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Background: Studies have shown that tumor cell amino acid metabolism is closely associated with lung adenocarcinoma (LUAD) development and progression. However, the comprehensive multi-omics features and clinical impact of the expression of genes associated with amino acid metabolism in the LUAD tumor microenvironment (TME) are yet to be fully understood., Methods: LUAD patients from The Cancer Genome Atlas (TCGA) database were enrolled in the training cohort. Using least absolute shrinkage and selection operator Cox regression analysis, we developed PTAAMG-Sig, a signature based on the expression of tumor-specific amino acid metabolism genes associated with overall survival (OS) prognosis. We evaluated its predictive performance for OS and thoroughly explored the effects of the PTAAMG-Sig risk score on the TME. The risk score was validated in two Gene Expression Omnibus (GEO) cohorts and further investigated against an original cohort of chemotherapy combined with immune checkpoint inhibitors (ICIs). Somatic mutation, chemotherapy response, immunotherapy response, gene set variation, gene set enrichment, immune infiltration, and plasma-free amino acids (PFAAs) profile analyses were performed to identify the underlying multi-omics features., Results: TCGA datasets based PTAAMG-Sig model consisting of nine genes, KYNU, PSPH, PPAT, MIF, GCLC, ACAD8, TYRP1, ALDH2 , and HDC , could effectively stratify the OS in LUAD patients. The two other GEO-independent datasets validated the robust predictive power of PTAAMG-Sig. Our differential analysis of somatic mutations in the high- and low-risk groups in TCGA cohort showed that the TP53 mutation rate was significantly higher in the high-risk group and negatively correlated with OS. Prediction from transcriptome data raised the possibility that PTAAMG-Sig could predict the response to chemotherapy and ICIs therapy. Our immunotherapy cohort confirmed the predictive ability of PTAAMG-Sig in the clinical response to ICIs therapy, which correlated with the infiltration of immune cells (e.g., T lymphocytes and nature killer cells). Corresponding to the concentrations of PFAAs, we discovered that the high PTAAMG-Sig risk score patients showed a significantly lower concentration of plasma-free α-aminobutyric acid., Conclusion: In patients with LUAD, the PTAAMG-Sig effectively predicted OS, drug sensitivity, and immunotherapy outcomes. These findings are expected to provide new targets and strategies for personalized treatment of LUAD patients., Competing Interests: Authors TTagami and AH were employed by the company Ajinomoto Co, Ltd. The remaining authors declare that the research was conducted in the absence of any commercial or financial relationship that could be construed as a potential conflict of interest., (Copyright © 2024 Xiang, Kasajima, Azuma, Tagami, Hagiwara, Nakahara, Saito, Igarashi, Wei, Ban, Yoshihara, Nakamura, Sato, Koizume, Tamura, Sasada and Miyagi.)

Background: Erdheim-Chester disease (ECD) is a rare form of non-Langerhans cell histiocytosis with diverse clinical manifestations, often associated with mutations in the mitogen-activated protein kinase/extracellular signal-regulated kinase (MAPK/ERK) pathway. BRAF and KRAS mutations, which are driver mutations of oncogenes, participate in the same signaling pathway (MAPK/ERK pathway) and are usually mutually exclusive. We report a case of ECD with concurrent BRAF V600E and KRAS G12R mutations treated using BRAF and MEK inhibitors., Case: A 70-year-old man was referred to our hospital with a mesenteric nodal lesion on computed tomography scan. The patient experienced symptoms consistent with ECD, including central diabetes insipidus. Biopsy revealed histiocytes positive for CD68 and CD163, negative for S100, CD1a, and CD21. Liquid-based comprehensive genomic profiling and tissue-based cancer gene panel test identified BRAF V600E and KRAS G12R mutations with different variant allele fraction. Additional immunohistochemistry with an antibody specific to mutant BRAF V600E protein stained some proliferating histiocytes, consistent with ECD. Based on the genomic profiling results, we hypothesized that there was a coexistence of a clone harboring BRAF V600E and another clone harboring KRAS G12R , and planned a combination therapy with BRAF and MEK inhibitors targeting each clone, respectively. The patient received oral encorafenib at 100 mg once daily and oral binimetinib at 15 mg twice daily. The combination therapy resulted in rapid resolution of symptoms and significant improvement in imaging findings., Conclusion: This case represents a unique presentation of ECD with concurrent BRAF V600E and KRAS G12R mutations. Combination therapy with encorafenib and binimetinib targeting each clone resulted in a remarkable therapeutic effect and was well-tolerated. This is the first reported case of ECD treated with encorafenib and binimetinib. The combination therapy with BRAF and MEK inhibitors is one of the rational treatment options for cases of ECD with a suspicion of multiple clones., (© 2024 The Author(s). Cancer Reports published by Wiley Periodicals LLC.)

Formalin-fixed paraffin-embedded (FFPE) tissue samples are important for genomic analysis of thyroid carcinomas, particularly for various molecularly targeted therapies. Therefore, this study developed and validated a technique for preparing FFPE tissue samples that preserves nucleic acid quality, which is fundamental for precise genomic analysis, more effectively than conventional methods. We analyzed surgically resected thyroid gland tumors, lymph node metastases, and separately fixed tumor samples to optimize formalin fixation and assess the influence of specimen type and preparation methods on nucleic acid quality. We assessed several quality indicators, including the DNA integrity number, cycle threshold ratio, RNA integrity number, and DV200. Separately fixed tumor samples consistently exhibited higher DNA and RNA quality than conventionally processed samples. Additionally, lymph node metastases often exhibit nucleic acid quality matching or exceeding that of thyroid gland tumors, highlighting their potential reliability for genomic analysis. These findings suggest the utility of various specimen types for the comprehensive genetic profiling of thyroid carcinomas. In conclusion, this study demonstrated that preparing separately fixed tumor samples is an effective method for preserving DNA and RNA quality for genomic analyses. Biopsy punches enable specimen collection at various facilities, including those without the ability to handle frozen specimens. This contributes to the development of a method for obtaining high-quality pathological samples that can be widely used in general medical practice. Moreover, lymph node metastases often exhibit nucleic acid quality equal to or superior to that of thyroid gland tumors, highlighting their potential as acceptable sources for genomic analyses., Competing Interests: Declarations. Ethics Approval and Consent to Participate: Written informed consent was obtained from all patients for participation and publication of the study data. This study was performed in accordance with the tenets of the Declaration of Helsinki and approved by the Ethics Review Committee of Kanagawa Cancer Center (Approval No.: 2022-Eki-121). Competing Interests: The authors declare no competing interests., (© 2024. The Author(s).)

Cancer stem cells (CSC) are thought to be responsible for cancer phenotypes and cellular heterogeneity. Here we demonstrate that the human colon cancer cell line DLD1 contains two types of CSC-like cells that undergo distinct morphogenesis in the reconstituted basement membrane gel Matrigel. In our method with cancer cell spheroids, the parent cell line (DLD1-P) developed grape-like budding structures, whereas the other (DLD1-Wm) and its single-cell clones dynamically developed worm-like ones. Gene expression analysis suggested that the former mimicked intestinal crypt-villus morphogenesis, while the latter mimicked embryonic hindgut development. The organoids of DLD1-Wm cells rapidly extended in two opposite directions by expressing dipolar proteolytic activity. The invasive morphogenesis required the expression of MMP-2 and CD133 genes and ROCK activity. These cells also exhibited gastrula-like morphogenesis even in two-dimensional cultures without Matrigel. Moreover, the two DLD1 cell lines showed clear differences in cellular growth, tumor growth and susceptibility to paclitaxel. This study also provides a simple organoid culture method for human cancer cell lines. HT-29 and other cancer cell lines underwent characteristic morphogenesis in direct contact with normal fibroblasts. Such organoid cultures would be useful for investigating the nature of CSCs and for screening anti-cancer drugs. Our results lead to the hypothesis that CSC-like cells with both invasive activity and a fetal phenotype, i. e. oncofetal CSCs, are generated in some types of colon cancers., Competing Interests: Declaration of competing interest The authors declare the following financial interests/personal relationships which may be considered as potential competing interests: K. M. acted an advisor for Kishikasei Co. Ltd (Yokohama Japan) for 2 years (2021–2022). All other authors declare no competing interests. Patent application: Japanese Patent application in pending: 2021-049395; Date: May 23, 2021; Patent Title: Methods for producing organoid structures of cancer cell lines, medium additives for producing the organoids, and a method for screening anticancer drug candidates using the organoids; Applicants: Kanagawa Prefectural Hospital Organization, Kishikasei and K. M.; Inventors: K. M., Y. M. and Daisuke Maezawa., (Copyright © 2024 The Authors. Published by Elsevier Inc. All rights reserved.)

Neutrophil extracellular traps (NETs) released from neutrophils are related to cancer progression. However, the relationship between the therapeutic effects of immune checkpoint inhibitors (ICIs) such as anti-PD-1 and anti-PD-L1 antibodies and plasma NET concentration in patients with non-small cell lung cancer (NSCLC) is poorly understood. In this study, concentrations of citrullinated histone H3 (CitH3), a surrogate marker of NETs, in plasma before/after treatment were examined in patients with advanced or recurrent NSCLC undergoing ICI treatment (n = 185). The clinical significances of NET levels before/after treatment and posttreatment changes were statistically evaluated. As a result, multivariate Cox analysis showed that high NET levels before treatment were statistically significant predictors of unfavorable overall survival (OS; p < 0.001, HR 1.702, 95% CI 1.356-2.137) and progression-free survival (PFS; p < 0.001, HR 1.566, 95% CI 1.323-1.855). The Kaplan-Meier curves showed significant separation between the high- and low-NET groups in OS ( p = 0.002) and PFS ( p < 0.001). Additionally, high NET levels after treatment were also significantly associated with worse OS ( p < 0.001) and PFS ( p < 0.001) by multivariate Cox analysis. Notably, the pretreatment NET levels were significantly correlated with the plasma levels of NET-related inflammatory cytokines, such as IL-6 and IL-8, and with NET-related gene expression and immune-suppressive profile in peripheral blood mononuclear cells. Our findings suggest that NETs released from activated neutrophils might reduce the clinical efficacy of ICIs in patients with NSCLC.

The selection of highly specific target antigens is critical for the development of clinically efficient and safe chimeric antigen receptors (CARs). In search of diagnostic marker for malignant mesothelioma (MM), we have established SKM9-2 monoclonal antibody (mAb) which recognizes a MM-specific molecule, sialylated Protein HEG homolog 1 (HEG1), with high specificity and sensitivity. In this study, to develop a novel therapeutic approach against MM, we generated SKM9-2 mAb-derived CARs that included the CD28 (SKM-28z) or 4-1BB (SKM-BBz) costimulatory domain. SKM-28z CAR-T cells showed continuous growth and enhanced Tim-3, LAG-3, and PD-1 expression in vitro, which might be induced by tonic signaling caused by self-activation; however, these phenotypes were not observed in SKM-BBz CAR-T cells. In addition, SKM-BBz CAR-T cells exhibited slightly stronger in vitro killing activity against MM cell lines than SKM-28z CAR-T cells. More importantly, only SKM-BBz CAR-T cells, but not SKM-28z CAR-T cells, significantly inhibited tumor growth in vivo in a MM cell line xenograft mouse model. Gene expression profiling and reporter assays revealed differential signaling pathway activation; in particular, SKM-BBz CAR-T cells exhibited enhanced NF-kB signaling and reduced NFAT activation. In addition, SKM-BBz CAR-T cells showed upregulation of early memory markers, such as TCF7 and CCR7, as well as downregulation of pro-apoptotic proteins, such as BAK1 and BID, which may be associated with phenotypical and functional differences between SKM-BBz and SKM-28z CAR-T cells. In conclusion, we developed novel SKM9-2-derived CAR-T cells with the 4-1BB costimulatory domain, which could provide a promising therapeutic approach against refractory MM., (© 2024 UICC.)

Delayed neutrophil recovery is an important limitation to the administration of cord blood transplantation (CBT) and leaves the recipient vulnerable to life-threatening infection and increases the risk of other complications. A predictive model for neutrophil recovery after single-unit CBT was developed by using a machine learning method, which can handle large and complex datasets, allowing for the analysis of massive amounts of information to uncover patterns and make accurate predictions. Japanese registry data, the largest real-world dataset of CBT, was selected as the data source. Ninety-eight variables with observed values for >80% of the subjects known at the time of CBT were selected. Model building was performed with a competing risk regression model with lasso penalty. Prediction accuracy of the models was evaluated by calculating the area under the receiver operating characteristic curve (AUC) using a test dataset. The primary outcome was neutrophil recovery at day (D) 28, with recovery at D14 and D42 analyzed as secondary outcomes. The final cord blood engraftment prediction (CBEP) models included 2991 single-unit CBT recipients with acute leukemia. The median AUC of a D28-CBEP lasso regression model run 100 times was .74, and those for D14 and D42 were .88 and .68, respectively. The predictivity of the D28-CBEP model was higher than that of 4 different legacy models constructed separately. A highly predictive model for neutrophil recovery by 28 days after CBT was constructed using machine learning techniques; however, identification of significant risk factors was insufficient for outcome prediction for an individual patient, which is necessary for improving therapeutic outcomes. Notably, the prediction accuracy for post-transplantation D14, D28, and D42 decreased, and the model became more complex with more associated factors with increased time after transplantation., (Copyright © 2024 The American Society for Transplantation and Cellular Therapy. Published by Elsevier Inc. All rights reserved.)

Purpose: Low-grade adenosquamous carcinoma (LGASC) is a rare type of metaplastic carcinoma of the breast (MBC) with an indolent clinical course. A few LGASC cases with high-grade transformation have been reported; however, the genetics underlying malignant progression of LGASC remain unclear., Methods: We performed whole-genome sequencing analysis on five MBCs from four patients, including one case with matching primary LGASC and a lymph node metastatic tumor consisting of high-grade MBC with a predominant metaplastic squamous cell carcinoma component (MSC) that progressed from LGASC and three cases of independent de novo MSC., Results: Unlike de novo MSC, LGASC and its associated MSC showed no TP53 mutation and tended to contain fewer structural variants than de novo MSC. Both LGASC and its associated MSC harbored the common GNAS c.C2530T:p.Arg844Cys mutation, which was more frequently detected in the cancer cell fraction of MSC. MSC associated with LGASC showed additional pathogenic deletions of multiple tumor-suppressor genes, such as KMT2D and BTG1. Copy number analysis revealed potential 18q loss of heterozygosity in both LGASC and associated MSC. The frequency of SMAD4::DCC fusion due to deletions increased with progression to MSC; however, chimeric proteins were not detected. SMAD4 protein expression was already decreased at the LGASC stage due to unknown mechanisms., Conclusion: Not only LGASC but also its associated high-grade MBC may be genetically different from de novo high-grade MBC. Progression from LGASC to high-grade MBC may involve the concentration of driver mutations caused by clonal selection and inactivation of tumor-suppressor genes., (© 2023. The Author(s).)

Background: Impact of RNA-binding motif protein 10 (RBM10) and programmed death-ligand 1 (PD-L1) on the postoperative prognosis of patients with epidermal growth factor receptor gene mutation ( EGFR -Mt) lung adenocarcinoma with pathological lymph node metastasis is still unclear., Methods: Patients who underwent curative surgery for pN1-N2 EGFR -Mt lung adenocarcinoma (n=129) harboring the EGFR exon 19 deletion mutation (Ex19) (n=66) or EGFR exon 21 L858R mutation (Ex21) (n=63) between January 2010 and December 2020 were included in this retrospective study. The prognoses of patients with low/high cytoplasmic RBM10 expression and PD-L1 negativity/positivity based on immunohistochemistry (IHC) of resected specimens were compared using the log-rank test. The effects of RBM10 and PD-L1 expression on overall survival (OS) were examined via multivariable analysis using the Cox proportional hazards regression model. The effects of RBM10 and PD-L1 expression on progression-free survival (PFS) of EGFR-tyrosine kinase inhibitors (TKIs) therapy among patients with recurrent pN1-N2 EGFR -Mt lung adenocarcinoma (n=67) were examined using log-rank tests., Results: The RBM10 low expression group showed significantly better 5-year OS than the RBM10 high expression group (89.4% vs . 71.5%, P=0.020), and the PD-L1 negative group tended to have longer 5-year OS than the PD-L1 positive group (86.4% vs . 68.4%, P=0.050). Multivariable analysis showed that high RBM10 expression [hazard ratio (HR), 3.12; 95% confidence interval (CI): 1.19-8.17; P=0.021] and PD-L1 positivity (HR, 3.80; 95% CI: 1.64-8.84; P=0.002) were independent poor prognostic factors for OS. PFS of patients with relapse and first-line EGFR-TKI treatment was significantly better in the PD-L1-negative group than in the PD-L1-positive group (34.5 vs . 12.1 months, P=0.045). PFS of patients with Ex21 relapse and first-line EGFR-TKI treatment was significantly better in the RBM10 low expression group than in the RBM10 high expression group (25.5 vs . 13.0 months, P=0.025)., Conclusions: High RBM10 expression and PD-L1 positivity are poor prognostic factors for OS in patients with pN1-N2 EGFR -Mt lung adenocarcinoma after curative surgery. In patients with recurrent pN1-N2 EGFR -Mt lung adenocarcinoma, PD-L1 and RBM10 expression may influence response to EGFR-TKIs., Competing Interests: Conflicts of Interest: All authors have completed the ICMJE uniform disclosure form (available at https://tlcr.amegroups.com/article/view/10.21037/tlcr-23-355/coif). TI reports receiving Grant from Kanagawa Cancer Foundation and JSPS KAKENHI (Grant Number JP21K16519). The other authors have no conflicts of interest to declare., (2023 Translational Lung Cancer Research. All rights reserved.)

Background: High-grade fetal adenocarcinoma of the lung (H-FLAC) is a rare variant of pulmonary adenocarcinoma. Our previous study showed a high frequency of KMT2C mutations in lung cancers with an H-FLAC component, showing that KMT2C dysfunction may be associated with the biological features of H-FLACs., Methods: In this study, we performed RNA sequencing and immunohistochemical analysis to identify the differentially expressed genes and corresponding pathways associated with H-FLACs, compared with common adenocarcinomas., Results: Ingenuity pathway analysis based on RNA sequencing data revealed that DNA homologous recombination repair (HRR) pathways were significantly inactivated in H-FLAC. Expression of KMT2C, ATM, ATR , and BRCA2 was significantly lower in H-FLACs than in common adenocarcinomas, and BRCA1 expression showed a decreasing trend. Pearson correlation analyses for all cases revealed that KMT2C expression showed a strong positive correlation (R>0.7) with the expression of ATR, BRCA1 , and BRCA2 genes and a moderately positive correlation with ATM expression (R=0.47). Immunohistochemical analysis showed significantly lower levels of KMT2C, ATM, ATR, and BRCA2 expression in H-FLACs than in common adenocarcinomas, and a trend of lower BRCA1 levels. Additionally, KMT2C expression showed a weak to moderate correlation with that of ATM, ATR, BRCA1, and BRCA2., Conclusions: Cancers containing H-FLAC components showed lower levels of KMT2C and HRR factors than common lung adenocarcinomas, and their levels exhibited a positive correlation. These results support the hypothesis that loss of KMT2C function decreases the expression of the HRR factors in H-FLACs. H-FLACs with low KMT2C expression may be a good indication for poly (ADP-ribose) polymerase (PARP) inhibitor-based therapy., Competing Interests: Conflicts of Interest: All authors have completed the ICMJE uniform disclosure form (available at https://tlcr.amegroups.com/article/view/10.21037/tlcr-23-137/coif). The authors have no conflicts of interest to declare., (2023 Translational Lung Cancer Research. All rights reserved.)

Bone metastasis occurs frequently in cancer patients. Conventional therapies have limited therapeutic outcomes, and thus, exploring the mechanisms of cancer progression in bone metastasis is important to develop new effective therapies. In the bone microenvironment, adipocytes are the major stromal cells that interact with cancer cells during bone metastasis. However, the comprehensive functions of bone marrow adipocytes in cancer progression are not yet fully understood. To address this, we investigated the role of bone marrow adipocytes on cancer cells, by focusing on an invasive front that reflects the direct effects of stromal cells on cancer. In comprehensive histopathological and genetic analysis using bone metastasis specimens, we examined invasive fronts in bone metastasis and compared invasive fronts with adipocyte-rich bone marrow (adipo-BM) to those with hematopoietic cell-rich bone marrow (hemato-BM) as a normal counterpart of adipocytes. We found morphological complexity of the invasive front with adipo-BM was significantly higher than that with hemato-BM. Based on immunohistochemistry, the invasive front with adipo-BM comparatively had a significantly increased cancer-associated fibroblast (CAF) marker-positive area and lower density of CD8 + lymphocytes compared to that with hemato-BM. RNA sequencing analysis of primary and bone metastasis cancer revealed that bone metastasized cancer cells acquired drug resistance-related gene expression phenotypes. Clearly, these findings indicate that bone marrow adipocytes provide a favorable tumor microenvironment for cancer invasion and therapeutic resistance of bone metastasized cancers through CAF induction and immune evasion, providing a potential target for the treatment of bone metastasis., (© 2023 The Authors. Cancer Science published by John Wiley & Sons Australia, Ltd on behalf of Japanese Cancer Association.)

Myxofibrosarcoma (MFS) and undifferentiated sarcoma (US) have been considered as tumors of the same lineage based on genetic/epigenetic profiling. Although MFS shows a notably better prognosis than US, there are no clear criteria for distinguishing between them. Here, we examined 85 patients with MFS/US and found that tumors with infiltrative growth patterns tended to have more myxoid areas and higher local recurrence rates but fewer distant metastases and better overall survival. Morphologically characteristic sickle-shaped blood vessels, which tended to have fewer αSMA-positive cells, were also observed in these tumors, compared with normal vessels. Based on the incidence of these sickle-shaped blood vessels, we subdivided conventionally diagnosed US into two groups. This stratification was significantly correlated with metastasis and prognosis. RNA sequencing of 24 tumors (9 MFS and 15 US tumors) demonstrated that the proteasome, NF-kB, and VEGF pathways were differentially regulated among these tumors. Expression levels of KDR and NFATC4, which encode a transcription factor responsible for the neuritin-insulin receptor angiogenic signaling, were elevated in the sickle-shaped blood vessel-rich US tumors. These findings indicate that further analyses may help elucidate the malignant potential of MFS/US tumors as well as the development of therapeutic strategies for such tumors., (© 2023. The Author(s).)

Background/aim: The prognosis of anaplastic thyroid carcinoma (ATC) is poor, and there is currently no established treatment to improve its outcome. We previously reported that enhancer of zeste homolog 2 (EZH2) was highly expressed in ATC, and may be a therapeutic target; however, the effects of EZH2 on ATC growth currently remain unknown., Materials and Methods: We investigated the effects of an EZH2 inhibitor (DZNep) on four ATC cell lines (8305C, KTA1, TTA1 and TTA2). We performed a gene panel analysis of all ATC cell lines to identify differences in DZNep sensitivity between the cell lines. To investigate the effects of DZNep on the recovery of differentiation, we assessed changes in thyroid differentiation markers (TDMs) before and after the DZNep treatment using PCR., Results: EZH2 was expressed in all ATC cell lines. The cell-reducing effects of DZNep were detected in all ATC cell lines, and were the strongest in KTA1 cells followed by TTA2 cells. The TTA1 and 8305C cell lines, which showed weak cell-reducing effects, had TP53 mutations. No changes in TDMs were observed in any ATC cell line., Conclusion: DZNep, an EZH2 inhibitor, exerted suppressive effects on the growth of ATC cell lines and has potential as a therapeutic strategy; however, its effects may be attenuated in ATC with TP53 mutations., (Copyright © 2023 International Institute of Anticancer Research (Dr. George J. Delinasios), All rights reserved.)

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); 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, and the tumor was diagnosed as a spindle cell tumor/suspicious adult fibrosarcoma. The tumor relapsed during postoperative follow-up, and showed size reduction with chemotherapy, prior to regrowth. 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. Furthermore, both the uterine adenosarcoma and intra-abdominal mass were partially positive for CD10 and BCOR staining., Conclusion: These results led to the conclusive identification of the abdominal tumor as a metastasis of the uterine adenosarcoma. The JAZF1-BCORL1 rearrangement is predominantly associated with uterine stromal sarcomas; thus far, ours is the second report of the same in an adenosarcoma. 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 the designing of the best-suited treatment protocol., (© 2023. The Author(s).)

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&#95;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., 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., (© 2023. The Author(s).)

Langerhans cell histiocytosis (LCH) and acute myeloid leukemia (AML) are distinct entities of blood neoplasms, and the exact developmental origin of both neoplasms are considered be heterogenous among patients. However, reports of concurrent LCH and AML are rare. Herein we report a novel case of concurrent LCH and AML which shared same the driver mutations, strongly suggesting a common clonal origin.An 84-year-old female presented with cervical lymphadenopathy and pruritic skin rash on the face and scalp. Laboratory tests revealed pancytopenia with 13% of blasts, elevated LDH and liver enzymes, in addition to generalised lymphadenopathy and splenomegaly by computed tomography. Bone marrow specimens showed massive infiltration of MPO-positive myeloblasts, whereas S-100 and CD1a positive atypical dendritic cell-like cells accounted for 10% of the atypical cells on bone marrow pathology, suggesting a mixture of LCH and AML. A biopsy specimen from a cervical lymph node and the skin demonstrated the accumulation of atypical cells which were positive for S-100 and CD1a. LCH was found in lymph nodes, skin and bone marrow; AML was found in peripheral blood and bone marrow (AML was predominant compared with LCH in the bone marrow). Next generation sequencing revealed four somatic driver mutations ( NRAS -G13D, IDH2 -R140Q, and DNMT3A -F640fs/-I715fs), equally shared by both the lymph node and bone marrow, suggesting a common clonal origin for the concurrent LCH and AML. Prednisolone and vinblastine were initially given with partial response in LCH; peripheral blood blasts also disappeared for 3 months. Salvage chemotherapy with low dose cytarabine and aclarubicin were given for relapse, with partial response in both LCH and AML. She died from pneumonia and septicemia on day 384. Our case demonstrates a common cell of origin for LCH and AML with a common genetic mutation, providing evidence to support the proposal to classify histiocytosis, including LCH, as a myeloid/myeloproliferative malignancy., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2022 Kazama, Yokoyama, Ueki, Kazumoto, Satomi, Sumi, Ito, Yusa, Kasajima, Shimizu, Yamaguchi, Imoto, Miyano, Tanaka, Denda, Ota, Tojo and Kobayashi.)

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 &gt;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)., (© The Author(s) 2022. Published by Oxford University Press.)

Background: Amino 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., Methods: We 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., Results: On 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 (p<0.05). In particular, the multivariate model, composed of a combination of serine, glycine, arginine, and quinolinic acid, could most efficiently stratify patient OS (concordance index=0.775, HR=3.23, 95% CI 2.04 to 5.26). From the transcriptome analysis in PBMCs, this multivariate model was significantly correlated with the gene signatures related to immune responses, such as CD8 T-cell activation/proliferation and proinflammatory immune responses, and 12 amino acid-related genes were differentially expressed between the high-risk and low-risk groups., Conclusions: The multivariate model with PFAAs and metabolites in plasma might be useful for stratifying patients who will benefit from PD-1 inhibitors., Competing Interests: Competing interests: KA reports receiving personal fees from AstraZeneka, MSD, Bristol Myers Squibb, Ono Pharmaceutical and Chugai Pharmaceutical. HI reports receiving personal fees from AstraZeneka, KyowaKirin, AMCO, Ono Pharmaceutical, and Chugai Pharmaceutical. TTa reports receiving personal fees from AstraZeneka, Ono Pharmaceutical, and Chugai Pharmaceutical. KM reports receiving personal fees from AstraZeneka, MSD, Taiho, Boehringer Ingelheim, Ono Pharmaceutical, and Chugai Pharmaceutical. TS reports receiving personal fees from Bristol Myers Squibb and Chugai Pharmaceutical. TTo, YK, SKa, SKi, and AI are employees of Ajinomoto Co. The remaining authors have no conflicts of interest to disclose., (© Author(s) (or their employer(s)) 2022. Re-use permitted under CC BY. Published by BMJ.)

Lineage switches in acute leukemia occur rarely, and the underlying mechanisms are poorly understood. Herein, we report the case of an elderly patient with leukemia in which the leukemia started as B-cell acute lymphoblastic leukemia (B-ALL) and later changed to B- and T-cell mixed phenotype acute leukemia (MPAL) and acute myeloid leukemia (AML) during consecutive induction chemotherapy treatments. A 65-year-old woman was initially diagnosed with Philadelphia chromosome-negative B-ALL primarily expressing TdT/CD34/HLA-DR; more than 20% of the blasts were positive for CD19/CD20/cytoplasmic CD79a/cytoplasmic CD22/CD13/CD71.The blasts were negative for T-lineage markers and myeloperoxidase (MPO). Induction chemotherapy with the standard regimen for B-ALL resulted in primary induction failure. After the second induction chemotherapy regimen, the blasts were found to be B/T bi-phenotypic with additional expression of cytoplasmic CD3. A single course of clofarabine (the fourth induction chemotherapy regimen) dramatically reduced lymphoid marker levels. However, the myeloid markers (e.g., MPO) eventually showed positivity and the leukemia completely changed its lineage to AML. Despite subsequent intensive chemotherapy regimens designed for AML, the patient's leukemia was uncontrollable and a new monoblastic population emerged. The patient died approximately 8 months after the initial diagnosis without experiencing stable remission. Several cytogenetic and genetic features were commonly identified in the initial diagnostic B-ALL and in the following AML, suggesting that this case should be classified as lineage switching leukemia rather than multiple simultaneous cancers (i.e., de novo B-ALL and de novo AML, or primary B-ALL and therapy-related myeloid neoplasm). A complex karyotype was persistently observed with a hemi-allelic loss of chromosome 17 (the location of the TP53 tumor suppressor gene). As the leukemia progressed, the karyotype became more complex, with the additional abnormalities. Sequential target sequencing revealed an increased variant allele frequency of TP53 mutation. Fluorescent in situ hybridization (FISH) revealed an increased number of mixed-lineage leukemia ( MLL ) genes, both before and after lineage conversion. In contrast, FISH revealed negativity for MLL rearrangements, which are well-known abnormalities associated with lineage switching leukemia and MPAL. To our best knowledge, this is the first reported case of acute leukemia presenting with lineage ambiguity and MLL gene amplification., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2022 Takeda, Yokoyama, Fukuyama, Kawamata, Ito, Yusa, Kasajima, Shimizu, Ohno, Uchimaru, Yamaguchi, Imoto, Miyano and Tojo.)

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., (Copyright © 2021 The Authors. Published by Elsevier GmbH.. All rights reserved.)

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., (© 2021. The Author(s), under exclusive licence to The Japan Society of Human Genetics.)

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.

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., (© 2021 Japanese Society of Pathology and John Wiley & Sons Australia, Ltd.)

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., Competing Interests: Conflicts of Interest: All authors have completed the ICMJE uniform disclosure form (available at http://dx.doi.org/10.21037/tlcr-20-1158). Dr. SM reports other from Fujitsu Laboratories, Ltd., other from Liquid Mine, Ltd., outside the submitted work. The other authors have no conflicts of interest to declare., (2021 Translational Lung Cancer Research. All rights reserved.)

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., (© 2020 The Authors. Thoracic Cancer published by China Lung Oncology Group and John Wiley & Sons Australia, Ltd.)

Background: Pleomorphic carcinoma (PC) of the lung is a rare type of lung cancer with aggressive characteristics and a poor prognosis. Because it is rare, the molecular characteristics of PC remain unclear., Methods: A gene mutation analysis was performed using next-generation sequencing (NGS) in patients with PC of the lung who had undergone surgical resection., Results: A total of nine patients were enrolled in the study. All the patients were male and eight had a history of smoking. Eight tumors contained spindle cells and three contained giant cells. Mutations considered significant were found in eight of the nine patients: in TP53 in five patients, in MET in two patients, and in ALK, ERBB2, PIK3CA, APC, NF1, and CDKN2A in one patient each. No EGFR mutation was detected in our analysis. Co-mutations were detected in three patients: TP53 with MET and NF1, TP53 with ERBB2, and PIK3CA with CDKN2A., Conclusions: TP53 mutations were detected most frequently in PC of the lung with NGS analysis. Different co-mutations were seen in several specimens., Key Points: Significant findings of the study This study demonstrates that mutations in the TP53 gene are frequently found and co-mutations are sometimes found in pleomorphic carcinoma of the lung using genomic profiling analysis. What this study adds Our results will help to analogize the genetic characteristics and potential target of molecular-targeted agents of pleomorphic carcinoma of the lung., (© 2020 The Authors. Thoracic Cancer published by China Lung Oncology Group and John Wiley & Sons Australia, Ltd.)

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., Competing Interests: Conflict of interest statement: S Ikeda, T Kato, H Kenmotsu, S Iwasawa, and T Yamanaka received honoraria from Chugai Pharmaceutical. T Kato, H Kenmotsu, S Iwasawa, T Yamanaka, and H Okamoto received research funding from Chugai Pharmaceutical. T Ogura, T Iwasawa, R Kasajima, Y Miyagi, and T Misumi declare no potential conflicts of interest with any companies or organizations whose products or services might be discussed in this article., (© The Author(s), 2020.)

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.

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., (Published by Elsevier B.V.)

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., (© 2019 by The American Society of Hematology.)

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., (© 2018 by The American Society of Hematology.)

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., (© 2018 The Authors. Pathology International published by Japanese Society of Pathology and John Wiley & Sons Australia, Ltd.)

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.

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/mm 2 to 73.9/mm 2 and LMVD values from 0/mm 2 to 22.9/mm 2 . 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.

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.

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., (© 2016 Old City Publishing, Inc.)

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.