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[Introduction] Proliferative lesions in the breast have been implicated in the development of breast cancer. Previous studies showed that some proliferative lesions and adjacent breast cancers shared common genetic alterations, suggesting that these originated from the same ancestral cell. However, the clonal structure of normal epithelia and their clonal history during evolution to cancer are poorly understood. In this study, we analyzed genetic profiles of normal epithelia and proliferative lesions in the cancer-borne breast to illustrate the clonal evolution of cancer from a normal epithelial cell. [Methods] Single cell-derived organoids (n=47) were established from breast milk of 4 healthy women aged 22–36 and normal breast tissue of 15 breast cancer patients aged 29–83 to evaluate somatic mutation rate in normal epithelial cells. Multiple normal lobules and proliferative lesions together with cancer lesions were collected using laser-capture micro-dissection (LCM) from fresh frozen (n=3) or formalin-fixed paraffin-embedded (n=5) surgical specimens in 9 premenopausal breast cancer patients. Somatic mutations and copy number alterations were evaluated using whole-genome sequencing. [Results] The mutation profile of single cell-derived organoids suggests that somatic mutations accumulate in normal mammary epithelial cells at a constant rate of 19.4/genome/year before menopause, and the mutation rate decreases to 6.9/genome/year after menopause. Parity was negatively associated with mutation number (-49.3 per life birth). In total, we analyzed 143 LCM samples, including those from 72 normal lobules, 43 proliferative lesions, and 19 non-invasive and 9 invasive cancer samples. Five cases showed a large expansion of proliferative lesions sharing a substantial number of somatic mutations with cancer. These lesions expanded over a distance of 35-90 mm, sharing tens to hundreds of mutations including those in breast cancer-related driver genes, such as PIK3CA, AKT1, GATA3, CBFB and PTEN, while harboring private mutations or copy number alterations of their own. Of interest, the cancers in 4 out of these 5 cases was luminal-A type invasive ducal carcinoma or ER-positive HER2-negative ductal carcinoma in situ, and characterized in common by the presence of der(1;16), concurrent whole-arm 1q gain and 16q loss, in both cancer and proliferative lesions. Phylogenetic analysis adapted with the mutation rate in normal cells predicted that der(1;16) had been acquired between puberty and early 20’s, and the common ancestors of non-cancerous and cancerous lesions emerged by early 30’s, >10 years earlier than at the time of cancer diagnosis. By contrast, analysis of non-cancerous lobules unrelated to cancer showed that der(1;16)-negative non-cancer clones that had emerged after puberty stayed within a single lobule or spatially confined to adjacent lobules and rarely expanded to a large area as observed for those carrying der(1;16), even if the clones had acquired mutations in driver genes such as PIK3CA and PIK3R1, which highlighted the role of der(1;16) in wide clonal expansion. [Conclusions] Our results suggest that in some breast cancer cases, particularly in those with der(1;16), a highly recurrent translocation accounting for the major subset of Luminal A breast cancer, the clones with the funder driver alterations expanded macroscopically long before the onset of cancer, in which further clonal evolutions recursively occur multi-focally, giving rise to multiple proliferative lesions and ultimately, invasive cancers. Our findings provide new insight into the early development of breast cancer. Citation Format: Tomomi Nishimura, Nobuyuki Kakiuchi, Kenichi Yoshida, Takaki Sakurai, Tatsuki R. Kataoka, Eiji Kondoh, Yoshitsugu Chigusa, Masahiko Kawai, Morio Sawada, Takuya Inoue, Yasuhide Takeuchi, Hirona Maeda, Satoko Baba, Yusuke Shiozawa, Ryunosuke Saiki, Masahiro M. Nakagawa, Yasuhito Nannya, Yotaro Ochi, Tomonori Hirano, Yukiko Inagaki-Kawata, Kosuke Aoki, Masahiro Hirata, Eiji Suzuki, Masahiro Takada, Masahiro Kawashima, Kosuke Kawaguchi, Kenichi Chiba, Yuichi Shiraishi, Junko Takita, Satoru Miyano, Masaki Mandai, Kengo Takeuchi, Hironori Haga, Masakazu Toi, Seishi Ogawa. Clonal evolution of mammary epithelial cells into breast cancers [abstract]. In: Proceedings of the 2022 San Antonio Breast Cancer Symposium; 2022 Dec 6-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2023;83(5 Suppl):Abstract nr P5-13-04.

Neuroblastoma is the most common extracranial solid tumor that occurs in childhood and the most common cancer in infancy. Fewer than 5% of neuroblastomas occur in adolescents and young adults (AYAs), for whom the disease has an indolent and fatal course. Chen et al. previously reported that missense mutations in the protein- tyrosine kinase domain of anaplastic lymphoma kinase (ALK) occur in approximately 10% of cases of sporadic neuroblastoma, and these missense mutations result in the activation of the ALK protein kinase domain, which plays a key role in the tumorigenic process as seen in ALK fusion proteins. Since the initial discovery of the NPM-ALK fusion protein in human anaplastic lymphoma cell lines, more than 24 different ALK fusion proteins have been discovered in various malignancies. Till date, ALK-containing chimeric proteins have not been reported in neuroblastoma. In this study, a translocation within chromosome 2p and 4q was found to bring about the formation of an in-frame fusion gene that was composed of portions of the teneurin transmembrane protein 3 (TENM3) gene and ALK gene in tumor cells from an AYA patient with neuroblastoma. The patient was 19-year-old female diagnosed with stage 4 neuroblastoma (unfavorable histology, poorly differentiated subtype, MYCN DNA not amplified). Computed tomography revealed a mass in the left adrenal gland with bone, spine, liver, and lung metastasis. ALK rearrangement was confirmed via FISH analysis. Crizotinib resulted in no disease progression for 5 months, except for metastatic spinal disease, but it did not exhibit high efficacy compared with its effects against lymphoma and lung cancer. This 5′ in-frame fusion partner gene in neuroblastoma has not yet been identified, and its role is not well-known in oncogenesis. We generated the cell line with the expression of the full length TENM3-ALK cDNA in NIH-3T3 cells led to the formation of a fusion protein. A cell-based evaluation of this predicted ALK protein variant revealed activation of downstream targets-STAT3, AKT and ERK- and ALK inhibitors, crizotinib or lorlatinib, treatment inhibited the growth of xenograft tumors with stable TENM3-ALK expression. We determined whether TENM3-ALK affects transformation and proliferation using a soft agar colony formation assay. As expected, the cells that expressed an empty vector and wild type ALK line did not produce a considerable number of colonies, whereas TENM3-ALK transfected cells showed anchorage-independent growth, resulting in the production of a higher number of colonies. The evidence for the role of ALK receptor signaling in stimulating neuroblastoma tumor cell growth and the demonstrated in vitro and in vivo efficacy of the ALK selective inhibitors in this study provide biological and clinical justification for the further exploration of this combination and testing in patients with recurrent or refractory neuroblastoma. Citation Format: Mitsuteru Hiwatari, Masafumi Seki, Ryosuke Matsuno, Kenichi Yoshida, Takeshi Nagasawa, Aiko Sato-Otsubo, Shohei Yamamoto, Motohiro Kato, Kentaro Watanabe, Masahiro Sekiguchi, Satoru Miyano, Seishi Ogawa, Junko Takita. Identification of the novel TENM3-ALK fusion in an AYA case with ALK rearranged neuroblastoma [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 6734.

Translocations of retinoic acid receptor-α (RARA), typically PML–RARA, are a genetic hallmark of acute promyelocytic leukemia (APL). However, because a small fraction of APL lack translocations of RARA, we focused here on APL cases without RARA translocation to elucidate the molecular etiology of RARA-negative APL. We performed whole-genome sequencing, PCR, and FISH for five APL cases without RARA translocations. Four of five RARA-negative APL cases had translocations involving retinoic acid receptor-β (RARB) translocations, and TBL1XR1–RARB was identified as an in-frame fusion in three cases; one case had an RARB rearrangement detected by FISH, although the partner gene could not be identified. When transduced in cell lines, TBL1XR1–RARB homodimerized and diminished transcriptional activity for the retinoic acid receptor pathway in a dominant-negative manner. TBL1XR1–RARB enhanced the replating capacity of mouse bone marrow cells and inhibited myeloid maturation of human cord blood cells as PML–RARA did. However, the response of APL with RARB translocation to retinoids was attenuated compared with that of PML–RARA, an observation in line with the clinical resistance of RARB-positive APL to ATRA. Our results demonstrate that the majority of RARA-negative APL have RARB translocations, thereby forming a novel, distinct subgroup of APL. TBL1XR1–RARB as an oncogenic protein exerts effects similar to those of PML–RARA, underpinning the importance of retinoic acid pathway alterations in the pathogenesis of APL. Significance: These findings report a novel and distinct genetic subtype of acute promyelocytic leukemia (APL) by illustrating that the majority of APL without RARA translocations harbor RARB translocations. Cancer Res; 78(16); 4452–8. ©2018 AACR.

Translocations of retinoic acid receptor-α (

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.

Neuroblastomas harbor mutations in the nonreceptor anaplastic lymphoma kinase (ALK) in 8% to 9% of cases where they serve as oncogenic drivers. Strategies to reduce ALK activity offer clinical interest based on initial findings with ALK kinase inhibitors. In this study, we characterized phosphotyrosine-containing proteins associated with ALK to gain mechanistic insights in this setting. Flotillin-1 (FLOT1), a plasma membrane protein involved in endocytosis, was identified as a binding partner of ALK. RNAi-mediated attenuation of FLOT1 expression in neuroblastoma cells caused ALK dissociation from endosomes along with membrane accumulation of ALK, thereby triggering activation of ALK and downstream effector signals. These features enhanced the malignant properties of neuroblastoma cells in vitro and in vivo. Conversely, oncogenic ALK mutants showed less binding affinity to FLOT1 than wild-type ALK. Clinically, lower expression levels of FLOT1 were documented in highly malignant subgroups of human neuroblastoma specimens. Taken together, our findings suggest that attenuation of FLOT1-ALK binding drives malignant phenotypes of neuroblastoma by activating ALK signaling. Cancer Res; 74(14); 3790–801. ©2014 AACR.

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] Hepatoblastoma (HB) is a rare and heterogeneous childhood malignancy of liver. It is mainly affecting young children in the first 2 years of life, underscoring the unique sensitivity of the immature liver to tumorigenesis. The unique age range affected by this embryonal tumor suggests that developmental factors contribute to the tumor development. On the other hands it is well recognized activation of Wnt/β-catenin signaling pathway was frequently occurred in HB through somatic mutation of CTNNB1 and germline mutation of APC; however, the exact mechanism that normal hepatic developmental processes are altered by mutational events is an area of current research and the definitive contribution to tumorigenesis is unclear mainly due to its rarity. [Methods] To elucidate the genetic aberration underlying development and progression of HB, we performed whole exome sequencing and RNA sequencing using Illumina HiSeq 2000 on 14 HB specimens. In addition, we employed detailed genomic analysis on 5 multiple samples obtained from primary, post-chemotherapy and relapsed HB with familial adenomatous polyposis (FAP). All the candidates for mutations and fusions were validated by target and Sanger sequencing. Detected genetic alterations were sequenced in additional 53 HB samples. [Results] The somatic mutation rate was 8.1 (range of 1-41) per tumor with median of 0.16 non-silent mutations per Mb. It is statistically significant that number of somatic mutations is correlated with age. Consistent with the previous reports, most frequent mutated gene in our cohort was CTNNB1 with frequency of 71.4%. Overall, HBs have low mutation rates in concordance with other pediatric solid tumors. In our cohort, recurrent somatic mutations with high frequency was not detected, but overlapping germline mutations in Wnt signaling pathway were found, even if they were not affected with FAP. In total, 42 (79.2%) of 53 HB had mutations in genes regulating WNT/β-catenin signaling pathway. In multisampling sequencing analysis based on 5 different sites of HB specimens, progression from normal hepatocyte to HB seemed to be accompanied by few trancal events including only APC mutation in Wnt/β-catenin signaling pathway and gain of 1q and loss of 4q in copy number analysis. The major clone of relapsed tumors derived from minor clone of the primary tumor after chemotherapy getting additional genomic aberrations. [Conclusion] Our results revealed that mutations in Wnt/βcatenin-signaling pathway were truncal and significant mutations. In addition, even if only one cases multisampling sequencing, it is useful for revealing clonal evolution of HB. This study disclosed comprehensive genetic features of HB and identified potentially important therapeutic targets, as well as insight into possible cancer predisposition genes. Citation Format: Noriko Hoshino, Masafumi Seki, Motohiro Kato, Kenichi Yoshida, Yusuke Sato, Atsuko Nakazawa, Satoru Miyano, Akira Oka, Tadashi Iwanaka, Yasuhide Hayashi, Seishi Ogawa, Junko Takita. Clonal evolution and integral analysis of hepatoblastoma. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 3294. doi:10.1158/1538-7445.AM2015-3294

Pleuropulmonary blastoma (PPB) is an extremely rare and highly aggressive pediatric pulmonary malignancy that is often part of inherited cancer syndrome. Recently, germline mutations of DICER1, a gene encoding an endoribonuclease type III critical to the generation of small noncoding regulatory RNAs were reported in the majority of familial PPB. However, little is known about other genetic changes than DICER1 mutations that are responsible for the development and/or progression of PPB. Therefore, to assess the genetic alterations underlying the pathogenesis of PPB, genome-wide analyses using whole-exome sequencing and 250K SNP array were performed in total of ten cases with non-familial PPB. Whole-exome sequencing was performed in two cases which primary tumor-relapse tumor pairs were available. Although most mutations were commonly detected in both the primary and relapse tumors, several alterations were private to either of both samples, suggesting that clonal evolution derives genetic divergence in relapsed/metastatic tumors of PPB. In accordance with this, SNP array analysis also revealed several shared or private copy change variations, the most common finding was gain of the long arm of chromosome 8 in 7 cases, which was previously reported common in PPB. Whole-exome sequencing disclosed that DICER1 mutation, major cause of familial PPB, was recurrently found in the primary and relapse tumors of one case, but not detected in the another case. In the validation cohort of 8 cases of non-familial PPB, all of them had DICER1 mutations, and 6 cases had compound heterozygous mutations of DICER1. Notably, homozygous DICER1 mutation was observed in one case, and subsequent SNP array analysis revealed uniparental disomy of 14q lesion harboring DICER1 in this case. Somatic origins of missense changes were confirmed in 2 of 10 cases, for which DNA was obtained from peripheral blood. Meanwhile, whole-exome sequencing identified 13 candidate genes including the cadherin-related gene and a negative regulator of cell cycle in one case without DICER1 mutation. These gene mutations shared in primary and relapse samples, suggests that these are driver mutations rather than passenger mutations or non-functional SNPs. In conclusion, DICER1 mutation might be a driver mutation not only in familial PPB, but also in non-familial PPB. While DICER1 mutation was reported only germline heterozygous mutation in familial PPB, we notably found compound heterozygous or homozygous DICER1 mutations in non-familial PPB. Furthermore, there were different clones between primary and relapse samples, suggests that clonal evolution contributes to relapse tumor progression. Citation Format: Masafumi Seki, Riki Nisimura, Yusuke Okuno, Kenichi Yoshida, Yuichi Shiraishi, Masashi Sanada, Keisuke Kato, Katsuyoshi Koh, Ryoji Hanada, Yasuhide Hayashi, Satoru Miyano, Seishi Ogawa, Junko Takita. Genome-wide approach to identify gene targets of pleuropulmonary blastoma. [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 3803. doi:10.1158/1538-7445.AM2013-3803

Recently, mutational and epigenetic profiling of a large acute myeloid leukemia (AML) patient cohort revealed that NADP+dependent isocitrate dehydrogenase (IDH)1/2-mutated AMLs display global DNA hypermethylation and a specific hypermethylation signature, suggesting a shared proleukemogenic effect. To explore the involvements of IDH1/2 in the pathogenesis in pediatric hematological malignancies. We analyzed mutations that involve the activation sites of IDH1/2 using polymerase chain reaction amplification/sequencing in a total of 244 samples of pediatric myeloid malignancies as well as infantile leukemia including 17 AML-derived cell lines, 115 primary cases of AML, 28 primary cases of MDS, 15 primary cases of juvenile myelomonocytic leukaemia (JMML), 6 chronic myeloid leukemia (CML)-derived cell line, 18 primary cases of CML and 45 infantile leukemia(6 AML and 39 acute lymphoblastic leukemia (ALL) patients). Moreover, to assess whetherIDH1/2 mutations overlap with known gene abnormalities, such as FLT3, c-KIT, and NPM1 mutations, mutational analyses of FLT3, c-KIT, and NPM1 were also performed. The common IDH2 R140Q mutation was detected in a single AML case, whereas no IDH1 mutation was detected in samples of myeloid malignancies. Although no IDH2 mutation was detected in infantile leukemias, novel P127S, H133I and I130V of IDH1 mutations were detected in 4 of 45 (8.9%) infantile ALL cases. No IDH1 and IDH2 mutations were detected in the JMML, MDS, or CML samples examined. Six AML samples including one cell line had c-KIT mutations (D816V, N822K, or D419fs), 12 AML cases had FLT3-ITD and 10 infantile leukemia cases had FLT3 mutations (D835E or I836). The NPM1 mutation was detected in 2 of 132 AML samples. The AML case harboring the IDH2 mutation, Case 39 was a 12-year-old boy diagnosed as AML-M2 according to the French-American-British cooperative group classification system having t(8;21)(q22;q22), showed no abnormalities of NPM1, c-KIT, and FLT3. Remarkably, among 4 infantile ALL cases with IDH1 mutations, 3 cases showed mixed lineage leukemia(MLL) rearrangements with t(4;11). The FLT3-D835 mutation was found in 1 of 4 patients with IDH1 mutations. These results suggest that although the involvements of IDH1/2 mutations in the pathogenesis of pediatric myeloid malignancies are extremely rare, closely positioned to near activation site, R132 IDH1, these IDH1 mutations are the candidate second genetic events in a subset of MLL-leukemia. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 71. doi:1538-7445.AM2012-71

Neuroblastoma (NBL) is one of the most common pediatric tumor arising from developing sympathetic nervous system. Its clinical and biological behavior is markedly heterogenous ranging from aggressive progression despite of intensive therapy to spontanous regression. NBL DNA shows complex patterns of genomic aberrations including MYCN amplification, deletion of chromosome 1p or 11q, and gain of chromosome 17q. We previously constructed the genomic subgrouping system of NBL based on the array CGH analysis for new tumor risk classification of NBL (Tomioka et al, Oncogene 2008). Analysis of 234 tumors by using 2,464 clones-BAC arrays demonstrated there exist three genomic groups (GG) of chromosomal aberrations in NBL: silent (S), partial gains and/or losses (P) and whole gains and/or losses (W). Each GG was further segregated into subgroups with different clinical outcomes clearly defined by MYCN amplification (MYCN-amp), 1p loss, 11q loss and 17q gain. Importantly, high-resolution array CGH analysis also led us to identify a novel genomic alteration in NBL, the activating mutation and amplification of anaplastic lymphoma kinase (ALK) oncogene on chromosome 2p23. In this study, to conduct the clinical validation of our GG risk classification system, array CGH and mutation analysis of ALK gene of 343 sporadic NBLs (stage 1: 48; stage2: 29; stage 4s: 27; stage 3: 60; stage 4: 179; median follow-up period is 78 months) were performed and the clinical impact of the genomic signatures was assessed. The results were fairly reproducible with our previous data, showing that GG-S or GG-W without MYCN-amp exhibited good prognosis (Ss and Ws, 8-year survival rates: >80%), while GG-P or GG-S accompanying MYCN-amp were very poor (Pa and Sa, 8-year survival rates: 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 801.