Machine Learning Prediction Model for Neutrophil Recovery after Unrelated Cord Blood Transplantation.

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.

Novel chimeric antigen receptor-expressing T cells targeting the malignant mesothelioma-specific antigen sialylated HEG1.

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.

Impact of RBM10 and PD-L1 expression on the prognosis of pathologic N1-N2 epidermal growth factor receptor mutant lung adenocarcinoma.

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.

KMT2C expression and DNA homologous recombination repair factors in lung cancers with a high-grade fetal adenocarcinoma component.

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.

Genetic analysis of low-grade adenosquamous carcinoma of the breast progressing to high-grade metaplastic carcinoma.

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.

Histological markers, sickle-shaped blood vessels, myxoid area, and infiltrating growth pattern help stratify the prognosis of patients with myxofibrosarcoma/undifferentiated sarcoma.

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.

Bone marrow adipocytes induce cancer-associated fibroblasts and immune evasion, enhancing invasion and drug resistance.

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.

Validation of EZH2 Inhibitor Efficiency in Anaplastic Thyroid Carcinoma Cell Lines.

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.

Differential diagnosis of uterine adenosarcoma: identification of JAZF1-BCORL1 rearrangement by comprehensive cancer genomic profiling.

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.

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).

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. 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.

Case report: Common clonal origin of concurrent langerhans cell histiocytosis and acute myeloid leukemia.

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.

Atezolizumab for Pretreated Non-Small Cell Lung Cancer with Idiopathic Interstitial Pneumonia: Final Analysis of Phase II AMBITIOUS Study.

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).

Clinical significance of plasma-free amino acids and tryptophan metabolites in patients with non-small cell lung cancer receiving PD-1 inhibitor: a pilot cohort study for developing a prognostic multivariate model.

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.

Repeated Lineage Switches in an Elderly Case of Refractory B-Cell Acute Lymphoblastic Leukemia With MLL Gene Amplification: A Case Report and Literature Review.

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.

Clinicopathological and genetic analyses of small cell neuroendocrine carcinoma of the prostate: Histological features for accurate diagnosis and toward future novel therapies.

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.

Possible Role of Cytochrome P450 1B1 in the Mechanism of Gemcitabine Resistance in Pancreatic Cancer.

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.

Clinicopathological and molecular characteristics of endometrial neuroendocrine carcinomas reveal preexisting endometrial carcinoma origin.

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.

Application of targeted nanopore sequencing for the screening and determination of structural variants in patients with Lynch syndrome.

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.

Comprehensive molecular analysis of genomic profiles and PD-L1 expression in lung adenocarcinoma with a high-grade fetal adenocarcinoma component.

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.

Lung adenocarcinoma in a patient with a cis EGFR L858R-K860I doublet mutation identified using NGS-based profiling test: Negative diagnosis on initial companion test and successful treatment with osimertinib.

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.