our data suggested that both dissemination and field effect might contribute to multifocal occurrence of UUTUC. These findings will provide a new insight into the pathophysiology of UUTUC. Citation Format: Yoichi Fujii, Yusuke Sato, Shigekatsu Maekawa, Hiromichi Suzuki, Tetsuichi Yoshizato, Kenichi Yoshida, Yuichi Shiraishi, Kenichi Chiba, Hiroko Tanaka, Tohru Nakagawa, Haruki Kume, Hiroaki Nishimatsu, Yoshikazu Hirano, Masashi Sanada, Hideki Makishima, Satoru Miyano, Yukio Homma, Seishi Ogawa. Genomic landscape and clonal expansions of upper urinary tract urothelial carcinoma. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 94.

Pleuropulmonary blastoma (PPB) is an extremely rare and aggressive pediatric pulmonary malignancy. Recently, DICER1 mutations were reported to be heterozygous frameshift or nonsense mutations of germline origin, suggesting an important role of haploinsufficiency of DICER1. On the other hand, Dicer1 heterozygous deficient mice did not develop PPB, suggesting that DICER1 haploinsufficiency alone may not be sufficient for tumor development. Thus, to identify genetic alterations underlying the pathogenesis of PPB, we performed an integrated molecular study of PPB analyzed by whole-exome, RNA and micro RNA sequencing in conjunction with SNP array analyses. Whole-exome sequencing of 7 cases with sporadic PPB and targeted deep sequencing of DICER1 was performed in additional 5 cases. DICER1 mutations were found in 11/12 cases by whole-exome or deep sequencing, in which 6 cases carried compound heterozygous mutations. Two cases carried homozygous mutations, which were caused by UPD involving the 14q harboring the DICER1 locus. In total, biallelic DICER1 mutations were found in 8/11 cases with DICER1 mutations. Four cases were confirmed that compound heterozygotes of a germline nonsense/frameshift and a somatic missense mutation, two were homozygous for somatic, missense mutations. Conspicuously, all the 9 missense mutations were located within the RNase IIIb domain with a mutational hotspot at G1809, for which somatic origin was confirmed in 7 mutations. To assess the impact of the DICER1 hotspot mutation on RNAase IIIb activity, we performed miRNA sequencing in 3 cases with G1809R mutations. miRNA of fetal lung was used as a control. Comparing the ratio of miRNA or miRNA* read count to pre-miRNA read count, the tumor with G1809R mutant showed significantly low levels of the miRNA product, but the effects were less clear in the levels of the miRNA*, suggesting that G1809R mutants result in reduced RNase IIIb activity but retention of RNase IIIa activity. We also performed RNA sequencing in 5 cases, but apparent breakpoint cluster region was not detected. Since 17p LOH was found in 8/11 cases, we checked the TP53 mutation status in 12 cases by deep sequencing. We detected TP53 mutations in 5/12 cases, in which all 5 cases were accompanied by 17p LOH and leading to biallelic TP53 inactivation. In conclusion, biallelic DICER1 mutations were quite common in PPB, invariably accompanied by a somatic RNase IIIb domain mutation. A majority of cases had mutated DICER1 alleles in germline with or without an additional RNase IIIb domain mutation in the remaining allele. Given that the novel hotspot mutation, G1809R, revealed reduced RNase IIIb activity, alternative RNase IIIb activity could be involved in the tumorigenesis of PPB. Recurrent mutations were rare in PPB, except for frequent TP53 deletions/mutations. Our results provide a novel insight into the critical role of DICER1 mutations and importance of TP53 inactivation in the pathogenesis of PPB. Citation Format: Masafumi Seki, Kenichi Yoshida, Yuichi Shiraishi, Yusuke Sato, Teppei Shimamura, Riki Nishimura, Kenichi Chiba, Hiroko Tanaka, Keisuke Kato, Motohiro Kato, Ryoji Hanada, Yuko Nomura, Myoung-Ja Park, Toshiaki Ishida, Akira Oka, Satoru Miyano, Yasuhide Hayashi, Seishi Ogawa. Biallelic DICER1 mutations in sporadic pleuropulmonary blastoma. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 3088. doi:10.1158/1538-7445.AM2014-3088

Recent genetic studies have revealed a number of novel gene mutations in myeloid malignancies, unmasking an unexpected role of deregulated histone modification and DNA methylation in myeloid neoplasms. However, our knowledge about the spectrum of gene mutations in myeloid neoplasms is still incomplete. So, we analyzed 50 paired tumor-normal samples of myeloid neoplasms using whole exome sequencing, among which we identified recurrent mutations involving STAG2, a core cohesin component, and two other cohesin components, including STAG1 and PDS5B. Cohesin is a multimeric protein complex which is composed of four core subunits (SMC1, SMC3, RAD21 and STAG proteins), and is engaged in cohesion of sister chromatids, DNA repair and transcriptional regulation. To extend the findings in the whole-exome analysis, an additional 534 primary samples of various myeloid neoplasms was examined for mutations and deletions in a total of 9 components of the cohesin complexes, using high-throughput sequencing and SNP arrays. In total, mutations/deletions were found in a variety of myeloid neoplasms, including AML (22/131), CMML (15/86), MDS (26/205), in a mutually exclusive manner. Cohesin mutations frequently coexisted with other common mutations in myeloid neoplasms, significantly associated with spliceosome mutations. Deep sequencing of these mutant alleles revealed that majority of the cohesin mutations existed in the major tumor populations, indicating their early origin during leukemogenesis. Next, we examined several myeloid leukemia cell lines with or without cohesin mutations for expression of each cohesin component and their chromatin-bound fractions. Interestingly, the chromatin-bound fraction of several components of cohesin was significantly reduced in cell lines having mutated or defective cohesin components, suggesting substantial loss of cohesin-bound sites on chromatin. Finally, we introduced the wild-type RAD21 allele into RAD21-mutated cell lines (Kasumi-1), which effectively suppressed the proliferation of Kasumi-1, supporting a leukemogenic role of compromised cohesin functions. Less frequent mutations of cohesin components have been described in other cancers, where impaired cohesion and consequent aneuploidy were implicated in oncogenic action. However, about half of cohesin-mutated cases in our cohort had completely normal karyotypes, suggesting that cohesin-mutated cells were not clonally selected because of aneuploidy. Of note, the number of mutations determined by our whole exome analysis was significantly higher in cohesin-mutated cases compared to non-mutated cases. Since cohesin participates in post-replicative DNA repair, this may suggest that compromised cohesin function could induce DNA hypermutability and contribute to leukemogenesis. In conclusion, our findings highlight a possible role of compromised cohesin functions in myeloid leukemogenesis. Citation Format: Ayana Kon, Lee-Yung Shih, Masashi Minamino, Masashi Sanada, Yuichi Shiraishi, Yasunobu Nagata, Kenichi Yoshida, Yusuke Okuno, Masashige Bando, Shunpei Ishikawa, Aiko Sato-Otsubo, Genta Nagae, Aiko Nishimoto, Claudia Haferlach, Daniel Nowak, Yusuke Sato, Tamara Alpermann, Teppei Shimamura, Hiroko Tanaka, Kenichi Chiba, Ryo Yamamoto, Tomoyuki Yamaguchi, Makoto Otsu, Naoshi Obara, Mamiko Sakata-Yanagimoto, Tsuyoshi Nakamaki, Ken Ishiyama, Florian Nolte, Wolf-Karsten Hofmann, Shuichi Miyawaki, Shigeru Chiba, Hiraku Mori, Hiromitsu Nakauchi, H. Phillip Koeffler, Hiroyuki Aburatani, Torsten Haferlach, Katsuhiko Shirahige, Satoru Miyano, Seishi Ogawa. Recurrent pathway mutations of multiple components of cohesin complex in myeloid neoplasms. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 4602. doi:10.1158/1538-7445.AM2013-4602

Adult T-cell leukemia/lymphoma (ATL) is an aggressive peripheral T-cell neoplasm, which is etiologically associated with human T-lymphotropic virus type I (HTLV-1) infection during early infancy. Although HTLV-1 can effectively immortalize T-cells, there is a long latency period of ∼50 years prior to the onset of ATL, suggesting that HTLV-1 infection alone is not sufficient for the development of ATL, but additional acquired genetic hits that occur in immortalized T-cells during the later life are essential for its pathogenesis. However, little has been known about the gene mutations commonly involved in ATL pathogenesis. So, in order to understand the genetic basis of ATL, we performed whole exome sequencing of paired-normal DNA from 24 patients with different ATL subtypes, including acute (N=13), chronic (N=3), lymphoma (N=8) types. With a mean coverage of 96, 83% of the target sequences were analyzed at more than 20 depth on average. The mutation rate of 108 (44-207) per sample was significantly higher than that in acute myeloid leukemia (7.3-13), myelodysplastic syndromes (9.2) and chronic lymphocytic leukemia (11.5). What immediately drew our attention were the recurrent mutations involving TET2 mutations. They are found in a wide variety of myeloid malignancies in high frequency and implicated in their pathogenesis. The TET families of proteins are thought to be involved in the epigenetic regulation of gene expression through catalyzing conversion of 5’-methyl cytosine to 5’-hydroxymethyl cytosine, which are supposed to be further converted to unmethylated cytosine. One of the recent interests in TET2 mutations was the recent report of frequent TET2 mutations in peripheral T cell neoplasms, including angioimmunoblastic T-cell lymphomas(AITL), peripheral T-cell lymphomas not otherwise specified(PTCL-NOS), as well as other B cell neoplasms. In addition, 145 samples of ATL were screened for mutations in TET2 and other epigenetic regulators commonly mutated in myeloid malignancies, including DNMT3A, IDH1/2 using target deep sequencing. In total, 17 TET2 mutations were identified in 14 (9.6%) out of the 145 ATL samples. Less frequent mutations of IDH2 and DNMT3A were also identified. Different subtypes of ATL were almost evenly affected with 6 out of 47 acute, 3 out of 36 chronic and 5 out of 46 lymphoma types having TET2 mutations. In most evaluable cases, the TET2 mutations harbored a major tumor population, while the mutations in two cases only involved a minor population and was suggested to represent a relatively late event during clonal evolution. Combined with the recent studies reporting similar AITL and PTCL-NOS, our finding suggested a common role of deregulated epigenetic machinery in the development of mature T-cell neoplasms including ATL. Citation Format: Yasunobu Nagata, Yusuke Okuno, Aiko Sato, Yuichi Shiraishi, Kenichi Chiba, Hiroko Tanaka, Kenichi Yoshida, Masashi Sanada, Ken Ishiyama, Shuichi Miyawaki, Akira Kitanaka, Kazuya Shimoda, Satoru Miyano, Seishi Ogawa. Whole exome analysis reveals spectrum of gene mutations in adult T-cell leukemia/lymphoma. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 2019. doi:10.1158/1538-7445.AM2013-2019