Search

Low-grade adenosquamous carcinoma (LGASC) is a rare type of metaplastic carcinoma of the breast (MBC) with an indolent clinical course. A few cases with high-grade transformation have been reported; however, their genetic alterations remain unclear. This study was designed to explore the somatic genetic characteristics of LGASC with transformation to high-grade MBC. Whole-genome sequencing analysis was performed on five MBCs from four patients, including one case with matching primary LGASC and lymph node metastatic tumor consisting of high-grade MBC with a predominant metaplastic squamous cell carcinoma pattern (MSC) that has progressed from LGASC, and three de novo MSC. Both LGASC and its associated MSC, unlike de novo MSC, had no TP53 mutation and tended to have fewer structural variants than de novo MSC. Both LGASC and its associated MSC had common mutations and copy number alterations, including GNAS R844C, which shows an increase in its allele frequency in MSC. These results indicated that LGASC and its associated MSC were ancestry clonal and that clonal selection occurred during progression. MSC associated with LGASC had additional pathogenic deletions of multiple tumor suppressor genes caused by, for example, SMAD4–DCC fusion. Reverse transcription polymerase chain reaction, followed by Sanger sequencing, confirmed this fusion transcript in both LGASC and its associated MSC; however, chimera proteins were not detected by Western blotting. SMAD4 protein expression had already decreased at the stage of LGASC, which may have contributed to the tumorigenesis of LGASC. In conclusion, not only LGASC but also its associated high-grade MBC may be genetically different from de novo high-grade MBC. The progression from LGASC to high-grade MBC may concern the concentration of the driver mutation caused by clonal selection and inactivation of tumor suppressor genes.

Background: Uterine adenosarcoma is a rare malignant tumor that accounts for 8% of all uterine sarcomas, and less than 0.2% of all uterine malignancies. However, it is frequently misdiagnosed in clinical examinations, including pathological diagnosis, and imaging studies owing to its rare and non-specific nature, which is further compounded by the lack of specific diagnostic markers. Case Presentation: We report a case of uterine adenosarcoma for which a comprehensive genomic profiling (CGP) test provided a chance to reach the proper diagnosis. The patient, a woman in her 60s with a history of uterine leiomyoma was diagnosed with an intra-abdominal mass post presentation with abdominal distention and loss of appetite. She was suspected to have gastrointestinal stromal tumor (GIST), and the laparotomically excised mass was found to comprise uniform spindle-shaped cells that grew in bundles with a herringbone architecture, and occasional myxomatous stroma. Immunostaining revealed no specific findings, resulting in the tumor being diagnosed as a spindle cell tumor/suspicious adult fibrosarcoma. The tumor relapsed during postoperative follow-up, and showed size reduction with chemotherapy, prior to regrowing. CGP was performed to identify a possible treatment, which resulted in detection of a JAZF1-BCORL1 rearrangement. Since the rearrangement has been reported in uterine sarcomas, we reevaluated specimens of the preceding uterine leiomyoma, which revealed the presence of adenosarcoma components in the corpus uteri. Further, both the uterine adenosarcoma and intra-abdominal mass were partially positive for CD10 and BCOR staining. Conclusions: These results conclusively identified the abdominal tumor as a metastasis of the uterine adenosarcoma. The JAZF1-BCORL1 rearrangement is predominantly associated with uterine stromal sarcomas, and ours is the second report of the same in an adenosarcoma thus far. Adenosarcomas are rare and difficult to diagnose, especially in atypical cases with scarce glandular epithelial components. Identification of rearrangements involving BCOR or BCORL1, will encourage BCOR staining analysis, thereby potentially resulting in better diagnostic outcomes. Given that platinum-based chemotherapy was proposed as the treatment choice for this patient post diagnosis with adenosarcoma, CGP also indirectly contributed to designing the best suited treatment protocol.

Mutation spectrum of TP53 in gastric cancer (GC) has been investigated world-widely, but a comparison of mutation spectrum among GCs from various regions in the world are still sparsely documented. In order to identify the difference of TP53 mutation spectrum in GCs in Eastern Europe and in East Asia, we sequenced TP53 in GCs from Eastern Europe, Lujiang (China), and Yokohama, Kanagawa (Japan) and identified the feature of TP53 mutations of GC in these regions.In total, 689 tissue samples of GC were analyzed: 288 samples from East European populations (25 from Hungary, 71 from Poland and 192 from Romania), 268 from Yokohama, Kanagawa, Japan and 133 from Lujiang, Anhui province, China. DNA was extracted from FFPE tissue of Chinese, East European cases; and from frozen tissue of Japanese GCs. PCR products were direct-sequenced by Sanger method, and in ambiguous cases, PCR product was cloned and up to 8 clones were sequenced. We used No. NC_000017.11(hg38) as the reference sequence of TP53. Mutation patterns were categorized into nine groups: six base substitutions, insertion, deletion and deletion-insertion. Within G:C A:T mutations the mutations in CpG and non-CpG sites were divided. The Cancer Genome Atlas data (TCGA, ver.R20, July, 2019) having somatic mutation list of GCs from Whites, Asians, and other ethnicities were used as a reference for our data.The most frequent base substitutions were G:C A:T transition in all the areas investigated. The G:C A:T transition in non-CpG sites were prominent in East European GCs, compared with Asian ones. Mutation pattern from TCGA data revealed the same trend between GCs from White (TCGA category) vs Asian countries. Chinese and Japanese GCs showed higher ratio of G:C A:T transition in CpG sites and A:T G:C mutation was more prevalent in Asian countries.The divergence in mutation spectrum of GC in different areas in the world may reflect various pathogeneses and etiologies of GC, region to region. Diversified mutation spectrum in GC in Eastern Europe may suggest GC in Europe has different carcinogenic pathway of those from Asia.

Endometrial neuroendocrine carcinoma is a rare disease with unknown clinicopathological and molecular characteristics. Therefore, we conducted the present study to elucidate the clinicopathological and molecular characteristics of endometrial neuroendocrine carcinoma, as compared to conventional endometrial carcinoma, and to determine the origin of the former. We analyzed 22 endometrial neuroendocrine carcinomas and 22 conventional endometrial neoplasia cases with respect to clinical, histological and genetic features. Of these, 21/22 neuroendocrine carcinoma cases were admixed carcinomas, with 15 admixed with endometrioid adenocarcinoma. Genetic analysis of hotspot mutations in 50 cancer-related genes revealed that the neuroendocrine carcinoma group carried mutations in PIK3CA (12/22 cases; 54%) and PTEN (8/22 cases; 36%), commonly encountered in endometrioid adenocarcinoma. Comparative statistical analysis of neuroendocrine carcinoma and conventional endometrial neoplasia cases showed a significant trend only in PIK3CA mutation. Moreover, in six mixed-type neuroendocrine carcinoma cases, macrodissection was used to separate the neuroendocrine carcinoma and endometrioid adenocarcinoma components for next-generation sequencing, which revealed several mutations common among the two. These findings suggest that endometrial neuroendocrine carcinoma could originate from conventional endometrial neoplasia, especially endometrioid adenocarcinoma.

Lynch syndrome is a hereditary disease characterized by an increased risk of colorectal and other cancers. Germline variants in the mismatch repair (MMR) genes are responsible for this disease. Previously, we screened the MMR genes in colorectal cancer patients who fulfilled modified Amsterdam II criteria, and multiplex ligation-dependent probe amplification (MPLA) identified 11 structural variants (SVs) of MLH1 and MSH2 in 17 patients. In this study, we have tested the efficacy of long read-sequencing coupled with target enrichment for the determination of SVs and their breakpoints. DNA was captured by array probes designed to hybridize with target regions including four MMR genes and then sequenced using MinION, a nanopore sequencing platform. Approximately, 1000-fold coverage was obtained in the target regions compared with other regions. Application of this system to four test cases among the 17 patients correctly mapped the breakpoints. In addition, we newly found a deletion across an 84 kb region of MSH2 in a case without the pathogenic single nucleotide variants. These data suggest that long read-sequencing combined with hybridization-based enrichment is an efficient method to identify both SVs and their breakpoints. This strategy might replace MLPA for the screening of SVs in hereditary diseases.

Lung cancer is by far the most common cancer-related fatality. In addition to the existing standard treatment methods, it is urgent to develop new therapy methods to improve the survival rate of patients. Amino acid metabolism plays a crucial role in tumor development. Cancer cells rely on amino acids for replication, energy source, redox homeostasis, and release metabolites that recruit and stimulate immunosuppressive cells. This study constructed a novel prognostic signature model of lung adenocarcinoma (LUAD) composed of amino acid metabolism-related genes and assessed immune cell infiltration characteristics and predictive value for patient therapy. We screened differentially expressed genes involved in amino acid metabolic pathways in LUAD tumor tissues versus normal tissues from RNA-seq data of The Cancer Genome Atlas. Then we performed a multivariate cox analysis of 9 genes significantly associated with patients’ overall survival, which established a new prognostic signature. Dividing the patients into high- and low-risk groups using a cutoff value of -0.36, the outcome showed that the risk score of this model could predict survival of LUAD patients with relatively high accuracy (ROC-AUC (max)=0.73). Subsequently, we utilized the ssGSEA method to quantify the infiltration of immune cells. We found that several immune cell subpopulations were significantly altered abundance between the high- and low-risk patients. Immune cell subpopulations, such as mast cell, plasmacytoid dendritic cell, and central memory CD4 T cell, were significantly abundant in the low-risk patients. After ESTIMATE algorithm evaluation, a method for estimating the proportion of stromal and immune cells in tumor tissues based on gene expression profiles, we observed that immune and stroma scores were markedly upregulated in low-risk patients, suggesting that the immune and stromal activity were increased. The key molecules of this signature exhibited a significant correlation with the expression of immune checkpoint molecules. Moreover, multivariate cox regression model analysis was performed with patients’ age, gender, clinical stage, T stage, M stage, N stage and risk score. The results indicated that the risk score signature was a significant and independent prognostic biomarker for the prognostic prevision of LUAD patients.Finally, the risk score based on the amino acid metabolism-related signature was successfully validated by the data of LUAD patients in the GEO dataset GES30219.These findings suggest that the prognostic signature based on the expression of genes involved in amino acid metabolism may be useful and effective. We further characterized one of the 9 genes involved in tryptophan metabolism for its biological implications in tumor progression and construction of immuno-suppressive tumor microenvironment by experimental procedures with human LUAD cell lines. Citation Format: Huihui Xiang, Rika Kasajima, Hiroyuki Ito, Tomoyuki Yokose, Takashi Oshima, Tetsuro Sasada, Yohei Miyagi. Transcriptomic profiling identifies an amino acid metabolism-related prognostic gene signature in lung adenocarcinoma patients [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 2200.

Aim: Mutation spectrum of TP53 in gastric cancer (GC) has been investigated world-widely, but a comparison of mutation spectrum among GCs from various regions in the world are still sparsely documented. In order to identify the difference of TP53 mutation spectrum in GCs in Eastern Europe and in East Asia, we sequenced TP53 in GCs from Eastern Europe, Lujiang (China), and Yokohama, Kanagawa (Japan) and identified the feature of TP53 mutations of GC in these regions. Subjects and Method: In total, 689 tissue samples of GC were analyzed: 288 samples from East European populations (25 from Hungary, 71 from Poland and 192 from Romania), 268 from Yokohama, Kanagawa, Japan and 133 from Lujiang, Anhui province, China. DNA was extracted from FFPE tissue of Chinese, East European cases; and from frozen tissue of Japanese GCs. PCR products were direct-sequenced by Sanger method, and in ambiguous cases, PCR product was cloned and up to 8 clones were sequenced. We used No. NC_000017.11(hg38) as the reference sequence of TP53. Mutation patterns were categorized into nine groups: six base substitutions, insertion, deletion and deletion-insertion. Within G:C>A:T mutations the mutations in CpG and non-CpG sites were divided. Furthermore, 40 cases of Rwanda GC were compared in the world for mutation spectrum. The Cancer Genome Atlas data (TCGA, ver.R20, July, 2019) having somatic mutation list of GCs from Whites, Asians, and other ethnicities were used as a reference for our data. Results: The most frequent base substitutions were G:C>A:T transition in all the areas investigated. The G:C>A:T transition in non-CpG sites were prominent in East European GCs, compared with Asian ones. Mutation pattern from TCGA data revealed the same trend between GCs from White (TCGA category) vs Asian countries. Chinese and Japanese GCs showed higher ratio of G:C>A:T transition in CpG sites and A:T>G:C mutation was more prevalent in Asian countries. Conclusion: The divergence in mutation spectrum of GC in different areas in the world may reflect various pathogeneses and etiologies of GC, region to region. Diversified mutation spectrum in GC in Eastern Europe may suggest GC in Europe has different carcinogenic pathway of those from Asia. Whether the Rwanda pattern belong to Asian or East European would be interesting. Citation Format: Hiroko Natsume, Kinga Szczepaniak, Hidetaka Yamada, Yuji Iwashita, Marta Gędek, Jelena Šuto, Rika Kasajima, Felix Manirakiza, Augustin Nzitakera, Rei Ishikawa, Eric Rutaganda, François Ngabonziza, Benoit Seminega, Placide Kamali, Vincent Dusabejambo, Belson Rugwizangoga, Nobuhito Nobuhito, Yaping Wang, Yohei Miyagi, Simona Gurzu, Haruhiko Sugimura. Non-CpG sites preference in G:C>A:T transition of TP53 in gastric cancer of Eastern Europe (Poland, Romania and Hungary) compared to East Asian countries (China and Japan) : gastric cancer in the world, mutation spectrum revisited [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 2609.

Tyrosine kinase inhibitors are used as first-line treatment for non-small cell lung cancer (NSCLC) patients harboring driver mutations in EGFR, ALK, ROS1, and BRAF. Currently, standard molecular testing approaches help identify single genes for such targetable driver mutations in NSCLC; however, next-generation sequencing (NGS)-based genetic profiling provides a more comprehensive approach and is hence strongly recommended. This case study aimed to highlight the benefits of NGS-based tests for the diagnosis of complex EGFR L858R mutations. A patient was diagnosed with stage IVB NSCLC using a government-approved in vitro diagnostic test and was noted to have a high programmed death-ligand 1 tumor proportion score. This patient was treated with pembrolizumab monotherapy followed by cisplatin and pemetrexed owing to the lack of actionable driver gene mutations, including EGFR mutations. After treatment failure, a sample harvested from the same transbronchial lung biopsy specimen (formalin-fixed and paraffin-embedded) used for the initial EGFR test was subjected to NGS-based broad genetic profiling. The NGS-based test identified an EGFR L858R-K860I cis doublet mutation; however, neither of these mutations was identified upon initial molecular testing. The patient was then successfully treated with a third-generation EGFR-tyrosine kinase inhibitor, osimertinib. In this study, we delved deeper into the realm of L858R and K860I mutations in NSCLC and discuss the potential causes underlying our initial negative diagnosis. Furthermore, this study highlighted the additional benefits of replacing typical molecular tests with NGS-based broad profiling approaches. KEY POINTS: SIGNIFICANT FINDINGS OF THE STUDY: The EGFR L858R-K860I cis doublet mutation was not detected by a PCR-based EGFR test. A next generation sequencing (NGS)-based test was able to identify the L858R-K860I cis doublet mutation. WHAT THIS STUDY ADDS: Osimertinib was effective in an NSCLC patient with EGFR L858R and K860I mutations.

Differentiating small cell neuroendocrine (NE) carcinoma (SCNC) of the prostate from adenocarcinoma with NE differentiation based on morphological features alone sometimes can be challenging. Given that treatment strategies vary depending on histological type, an accurate diagnosis is critical. This study aimed to identify the accurate diagnostic factors for SCNC of the prostate. Furthermore, the possibility of novel treatment strategies through genetic analysis was also investigated. Prostate biopsies conducted in our hospital between January 2017 and May 2020 were included. Consequently, seven cases of SCNC and four cases of adenocarcinoma with NE differentiation were identified. No significant differences in the serum neuron-specific enolase, pro-gastrin-releasing peptide, and prostate-specific antigen (PSA) levels were observed between both tumors. The Ki-67 labeling index was significantly higher, and PSA immunoreactivity tended to be lower in SCNC. Although the morphology was undetectable, genetic analysis confirmed several mutations, including those of PIK3CA and TP53. The fact that morphological findings are not apparent indicates that genetic investigation rather than only morphological findings would be important in the future. In conclusion, given the heterogeneity of serum NE markers in SCNC, diagnosis based on these markers alone is challenging. A high Ki-67 labeling index and low PSA immunoreactivity may be useful for diagnosis, but p53 immunoreactivity is insufficient in distinguishing. Although further studies are required to interpret the results of the genetic analysis involving ALK, PIK3CA, and TP53 mutations, the results of our genetic analysis suggest that PIK3CA mutations in SCNC of the prostate may provide a novel therapeutic strategy.

Patient-derived xenograft models reportedly represent original tumor morphology and gene mutation profiles. In addition, patient-derived xenografts are expected to recapitulate the parental tumor drug responses. In this study, we analyzed the pathways involved in gemcitabine resistance using patient-derived xenograft models of pancreatic cancer. The patient-derived xenograft models were established using samples from patients with pancreatic cancer. The models were treated with gemcitabine to better understand the mechanism of resistance to this anti-cancer drug. We performed comparative gene analysis through the next-generation sequencing of tumor tissues from gemcitabine-treated or non-treated patient-derived xenograft mice and gene set enrichment analysis to analyze mRNA profiling data. Pathway analysis of gemcitabine-treated patient-derived xenografts disclosed the upregulation of multiple gene sets and identified several specific gene pathways that could potentially be related to gemcitabine resistance in pancreatic cancer. Further, we conducted an in vitro analysis to validate these results. The mRNA expression of cytochrome P450 1B1 and cytochrome P450 2A6 was upregulated in a concentration-dependent manner following gemcitabine treatment. Moreover, the sensitivity to gemcitabine increased, and viable cells were decreased by the cytochrome P450 1B1 inhibitor, indicating that the cytochrome P450 1B1 pathway may be related to gemcitabine resistance in pancreatic cancer.

Differentiating small cell neuroendocrine carcinoma (SCNC) of the prostate from prostatic cancer with diffuse neuroendocrine (NE) differentiation based on morphological features alone can be challenging. Given that treatment strategies vary depending on histological type, accurate diagnosis is critical. This study firstly aimed to identify the accurate diagnostic factors for primary prostate NE tumors. Furthermore, the possibility of novel treatment strategies through genetic analysis is also an aim. Specifically, prostate biopsies conducted in our hospital between January 2017 and May 2020 were included. As a result seven cases of SCNC and four cases of prostatic cancer with diffuse NE differentiation were identified. No significant differences in serum neuron-specific enolase (NSE), pro-gastrin-releasing peptide (ProGRP), and prostate-specific antigen (PSA) levels were observed between SCNC and adenocarcinoma with diffuse NE differentiation. The Ki-67 labeling index was significantly higher and PSA immunoreactivity tended to be lower in SCNC. Genetic analysis did not detect any ALK mutations but confirmed several other mutations, including those of PIK3CA and TP53. In conclusion, given the heterogeneity in serum NE markers in SCNC, diagnosis based on these markers alone is difficult. A high Ki-67 labeling index and low PSA immunoreactivity may be useful for diagnosing, but p53 immunoreactivity is insufficient to distinguish these tumors. Although further studies are required to interpret the results of the genetic analysis involving ALK, PIK3CA, and TP53 mutations, the results of our genetic analysis suggest that PIK3CA mutations in SCNC of the prostate may provide a novel therapeutic strategy.

This study was performed to assess the utility of tumor-derived fragmentary DNA, or circulating tumor DNA (ctDNA), for identifying high-risk patients for relapse of acute myeloid leukemia and myelodysplastic syndrome (AML/MDS) after undergoing myeloablative allogeneic hematopoietic stem cell transplantation (alloSCT). We retrospectively collected tumor and available matched serum samples at diagnosis and 1 and 3 months post-alloSCT from 53 patients with AML/MDS. After identifying driver mutations in 51 patients using next-generation sequencing, we designed at least 1 personalized digital polymerase chain reaction assay per case. Diagnostic ctDNA and matched tumor DNA exhibited excellent correlations with variant allele frequencies. Sixteen patients relapsed after a median of 7 months post-alloSCT. Both mutation persistence (MP) in bone marrow (BM) at 1 and 3 months post-alloSCT and corresponding ctDNA persistence (CP) in the matched serum (MP1 and MP3; CP1 and CP3, respectively) were comparably associated with higher 3-year cumulative incidence of relapse (CIR) rates (MP1 vs non-MP1, 72.9% vs 13.8% [P = .0012]; CP1 vs non-CP1, 65.6% vs 9.0% [P = .0002]; MP3 vs non-MP3, 80% vs 11.6% [P = .0002]; CP3 vs non-CP3, 71.4% vs 8.4% [P < .0001]). We subsequently evaluated whether subset analysis of patients with 3 genes associated with clonal hematopoiesis, DNMT3A, TET2, and ASXL1 (DTA), could also be helpful in relapse prediction. As a result, CP based on DTA gene mutations also had the prognostic effect on CIR. These results, for the first time, support the utility of ctDNA as a noninvasive prognostic biomarker in patients with AML/MDS undergoing alloSCT.

Background Interstitial pneumonia (IP) is a poor prognostic comorbidity in patients with non-small cell lung cancer (NSCLC) and is also a risk factor for pneumonitis. The TORG1936/AMBITIOUS trial, the first known phase II study of atezolizumab in patients with NSCLC with comorbid IP, was terminated early because of the high incidence of severe pneumonitis. Methods This study included patients with idiopathic chronic fibrotic IP, with a predicted forced vital capacity (%FVC) of >70%, with or without honeycomb lung, who had previously been treated for NSCLC. The patients received atezolizumab every 3 weeks. The primary endpoint was the 1-year survival rate. Results A total of 17 patients were registered; the median %FVC was 85.4%, and 41.2% had honeycomb lungs. The 1-year survival rate was 53.3% (95% CI, 25.9-74.6). The median overall and progression-free survival times were 15.3 months (95% CI, 3.1-not reached) and 3.2 months (95% CI, 1.2-7.4), respectively. The incidence of pneumonitis was 29.4% for all grades, and 23.5% for grade ≥3. Tumor mutational burden and any of the detected somatic mutations were not associated with efficacy or risk of pneumonitis. Conclusion Atezolizumab may be one of the treatment options for patients with NSCLC with comorbid IP, despite the high risk of developing pneumonitis. This clinical trial was retrospectively registered in the Japan Registry of Clinical Trials on August 26, 2019, (registry number: jRCTs031190084, https://jrct.niph.go.jp/en-latest-detail/jRCTs031190084).

Background Fetal adenocarcinoma of the lung is a rare variant of lung adenocarcinoma and is subcategorized into low-grade and high-grade (H-FLAC) fetal adenocarcinoma. We previously reported poor prognosis in pulmonary adenocarcinomas with an H-FLAC component; however, the genetic abnormalities involved in H-FLAC remain unclear. Therefore, this study aimed to elucidate molecular abnormalities as potential therapeutic targets for H-FLACs. Methods We performed immunohistochemical analysis and comprehensive genetic analyses using whole-exome sequencing in 16 lung cancer samples with an H-FLAC component. DNA was extracted from formalin-fixed paraffin-embedded tissues after macrodissection of the H-FLAC component. Results Cancer-related mutations were identified in TP53 (7/16 cases), KMT2C (6/16 cases), KRAS (4/16 cases), NF1 (3/16 cases), STK11 (3/16 cases), CTNNB1 (2/16 cases), and EGFR (1/16 cases). A high tumor mutation burden of ≥10 mutations per megabase was observed in 3/16 cases. A high microsatellite instability was not detected in any case. Based on the cosine similarity with the Catalogue of Somatic Mutations in Cancer mutational signatures, H-FLACs were hierarchically clustered into three types: common adenocarcinoma-like (five cases), surfactant-deficient (ten cases), and signatures 2 and 13-related (one case). All common adenocarcinoma-like cases presented thyroid transcription factor-1 (TTF-1) expression, whereas surfactant-deficient cases often presented loss of TTF-1 and surfactant protein expression and included cases with mutations in the surfactant system genes NKX2-1 and SFTPC. H-FLACs displayed low programmed death ligand-1 (PD-L1) expression (1-49% of tumor cells) in 5/16 cases, and no case displayed high PD-L1 expression (≥50% of tumor cells). Conclusions This study indicates that lung cancers with an H-FLAC component rarely harbor currently targetable driver gene mutations for lung cancer but display a high frequency of KMT2C mutations. The microsatellite instability, tumor mutation burden, and PD-L1 expression status suggest a poor response to immune checkpoint therapy.

Lynch syndrome is a hereditary disease characterized by an increased risk of colorectal and other cancers. Germline variants in the mismatch repair (MMR) genes are responsible for this disease. Previously, we screened the MMR genes in colorectal cancer patients who fulfilled modified Amsterdam II criteria, and multiplex ligation-dependent probe amplification (MPLA) identified 11 structural variants (SVs) of MLH1 and MSH2 in 17 patients. In this study, we have tested the efficacy of long read-sequencing coupled with target enrichment for the determination of SVs and their breakpoints. DNA was captured by array probes designed to hybridize with target regions including four MMR genes and then sequenced using MinION, a nanopore sequencing platform. Approximately, 1000-fold coverage was obtained in the target regions compared with other regions. Application of this system to four test cases among the 17 patients correctly mapped the breakpoints. In addition, we newly found a deletion across an 84 kb region of MSH2 in a case without the pathogenic single nucleotide variants. These data suggest that long read-sequencing combined with hybridization-based enrichment is an efficient method to identify both SVs and their breakpoints. This strategy might replace MLPA for the screening of SVs in hereditary diseases.

In personalized cancer genomic medicine, characterizing a patient’s molecular profile based on comprehensive information is important for maximizing treatment benefits. However, current cancer genome analysis is centered on single nucleotide variation (SNV), gene expression, and copy number variation (CNV) but places little emphasis on structural variations (SV) beside fusions. To date, investigation of SVs has been limited because SV analysis entails a cumbersome annotation process. This study describes the design, development, and implementation of an annotation tool for SV, termed SVAnnotator. Detailed annotation was performed on the results of SV detection of 2,781 whole genome samples from the ICGC/TCGA PanCancer Analysis of Whole Genomes (PCAWG) with identifications of fusion, exon skipping, gene disruption, and tandem duplication SVs. These annotations of SVs will facilitate understanding of molecular events and further enhance utilities of precision medicine in stratifications, pathogenicity assessments and drug responses. Frequent novel SV events in MACROD2, FHIT, WWOX and CCSER1 were observed across many cancers. Importantly, SV events were frequently identified in well-established tumor suppressor genes including RB1, NF1, PTEN and TP53. As such, it is plausible that potential therapeutic opportunities are overlooked when SV analysis is not appropriately performed. Given the frequency of SVs detected in our study, SVanalysis with detailed annotation should be a routine part of comprehensive precision medicine analysis, and further studies are warranted to enhance clinical benefits as well as our understanding of uncharacterized SV events.

BackgroundAmino acid metabolism is essential for tumor cell proliferation and regulation of immune cell function. However, the clinical significance of free amino acids (plasma-free amino acids (PFAAs)) and tryptophan-related metabolites in plasma has not been fully understood in patients with non-small cell lung cancer (NSCLC) who receive immune checkpoint inhibitors.MethodsWe conducted a single cohort observational study. Peripheral blood samples were collected from 53 patients with NSCLC before treatment with PD-1 (Programmed cell death-1) inhibitors. The plasma concentrations of 21 PFAAs, 14 metabolites, and neopterin were measured by liquid chromatography–mass spectrometry. Using Cox hazard analysis with these variables, a multivariate model was established to stratify patient overall survival (OS). Gene expression in peripheral blood mononuclear cells (PBMCs) was compared between the high-risk and low-risk patients by this multivariate model.ResultsOn Cox proportional hazard analysis, higher concentrations of seven PFAAs (glycine, histidine, threonine, alanine, citrulline, arginine, and tryptophan) as well as lower concentrations of three metabolites (3h-kynurenine, anthranilic acid, and quinolinic acid) and neopterin in plasma were significantly correlated with better OS (pConclusionsThe multivariate model with PFAAs and metabolites in plasma might be useful for stratifying patients who will benefit from PD-1 inhibitors.

Background: Approximately 10% of patients with non-small cell lung cancer (NSCLC) are complicated with comorbid interstitial pneumonia (IP) with a poor prognosis. The pharmacotherapy for advanced lung cancer occasionally induces fatal acute exacerbation of pre-existing IP. Due to the lack of prospective studies, there is an urgent need to establish a safe and effective pharmacotherapy, especially for second-line or later settings. Atezolizumab, an anti-programmed cell death-ligand 1 antibody, is thought to be the safest candidate for second-line therapy among various immune checkpoint inhibitors. Moreover, compared with patients without IP, the patients with comorbid IP may have higher tumor mutation burden (TMB) or microsatellite instability (MSI), which are partly associated with a more favorable response to immune checkpoint inhibitors. Methods: The Thoracic Oncology Research Group 1936/AMBITIOUS study is an ongoing, multicenter, single-arm, phase II trial to assess the safety and efficacy of atezolizumab for pretreated advanced/recurrent patients with NSCLC complicated with idiopathic, chronic fibrotic IP with a forced vital capacity of >70%. The patients will receive atezolizumab (1200 mg, day 1) every 3 weeks until the discontinuation criteria are met. The primary end point of this study is the 1-year survival rate, and a sample size of 38 patients is set. As a translational research, we will perform the analysis of TMB, somatic mutations, and MSI for nucleic acids extracted from archival tumor samples. Discussion: Since there is no standard second-line or later therapy of advanced NSCLC with IP, the results of this study are expected to have a major impact on clinical practice. Trial registration: Japan Registry of Clinical Trials, jRCTs031190084, registered 26 August 2019 - retrospectively registered, https://jrct.niph.go.jp/en-latest-detail/jRCTs031190084

A growing body of evidence suggests that tumor-derived fragmentary DNA, known as circulating tumor DNA (ctDNA), has the potential to serve as a non-invasive biomarker for disease monitoring. However, in the setting of hematological malignancy, few published studies support the utility of ctDNA. We retrospectively investigated ctDNA levels of 17 patients with various hematological malignancies who had achieved remission after first-line therapy. We identified somatic driver mutations by next-generation sequencing, and designed droplet digital PCR assays for each mutation to measure ctDNA. Variant allele frequencies of ctDNA changed in association with clinical response in all patients. Eight patients clinically relapsed after a median of 297 days post-first-line therapy (termed, "relapsed group"); the remaining nine patients remained disease-free for a median of 332 days (termed, "remission group"). Among patients in the relapsed group, ctDNA levels increased more than twofold at paired serial time points. In marked contrast, ctDNA levels of all patients in the remission group remained undetectable or stable during clinical remission. Notably, ctDNA-based molecular relapse demonstrated a median 30-day lead time over clinical relapse. In summary, ctDNA monitoring may help identify hematologic cancer patients at risk for relapse in advance of established clinical parameters.

Genome research using appropriately collected pathological tissue samples is expected to yield breakthroughs in the development of biomarkers and identification of therapeutic targets for diseases such as cancers. In this connection, the Japanese Society of Pathology (JSP) has developed "The JSP Guidelines on the Handling of Pathological Tissue Samples for Genomic Research" based on an abundance of data from empirical analyses of tissue samples collected and stored under various conditions. Tissue samples should be collected from appropriate sites within surgically resected specimens, without disturbing the features on which pathological diagnosis is based, while avoiding bleeding or necrotic foci. They should be collected as soon as possible after resection: at the latest within about 3 h of storage at 4°C. Preferably, snap-frozen samples should be stored in liquid nitrogen (about -180°C) until use. When intending to use genomic DNA extracted from formalin-fixed paraffin-embedded tissue, 10% neutral buffered formalin should be used. Insufficient fixation and overfixation must both be avoided. We hope that pathologists, clinicians, clinical laboratory technicians and biobank operators will come to master the handling of pathological tissue samples based on the standard operating procedures in these Guidelines to yield results that will assist in the realization of genomic medicine.

The pancreatic cancer microenvironment is crucial in cancer development, progression and drug resistance. Cancer-stromal interactions have been recognized as important targets for cancer therapy. However, identifying relevant and druggable cancer-stromal interactions is challenging due to the lack of quantitative methods to analyze the whole cancer-stromal interactome. Here we studied 14 resected pancreatic cancer specimens (8 pancreatic adenocarcinoma (PDAC) patients as a cancer group and 6 intraductal papillary-mucinous adenoma (IPMA) patients as a control). Shotgun proteomics of the stromal lesion dissected with laser captured microdissection (LCM) was performed, and identified 102 differentially expressed proteins in pancreatic cancer stroma. Next, we obtained gene expression data in human pancreatic cancer and normal pancreatic tissue from The Cancer Genome Atlas database (n = 169) and The Genotype-Tissue Expression database (n = 197), and identified 1435 genes, which were differentially expressed in pancreatic cancer cells. To identify relevant and druggable cancer-stromal-interaction targets, we applied these datasets to our in-house ligand-receptor database. Finally, we identified 9 key genes and 8 key cancer-stromal-interaction targets for PDAC patients. Furthermore, we examined FN1 and ITGA3 protein expression in pancreatic cancer tissues using tissue microarrays (TMAs) of 271 PDAC cases, and demonstrated that FN1-ITGA3 had unfavorable prognostic impact for PDAC patients.

There are regional and/or ethnic differences in tumorigenic pathways among several types of cancer, including prostate cancer (PCa). However, information on genome‑wide gene alterations and the transcriptome is currently only available for PCa patients from Western countries. In order to profile the genetic alterations in Japanese patients with PCa, new panels were created to examine nucleotide sequence variations in 71 selected PCa‑related genes (KCC71) and to detect all fusion RNA transcripts known in PCa (PCaFusion). An analysis of 21 Japanese PCa cases identified 33 different somatic variants in 24 genes in the KCC71 panel, including 2 in SPOP (F102V and F133L), 2 in BRCA2 (I1859fs and R2318ter, resulting in premature termination of the polypeptide), and 1 each in BRAF (K601E), CDH1 (E880K) and RB1 (R621S), as pathogenic alterations. Unexpectedly, the TMPRSS2‑ERG fusion transcript was detected in only 1 case, although the SLC45A3‑ELK4 and USP9Y‑TTTY15 fusion transcripts, known as transcription‑mediated chimeric RNAs, were detected in all examined cases. A new pathway analysis with The Cancer Network Galaxy (TCNG), a cancer gene regulatory network database, was also applied in an attempt to predict molecular pathways implicated in PCa in the Japanese population. Based on the 24 genes having somatic variants identified by the panel analysis as initial seed genes, a putative core network was finally established, including 5 identified genes, namely TNK2, SOX9, CDH1, FOXA1 and TP53, with high commonality from TCNG datasets. These genes are expected to be involved in tumor development, as revealed by the results of an enrichment analysis with Gene Ontology terms. This analysis must be further extended to include more cases in order to verify this method and also to elucidate the characteristics of PCa in Japanese patients.

A 61-year-old Japanese woman was diagnosed with FIGO Stage IB endometrioid cancer (EC) combined with large cell neuroendocrine carcinoma (LCNEC). Metastasis to the lymph nodes in the right bronchopulmonary area, mediastinum and brain were also identified. The patient eventually developed pleuritis and pericarditis carcinomatosa, and died of cancer at 51 months after surgery. Because gene aberrations in uterine neuroendocrine carcinoma are still not well understood, we examined alterations in the mutational hotspots of 50 selected cancer-associated genes. The EC and LCNEC components shared identical alterations in PTEN, PIK3CA and FGFR3. Both the EC and LCNEC components had heterozygous SBSs on CTNNB1 but at different codons (G34R in EC, and T41A in LCNEC). The altered gene signature raised a possibility that the EC and LCNEC components were derived from a common precursor lesion. The LCNEC independently obtained a significant CTNNB1 mutation and the lymph node metastasis originated from this component. Because the LCNEC component seemed to bring about the aggressive course of the disease and defined the patient outcome, further investigations are needed to elucidate the mechanism of NE carcinoma development in the endometrium. ELECTRONIC SUPPLEMENTARY MATERIAL: The online version of this article (doi:10.1007/s13691-016-0263-9) contains supplementary material, which is available to authorized users.

Acute myeloid leukemia (AML) is a clonal myeloid neoplasm that typically arises de novo; however, some cases evolve from a preleukemic state, such as myelodysplastic syndrome (MDS). Such secondary AMLs and those with typical MDS-related clinical features are known as AMLs with myelodysplasia-related changes (AML-MRC). Because patients with AML-MRC have poor prognosis, more accurate diagnostic approaches are required. In this study, we performed targeted sequencing of 54 genes in 3 cell populations (granulocyte, blast, and T-cell fractions) using samples from 13 patients with MDS, 16 patients with clinically diagnosed AML-MRC, 4 patients with suspected AML-MRC but clinically diagnosed as AML not otherwise specified (AML-NOS), and 11 patients with de novo AML. We found that overlapping mutations, defined as those shared at least by the blast and granulocyte fractions, were significantly enriched in patients with MDS and AML-MRC, including those with suspected AML-MRC, indicating a substantial history of clonal hematopoiesis. In contrast, blast-specific nonoverlapping mutations were significantly enriched in patients with de novo AML. Furthermore, the presence of overlapping mutations, excluding DNMT3A, TET2, and ASXL1, effectively segregated patients with MDS and AML-MRC or suspected AML-MRC from patients with de novo AML. Additionally, the presence of ≥3 mutations in the blast fraction was useful for distinguishing patients with AML-MRC from those with MDS. In conclusion, our approach is useful for classifying clinically diagnosable AML-MRC and identifying clinically diagnosed AML-NOS as latent AML-MRC. Additional prospective studies are needed to confirm the utility of this approach.

Background Interstitial pneumonia (IP) is one of the most common and poor prognostic comorbidities in patients with non-small cell lung cancer (NSCLC) and is also a known risk factor for pneumonitis. Approximately 10% of patients have concomitant IP diagnosed at the time of cancer diagnosis. IP is associated with smoking, microsatellite instability (MSI) and higher tumour mutation burden (TMB). Atezolizumab monotherapy is an established treatment for recurrent NSCLC. PD-L1 inhibitors are reported to have a lower risk of pneumonitis than PD-1 inhibitors. This study aims to assess the safety and efficacy of atezolizumab monotherapy for pretreated advanced or recurrent NSCLC patients with idiopathic IP. Trial design The Thoracic Oncology Research Group (TORG) 1936/AMBITIOUS study is a multicentre, single-arm, phase II trial. The key inclusion criteria are (1) histologically or cytologically proven NSCLC, (2) unresectable stage III/IV or recurrent disease, (3) prior chemotherapy, including platinum doublet chemotherapy, (4) chronic, fibrotic and idiopathic IP with a predicted vital capacity ≥ 70%, (5) age ≥ 20 years and (6) ECOG performance status 0, 1. The enrolled patients will receive atezolizumab (1200 mg) every 3 weeks until the discontinuation criteria are satisfied. The primary end point is the 1-year survival rate. We set an expected value of 40% and a threshold value of 15%. Taking statistical points (two-sided α = 0.05; 1 − β = 0.9) and ineligible patients into account, the sample size was set at 38 based on the exact binomial test. The key secondary end points are the incidence of acute exacerbation of IP within 1 year after treatment initiation, overall survival, progression-free survival, objective response rate and safety. As a translational research, we will perform analysis of TMB, somatic mutations and MSI from tumour samples. Legal entity responsible for the study Thoracic Oncology Research Group (TORG). Funding Chugai Pharmaceutical Co., Ltd. Disclosure S. Ikeda: Honoraria (self), Research grant / Funding (institution): Chugai Pharmaceutical; Honoraria (self): Boehringer Ingelheim; Honoraria (self): AstraZeneca; Honoraria (self): Taiho Pharmaceutical; Honoraria (self): Ono Pharmaceutical. T. Kato: Honoraria (self), Research grant / Funding (institution): AbbVie; Research grant / Funding (institution): Astellas; Honoraria (self), Research grant / Funding (institution): AstraZeneca; Honoraria (self): Boehringer Ingelheim; Honoraria (self), Research grant / Funding (institution): Bristol-Myers Squibb; Honoraria (self), Research grant / Funding (self): Chugai Pharmaceutical; Honoraria (self), Research grant / Funding (self): Eli Lilly; Research grant / Funding (institution): Kyorin Pharmaceutical; Honoraria (self), Research grant / Funding (institution): Kyowa Hakko Kirin; Honoraria (self), Research grant / Funding (institution): Merck Serono; Honoraria (self), Research grant / Funding (institution): Merck Sharp and Dohme; Honoraria (self): Nitto Denko; Honoraria (self), Research grant / Funding (institution): Novartis; Honoraria (self), Research grant / Funding (institution): Ono Pharmaceutical; Honoraria (self), Research grant / Funding (institution): Pfizer; Honoraria (self): Quintiles; Research grant / Funding (institution): Regeneron; Honoraria (self): Sumitomo Dainippon Pharma; Honoraria (self), Research grant / Funding (institution): Taiho Pharmaceutical; Honoraria (self): Takeda Pharmaceutical; Honoraria (self): F. Hoffmann-La Roche. H. Kenmotsu: Honoraria (self), Research grant / Funding (institution): Chugai Pharmaceutical; Honoraria (self): Ono Pharmaceutical; Honoraria (self), Research grant / Funding (institution): Boehringer Ingelheim; Honoraria (self): Eli Lilly ; Honoraria (self): Kyowa Hakko Kirin; Honoraria (self): Bristol-Myers Squibb; Honoraria (self): Merck Sharp and Dohme; Honoraria (self): Novartis Pharma; Honoraria (self): Daiichi-Sankyo; Honoraria (self), Research grant / Funding (institution): AstraZeneca. T. Ogura: Honoraria (self), Research grant / Funding (institution): Boehringer Ingelheim; Honoraria (self): Meiji Seika Pharma; Honoraria (self): Shionogi Pharmaceutical; Honoraria (self): Toray; Honoraria (self): AstraZeneca; Honoraria (self): Eisai; Honoraria (self): Astellas Pharma; Honoraria (self): Kyorin Pharmaceutical; Honoraria (self): Ono Pharmaceutical; Honoraria (self): Actelion Pharmaceuticals; Honoraria (self): Novartis. S. Iwasawa: Honoraria (self), Research grant / Funding (institution): Chugai Pharmaceutical; Honoraria (self), Research grant / Funding (institution): Ono Pharmaceutical; Honoraria (self): AstraZeneca. T. Iwasawa: Honoraria (self): Ono pharmaceutical; Honoraria (self): Boehringer Ingelheim; Honoraria (self): Shionogi. T. Yamanaka: Honoraria (self), Research grant / Funding (institution): Takeda Pharmaceutical; Honoraria (self), Research grant / Funding (institution): Chugai Pharmaceutical; Honoraria (self), Research grant / Funding (institution): Boehringer Ingelheim; Honoraria (self), Research grant / Funding (institution): Taiho Pharmaceutical; Honoraria (self), Research grant / Funding (institution): Daiichi-Sankyo; Research grant / Funding (institution): Ono Pharmaceutical; Honoraria (self), Research grant / Funding (institution): Bayer; Research grant / Funding (institution): Merck Serono; Research grant / Funding (institution): Astellas; Research grant / Funding (institution): Eli Lilly; Honoraria (self): Pfizer; Honoraria (self): Sysmex; Honoraria (self): Huya Biosciences; Honoraria (self): Gilead Sciences. H. Okamoto: Research grant / Funding (institution): Chugai Pharmaceutical; Research grant / Funding (institution): Takeda Pharmaceutical; Research grant / Funding (institution): AstraZeneca; Research grant / Funding (institution): Merck Sharp and Dohme; Research grant / Funding (institution): Ono Pharmaceutical; Research grant / Funding (institution): Taiho pharmaceutical; Research grant / Funding (institution): Bristol-Myers Squibb; Research grant / Funding (institution): Eli Lilly; Research grant / Funding (institution): Daiichi Sankyo. All other authors have declared no conflicts of interest.

Expression of the protein deacetylase SIRT1 is associated with either poor or favorable prognosis in cancer patients, depending on the cancer type. In head and neck squamous cell carcinoma (HNSCC), SIRT1 expression is associated with favorable prognosis. However, the molecular mechanism underlying the tumor-suppressive function of SIRT1 in HNSCC is unknown. SIRT1 promotes differentiation in epithelial cells; therefore, we investigated whether SIRT1 promotes differentiation in HNSCC cells by studying the correlations between the expression of SIRT1 and several genes implicated in stemness or differentiation in HNSCC-derived cell lines. Our results suggest that SIRT1 does not contribute to differentiation in HNSCC cells. RNA interference-mediated reduction of SIRT1 revealed that SIRT1 supports the expression of TAp63, which has been implicated in tumor suppression, in addition to epithelial differentiation. A positive correlation was observed between SIRT1 and TAp63 expression in HNSCC tissues, as determined by quantitative reverse transcription-polymerase chain reaction analysis of RNA extracted from formalin-fixed paraffin-embedded biopsy samples. Together, these results suggest that although SIRT1 does not regulate differentiation of HNSCC cells, it functions as a tumor suppressor in HNSCC by supporting the transcription of tumor-suppressive TAp63. This finding supports the notion that SIRT1-activating drugs could be useful for the treatment of HNSCC.

Background The worldwide incidence of neuroendocrine tumors (NETs) has increased remarkably, with the hindgut being the second most common site for such tumors. However, the mechanisms underlying progression and metastasis of hindgut NETs are unclear. A retrospective study was conducted to elucidate these mechanisms. Methods Clinicopathological data of cases of hindgut NET between April 1996 and September 2015 were analyzed, retrospectively. Patients with neuroendocrine carcinoma were excluded. Formalin-fixed paraffin-embedded tissues of hindgut NET cases were subjected to detailed morphometric and immunohistochemical analyses. Statistical analyses were performed using the non-parametric Mann-Whitney U test, Spearman’s correlation coefficient, and chi-squared test. Multivariate logistic regression analysis was conducted as appropriate for the data set. Results Fifty-six hindgut NET cases were considered. Microvessel density and lymphatic microvessel density were identified as significant risk factors for venous and lymphatic invasion. There was a positive correlation between microvessel density and the maximum tumor diameter. Multivariate logistic regression analysis revealed that the maximum tumor diameter alone was an independent predictor of lymph node metastasis, whereas lymphovascular invasion and MVD was not the predictor of lymph node metastasis. There were no significant correlations between the Ki-67 labeling index and any of the parameters evaluated including age, sex, the maximum tumor diameter, venous invasion, lymphatic invasion, microvessel density, lymphatic microvessel density, and lymph node metastasis. Conclusions Angiogenic mechanisms may play important roles in the progression of hindgut NET. Otherwise, the maximum tumor diameter alone was an independent predictor of lymph node metastasis in hindgut NETs. Moreover, our study raises the question of whether the presence of lymphovascular invasion, in endoscopically obtained hindgut NET tissues, is an absolute indication for additional surgery or not.

Genome research using appropriately collected pathological tissue samples is expected to yield breakthroughs in the development of biomarkers and identification of therapeutic targets for diseases such as cancers. In this connection, the Japanese Society of Pathology (JSP) has developed "The JSP Guidelines on the Handling of Pathological Tissue Samples for Genomic Research" based on an abundance of data from empirical analyses of tissue samples collected and stored under various conditions. Tissue samples should be collected from appropriate sites within surgically resected specimens, without disturbing the features on which pathological diagnosis is based, while avoiding bleeding or necrotic foci. They should be collected as soon as possible after resection: at the latest within about 3 h of storage at 4°C. Preferably, snap-frozen samples should be stored in liquid nitrogen (about -180°C) until use. When intending to use genomic DNA extracted from formalin-fixed paraffin-embedded tissue, 10% neutral buffered formalin should be used. Insufficient fixation and overfixation must both be avoided. We hope that pathologists, clinicians, clinical laboratory technicians and biobank operators will come to master the handling of pathological tissue samples based on the standard operating procedures in these Guidelines to yield results that will assist in the realization of genomic medicine.

Background As the World Health Organization grading system for gastroenteropancreatic-neuroendocrine tumors (GEP-NETs) may not always correlate with tumor progression, it is imperative that other independent predictors of tumor progression be established. To identify such predictors, we conducted a retrospective histopathological study of hindgut NETs, obtained from endoscopic procedures, and used statistical analyses to evaluate predictive factors. Methods We first obtained clinicopathological data of cases of hindgut NETs. Tissue sections from tumor samples were prepared and subjected to pathological examination. In particular, we calculated the microvessel density (MVD) and lymphatic microvessel density (LMVD) values, and performed appropriate statistical analyses. Results A total of 42 cases of hindgut NETs were selected for the study, 41 from the rectum and 1 from the sigmoid colon. Based on the Ki-67 labeling index, 34 cases were classified as NET G1 tumors and 8 as NET G2 tumors. MVD values ranged from 1.4/mm2 to 73.9/mm2 and LMVD values from 0/mm2 to 22.9/mm2. MVD and LMVD were identified as risk factors for venous and lymphatic invasion of hindgut NETs. Moreover, MVD positively correlated with the maximum diameter of the tumor. Conclusions Tumor progression of NETs may cause angiogenesis and lymphangiogenesis, via an unknown mechanism, as well as lymphovascular invasion. Angiogenesis likely plays an important role in occurrence and progression in the initial phase of hindgut NETs.

We evaluated multiple oncogenic mutations and fusion genes in small specimen obtained by bronchoscopy. Eight patients with lung cancer were recruited, 3 small cell lung cancer, 3 non-small cell lung cancer, 1 adenocarcinoma and 1 squamous cell carcinoma. A median value of extracted RNA and DNA amounts from specimen was 1573 ng (range 367.5 to 8900) and 6700 ng (range 550 to 68000 ng), respectively. We applied amplicon sequencing panels that cover exon regions of 41 genes related to lung tumorigenesis as well as total 61 major variants of ALK, ROS, RET or NTRK1 fusion transcripts. Nineteen of 41 gene mutations were detected in our isolated DNAs of 8 patients. We could detect four to eleven mutations in each specimen; however the mutation combination in each 8 patients were different. The most common genetic alterations were TP53, KMT2D, MET, NOTCH2 and SETD2, which were detected in 4 to 6 patients. We did not detect fusion transcripts of ALK, ROS, RET and NTRK1 in every specimen. In conclusion, multiplex genomic test was performed on small amounts specimen of bronchoscopy biopsy with a 100% success rate. Such testing is considered to be able to assist physicians in matching patients with approved or experimental targeted treatments.

Background: Allogeneic hematopoietic stem cell transplantation (alloSCT) is the only curative option for patients with high risk acute myeloid leukemia (AML) and myelodysplastic syndrome (MDS). Whereas, relapse is the main event in therapeutic failure for these patients. We previously reported the utility of residual circulating tumor DNA (ctDNA) status for identifying patients with AML and MDS at high risk for relapse post myeloablative alloSCT (Nakamura et al, ASH. 2017). However, it remains to be elucidated whether persistent mutation status in serum and bone marrow (BM) have comparable ability to identify patients at high risk for relapse. Additionally, recent reports indicated mutation persistence (MP) in BM based on three genes regarding clonal hematopoiesis, DNMT3A, TET2, and ASXL1 (DTA), was not informative for relapse prediction of patients with AML in the setting of chemotherapy (Jongen-Lavrencic et al, N Engl J Med. 2018). Therefore, the prognostic impact of residual ctDNA status based on DTA genes should also be tested. Methods: To address these questions, we retrospectively collected tumor and matched serum samples at diagnosis and 1 and 3 months post-alloSCT from 53 patients with AML and MDS. Cell-free DNA was extracted from serum samples. We subjected tumor DNA, extracted from BM or peripheral blood, and buccal swab DNA, to next-generation sequencing (NGS), identifying candidate driver mutations. After identifying driver mutations, we designed droplet digital PCR (ddPCR) assay. The primary endpoint was the cumulative incidence of relapse (CIR) rate, and the secondary endpoint was the overall survival (OS) rate. We used DeLong's test to compare the performance between two assays based on the area under the curve (AUC) of receiver operating characteristics (ROC) curves. Results: Driver mutations were identified in 51 of 53 patients by NGS, and our cohort consisted of 37 patients with AML and 14 patients with MDS. The median age of the patients was 53 years. The conventional cytogenetic risk category was an adverse or high risk in 39.2% of patients, and 49.0% were in relapse or refractory disease status at alloSCT, and all patients received myeloablative conditioning; in most cases, the stem cell source was cord blood. The most frequent mutations found involved epigenetic regulators (DNMT3A/TET2/ASXL1, mutated in 32.1%), followed by signal transduction proteins (NRAS/FLT3, 31.4%). We could design at least one representative ddPCR assay for 51 patients. There was a clear correlation of variant allele frequency measurement between diagnostic ctDNA and matched tumor DNA (r2 = 0.67; P < 0.0001). Sixteen patients relapsed after a median of 7 months post-alloSCT. Both MP in BM at 1 and 3 months post-alloSCT and corresponding ctDNA persistence (CP) in serum (MP1 and MP3; CP1 and CP3, respectively) were comparably associated with higher 3-year CIR rates and inferior OS rates [3-year CIR (3-year OS): MP1 vs. non-MP1: 72.9 (50.0)% vs. 13.8 (88.0)%; P = 0.0012 (.0304) (Figure 1A); CP1 vs. non-CP1: 65.6 (45.8)% vs. 9.0 (91.7)%; P = 0.0002 (.0014) (Figure 1B); MP3 vs. non-MP3; 80.0 (30.0)% vs. 11.6 (94.1)%; P = 0.0002 (.0007); CP3 vs. non-CP3: 71.4 (53.4)% vs. 8.4 (92.5)%; P < 0.0001 (.0021)]. We next tested whether CP based on DTA could also be helpful in relapse prediction, we performed a subset analysis of patients with DTA based ddPCR assays (n=12). As a result, CP based on DTA genes also had the prognostic impact on CIR (Figure 1C). Finally, we compared the discriminatory ability of CP with those of MP. There was no significant difference between either CP and MP (Figure 1D). Additionally, when CP1 was compared with CP3, CP3 was found to be a better indicator of CIR and OS. Conclusions: In summary, we, for the first time, demonstrated that non-invasive serum ctDNA-testing, regardless of DTA genes, had comparable utility to molecular MRD testing of BM with regard to identifying patients at high risk for relapse in AML and MDS undergoing myeloablative alloSCT. Although prospective large-scale analyses are needed to confirm our findings, such non-invasive ctDNA-testing might allow for rapid clinical decision-making and, ultimately, subsequent risk-adapted therapeutic interventions post-alloSCT in AML and MDS. Figure 1. CIR based on the 1 month (A) MP and (B) CP status. (C) CIR based on the 1month CP status according to DTA subset. (D) Comparison of ROC curves for relapse prediction between 1 month CP and MP. Disclosures No relevant conflicts of interest to declare.

Background: Next-generation sequencing (NGS) is an attractive tool for prospective use in the field of precision medicine. Using NGS to guide therapy has provided a large volume of genomic data and therapeutic actionability of somatic NGS results. These data are evolving too rapidly to rely solely on human curation. So the interpretation of the clinical significance of such large amounts of genetic data remains the most severe bottleneck preventing the realization of precision medicine. Watson for Genomics (WfG) is a representative artificial intelligence (AI) software, which analyzes and categorizes genetic alterations that are related to disease progression and provides a list of potential therapeutic options within 3 minutes per sample. Recent reports suggested that WfG could empower tumor boards and improve patient care by providing a rapid, comprehensive approach for data analysis and consideration of the up-to-date availability of clinical trials (Patel NM, et.al. Oncologist. 2018). However, only limited data are available regarding the utility of AI-guided precision medicine approach in the field of hematological disease. The purpose of this study is to test the utility of AI in assisting the interpretation of high throughput genomic data from patients with the hematological disease. Methods: After obtaining written informed consent, we enrolled patients with hematological disease at our research hospital between May 2015 to June 2018. Genomic DNA was prepared from malignant cell fractions and normal tissues in each patient and subjected to comparative NGS, mainly targeted deep sequencing (TDS) with ready-made panels and, on demand, whole exome sequencing (WES). Sequence data was analyzed using a pipeline of in-house semi-automated medical informatics. After initial bioinformatics filtering, we used WfG to identify potential driver mutations, which were annotated as "pathogenic" or "likely pathogenic" (WfG version 39.132 and 39.135 as of July 2018). The results were compared with the findings of expert hematologists. Results: 247 paired samples (TDS, n= 143; WES, n= 104) collected from 187 patients were analyzed. Our cohort consisted of 63 patients with acute myeloid leukemia, 40 with myelodysplastic syndromes (MDS), 19 with myeloproliferative neoplasms (MPN), 9 with MDS/MPN, 10 with acute lymphoblastic leukemia/lymphoma, 17 with non Hodgkin lymphoma, 6 with multiple myeloma (MM) and others. In 151 of 187 patients, a total of 290 somatic driver mutations were identified by human curation. The frequently mutated genes were TP53 (n=31), NRAS (n=17), TET2 (n=16), U2AF1 (n=14), FLT3/ASXL1/WT1 (n=13 each), and DNMT3A/RUNX1 (n=12 each). WfG identified 79% (n=229) of driver mutations which human experts also did. There was some discordance between WfG and the human (Figure 1): Sixteen mutations were interpreted as "variant of unknown significance" by WfG, but these mutations were deduced as driver mutation by the human. Conversely, in two representative cases, WfG identified a relevant driver mutation that the human did not: FAM46C and SOCS1, from a patient with MM and with primary mediastinal large-B cell lymphoma, respectively. These examples indicate the potential for a mutually complementary or cooperative relationship between AI "software" and the human expert "hardware" in the interpretation of high throughput genomic data. Conclusion: Combing AI "software" and the human expert "hardware" will allow for the quick delivery of comprehensive information needed for patient care that outperforms what either can achieve individually in the field of hematological disease. Figure1. Comparison of potential driver mutations between human curation and Watson for Genomics. The size of the gene symbol indicates the total number of mutations identified Disclosures No relevant conflicts of interest to declare.

Acute myeloid leukemia (AML) is characterized by unregulated clonal expansion and maturation arrest of myeloid committed progenitors (MP). AML generally represents de novo onset or evolves from preceding myelodysplastic syndrome (MDS), which is defined by refractory cytopenias, clonal hematopoiesis, and/or multi-lineage dysplasia. The WHO classification 2008 includes this entity as “AML with myelodysplasia-related changes (AML-MRC)”, and currently, diagnosis of AML-MRC is based on either previous history of MDS, multi-lineage dysplasia, or MDS-related cytogenetic abnormality. However, AML-MRC often represents de novo onset without these MDS-compatible clinical features. Considering that AML-MRC exhibits rather poor prognosis with refractoriness to conventional chemotherapy against AML, more accurate and objective diagnostic approach is requisite to unveil hidden “MDS signatures” in patients with apparently de novo AML. A certain set of gene mutations is specific and recurrent in MDS. Given the pre-existing “MDS signatures”, the founder gene mutations might be detected in not only blast cells but also neutrophils and/or T cells in AML-MRC. To test this hypothesis, we performed FACS sorting of neutrophils, T cells, and blasts fractions, respectively, followed by mutation screening using targeted deep sequencing, namely, cell lineage-oriented sequencing (CLS). Genomic DNA both from each cell fraction and buccal swab was subjected to screening mutations in 54 genes which are tightly involved in MDS and AML. Pair-end deep sequencing was performed on an Illumina MiSeq, using library prepared by TruSight Myeloid Panel (Illumina, San Diego, CA). Bioinformatic analysis was performed by in-house pipeline. We performed CLS of clinically diagnosed AML-MRC (n=7), suspected AML-MRC (n=2), de novo AML (AML with t(15;17) or AML with inv16, n=4), MDS (RAEB-1 and RAEB-II, n=3), and familial MDS (n=1). As expected, in a familial MDS case, overlapping germline RUNX1 driver mutation was demonstrated in granulocytes, blast cells and T cells, supporting that it would be originated from a hematopoietic stem cell. Notably, in MDS, AML-MRC, and suspected AML-MRC cases with no germ-line mutations, the founder mutations present in neutrophils were also retained in the AML blast cells, irrespective of a history of MDS, suggesting that these are derived from a myeloid progenitor cell. In marked contrast, there were no overlapping driver mutations between blast cell and neutrophil fractions in de novo AML characterized by recurrent chromosomal abnormalities. In summary, CLS revealed that founder mutations are shared by neutrophils and AML blast cells in AML-MRC, but not in de novo AML. Although our data should be validated in a larger cohort of AML cases, CLS is a promising approach to molecular diagnosis of latent AML-MRC which require distinct therapeutic options from de novo AML. Citation Format: Kazuaki Yokoyama, Nozomi Yusa, Sousuke Nakamura, Mika Ito, Asako Kobayashi, Masayuki Kobayashi, Rika Kasajima, Hiroaki Yui, Eigo Shimizu, Atushi Niida, Rui Yamaguti, Tsuneo Ikenoue, Seiya Imoto, Yoichi Frukawa, Satoru Miyano, Arinobu Tojo. Cell lineage-oriented clinical sequencing unveils distinct clonal ontogeny of acute myeloid leukemia with myelodysplasia-related changes [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 736. doi:10.1158/1538-7445.AM2017-736

Next-generation sequencing (NGS) is an attractive tool for prospective use in the field of clinical oncology. However, for this purpose, further innovations are necessary including medical informatics which links somatic mutations to clinical intervention. This process is currently labor-intensive, involving experienced curators who pick up the relevant evidence among a growing body of knowledge and translate it into medical practice. We organized a clinical sequencing team, called as IMSUT Tumor Board, and have been integrating clinical and genomic information in hematological malignancies with the aid of a cognitive computing (CC) system. Genomic DNA was prepared from malignant cell fractions and normal tissues in each patient, and subjected to comparative NGS, mainly targeted deep sequencing with ready-made panels and, on demand, whole exome sequencing. Sequence data was analyzed using a pipeline of in-house semi-automated medical informatics, namely YOKOMON-GO. CC was used to identify candidate driver mutations and pathways in each patient, from which pathogenic information as well as applicable drug information was deduced. A summary of NGS data was reported and discussed in IMSUT Tumor Board to deliberate upon potentially actionable findings. Up to date, we have performed NGS analysis on 90 patients with AML, MDS, MPN, et al., among which informative and actionable findings could be obtained in 50 and 18 patients, respectively. Six patients actually received treatments motivated in IMSUT Tumor Board. Our preliminary results indicate that CC can be well suited to clinical sequencing. Disclosures Koyama: IBM: Employment.

553 Background: Targeted therapies of monoclonal antibodies have changed the treatment of metastatic colorectal cancer (mCRC). A target therapy with chemotherapy regimen for mCRC was decided by KRAS mutation status (KRAS exon2 [codon12, codon13]). Currently, there are many reports suggesting that in addition to analysis of KRAS mutation status, the evaluation of EGFR gene copy number, levels of EGFR ligands, BRAF, NRAS, PIK3CA mutations could be helpful to have a more accurate selection of patients who may have a benefit from anti-EGFR targeted drugs. Methods: Mutation status of 50 oncogenes were analysed in 35 mCRC patients with all RAS wild type, using next-generation sequencing technology. The response for chemotherapy was classified response group (R group) and non-response group (N group) by RECIST. The relation between mutation status of 50 oncogenes and the response for chemotherapy was assessed. Results: There were 25 oncogene mutations in the 50 genes. Driver mutation associated with oncogenic mutation deeply were 5 oncogenes, which were PIK3CA, AKT1, BRAF, PDGFRA and TP53. Only BRAF mutation was significantly associated with poor chemo response in the 5 oncogenes. A case which had two driver mutations was only in the N group. One of the two driver mutations was tumor suppressor gene, TP53. Conclusions: BRAF mutation and the number of driver mutations are key predictors of chemosensitivity in the mCRC cases with all RAS wild type.