Comprehensive genetic profiling reveals frequent alterations of driver genes on the X chromosome in extranodal NK/T-cell lymphoma.

Extranodal NK/T-cell lymphoma (ENKTCL) is an Epstein-Barr virus (EBV)-related neoplasm with male dominance and a poor prognosis. A better understanding of the genetic alterations and their functional roles in ENKTCL could help improve patient stratification and treatments. Here, we performed comprehensive genetic analysis of 177 ENKTCL cases to delineate the landscape of mutations, copy number alterations (CNAs), and structural variations, identifying 34 driver genes including six previously unappreciated ones, namely HLA-B, HLA-C, ROBO1, CD58, POT1, and MAP2K1. Among them, CD274 (24%) was the most frequently altered, followed by TP53 (20%), CDKN2A (19%), ARID1A (15%), HLA-A (15%), BCOR (14%), and MSN (14%). Chromosome X (chrX) losses were the most common arm-level CNAs in females (~40%), and alterations of four X-linked driver genes (MSN, BCOR, DDX3X, and KDM6A) were more frequent in males and females harboring chrX losses. Among X-linked drivers, MSN was the most recurrently altered, and its expression was lost in approximately one-third of cases using immunohistochemical analysis. Functional studies of human cell lines demonstrated that MSN disruption promoted cell proliferation and NF-κB activation. Moreover, MSN inactivation increased sensitivity to NF-κB inhibition in vitro and in vivo. In addition, recurrent deletions were observed at the origin of replication in the EBV genome (6%). Finally, by integrating the 34 drivers and 19 significant arm-level CNAs, non-negative matrix factorization and consensus clustering identified two molecular groups with different genetic features and prognosis irrespective of clinical prognostic factors. Together, these findings could help improve diagnostic and therapeutic strategies in ENKTCL.

Pan-Cancer Comparative and Integrative Analyses of Driver Alterations Using Japanese and International Genomic Databases.

Using 48,627 samples from the Center for Cancer Genomics and Advanced Therapeutics (C-CAT), we present a pan-cancer landscape of driver alterations and their clinical actionability in Japanese patients. Comparison with White patients in Genomics Evidence Neoplasia Information Exchange (GENIE) demonstrates high TP53 mutation frequencies in Asian patients across multiple cancer types. Integration of C-CAT, GENIE, and The Cancer Genome Atlas data reveals many cooccurring and mutually exclusive relationships between driver mutations. At pathway level, mutations in epigenetic regulators frequently cooccur with PI3K pathway molecules. Furthermore, we found significant cooccurring mutations within the epigenetic pathway. Accumulation of mutations in epigenetic regulators causes increased proliferation-related transcriptomic signatures. Loss-of-function of many epigenetic drivers inhibits cell proliferation in their wild-type cell lines, but this effect is attenuated in those harboring mutations of not only the same but also different epigenetic drivers. Our analyses dissect various genetic properties and provide valuable resources for precision medicine in cancer. SIGNIFICANCE: We present a genetic landscape of 26 principal cancer types/subtypes, including Asian-prevalent ones, in Japanese patients. Multicohort data integration unveils numerous cooccurring and exclusive relationships between driver mutations, identifying cooccurrence of multiple mutations in epigenetic regulators, which coordinately cause transcriptional and phenotypic changes. These findings provide insights into epigenetic regulator-driven oncogenesis. This article is featured in Selected Articles from This Issue, p. 695.

Inter- and intra-tumor heterogeneity of genetic and immune profiles in inherited renal cell carcinoma.

Patients with von Hippel-Lindau disease (vHL) are at risk of developing spatially and temporally multiple clear cell renal cell carcinomas (ccRCCs), which offers a valuable opportunity to analyze inter- and intra-tumor heterogeneity of genetic and immune profiles within the same patient. Here, we perform whole-exome and RNA sequencing, digital gene expression, and immunohistochemical analyses for 81 samples from 51 ccRCCs of 10 patients with vHL. Inherited ccRCCs are clonally independent and have less genomic alterations than sporadic ccRCCs. Hierarchical clustering of transcriptome profiles shows two clusters with distinct immune signatures: immune hot and cold clusters. Interestingly, not only samples from the same tumors but also different tumors from the same patients tend to show a similar immune signature, whereas samples from different patients frequently exhibit different signatures. Our findings reveal the genetic and immune landscape of inherited ccRCCs, demonstrating the relevance of host factors in shaping anti-tumor immunity.

Precise characterization of somatic complex structural variations from tumor/control paired long-read sequencing data with nanomonsv.

We present our novel software, nanomonsv, for detecting somatic structural variations (SVs) using tumor and matched control long-read sequencing data with a single-base resolution. The current version of nanomonsv includes two detection modules, Canonical SV module, and Single breakend SV module. Using tumor/control paired long-read sequencing data from three cancer and their matched lymphoblastoid lines, we demonstrate that Canonical SV module can identify somatic SVs that can be captured by short-read technologies with higher precision and recall than existing methods. In addition, we have developed a workflow to classify mobile element insertions while elucidating their in-depth properties, such as 5' truncations, internal inversions, as well as source sites for 3' transductions. Furthermore, Single breakend SV module enables the detection of complex SVs that can only be identified by long-reads, such as SVs involving highly-repetitive centromeric sequences, and LINE1- and virus-mediated rearrangements. In summary, our approaches applied to cancer long-read sequencing data can reveal various features of somatic SVs and will lead to a better understanding of mutational processes and functional consequences of somatic SVs.

Integrated genetic and clinical prognostic factors for aggressive adult T-cell leukemia/lymphoma.

The prognosis of aggressive adult T-cell leukemia/lymphoma (ATL) is poor, and allogeneic hematopoietic stem cell transplantation (allo-HSCT) is a curative treatment. In order to identify favorable prognostic patients after intensive chemotherapy, and who therefore might not require upfront allo-HSCT, we aimed to improve risk stratification of aggressive ATL patients aged <70 years. The clinical risk factors and genetic mutations were incorporated into risk modeling for overall survival (OS). We generated the m7-ATLPI, a clinicogenetic risk model for OS, that included the ATL prognostic index (PI) (ATL-PI) risk category, and non-silent mutations in seven genes, namely TP53, IRF4, RHOA, PRKCB, CARD11, CCR7, and GATA3. In the training cohort of 99 patients, the m7-ATLPI identified a low-, intermediate-, and highrisk group with 2-year OS of 100%, 43%, and 19%, respectively (hazard ratio [HR] =5.46; P<0.0001). The m7-ATLPI achieved superior risk stratification compared to the current ATL-PI (C-index 0.92 vs. 0.85, respectively). In the validation cohort of 84 patients, the m7-ATLPI defined low-, intermediate-, and high-risk groups with a 2-year OS of 81%, 30%, and 0%, respectively (HR=2.33; P=0.0094), and the model again outperformed the ATL-PI (C-index 0.72 vs. 0.70, respectively). The simplified m7-ATLPI, which is easier to use in clinical practice, achieved superior risk stratification compared to the ATLPI, as did the original m7-ATLPI; the simplified version was calculated by summing the following: high-risk ATL-PI category (+10), low-risk ATL-PI category (-4), and non-silent mutations in TP53 (+4), IRF4 (+3), RHOA (+1), PRKCB (+1), CARD11 (+0.5), CCR7 (-2), and GATA3 (-3).

CARD11 mutation and HBZ expression induce lymphoproliferative disease and adult T-cell leukemia/lymphoma.

Adult T-cell leukemia/lymphoma (ATL) is caused by human T-cell leukemia virus type 1 (HTLV-1). In addition to HTLV-1 bZIP factor (HBZ), a leukemogenic antisense transcript of HTLV-1, abnormalities of genes involved in TCR-NF-κB signaling, such as CARD11, are detected in about 90% of patients. Utilizing mice expressing CD4+ T cell-specific CARD11(E626K) and/or CD4+ T cell-specific HBZ, namely CARD11(E626K)CD4-Cre mice, HBZ transgenic (Tg) mice, and CARD11(E626K)CD4-Cre;HBZ Tg double transgenic mice, we clarify these genes' pathogenetic effects. CARD11(E626K)CD4-Cre and HBZ Tg mice exhibit lymphocytic invasion to many organs, including the lungs, and double transgenic mice develop lymphoproliferative disease and increase CD4+ T cells in vivo. CARD11(E626K) and HBZ cooperatively activate the non-canonical NF-κB pathway, IRF4 targets, BATF3/IRF4/HBZ transcriptional network, MYC targets, and E2F targets. Most KEGG and HALLMARK gene sets enriched in acute-type ATL are also enriched in double transgenic mice, indicating that these genes cooperatively contribute to ATL development.

Feasibility and clinical utility of comprehensive genomic profiling of hematological malignancies.

Identification of genetic alterations through next-generation sequencing (NGS) can guide treatment decision-making by providing information on diagnosis, therapy selection, and prognostic stratification in patients with hematological malignancies. Although the utility of NGS-based genomic profiling assays was investigated in hematological malignancies, no assays sufficiently cover driver mutations, including recently discovered ones, as well as fusions and/or pathogenic germline variants. To address these issues, here we have devised an integrated DNA/RNA profiling assay to detect various types of somatic alterations and germline variants at once. Particularly, our assay can successfully identify copy number alterations and structural variations, including immunoglobulin heavy chain translocations, IKZF1 intragenic deletions, and rare fusions. Using this assay, we conducted a prospective study to investigate the feasibility and clinical usefulness of comprehensive genomic profiling for 452 recurrently altered genes in hematological malignancies. In total, 176 patients (with 188 specimens) were analyzed, in which at least one alteration was detected in 171 (97%) patients, with a median number of total alterations of 7 (0-55). Among them, 145 (82%), 86 (49%), and 102 (58%) patients harbored at least one clinically relevant alteration for diagnosis, treatment, and prognosis, respectively. The proportion of patients with clinically relevant alterations was the highest in acute myeloid leukemia, whereas this assay was less informative in T/natural killer-cell lymphoma. These results suggest the clinical utility of NGS-based genomic profiling, particularly for their diagnosis and prognostic prediction, thereby highlighting the promise of precision medicine in hematological malignancies.

Heterozygous Dnmt3a R878C induces expansion of quiescent hematopoietic stem cell pool.

Somatic mutation of DNMT3A (DNA methyltransferase 3 alpha) is implicated in the development of a wide range of hematological disorders, including clonal hematopoiesis of indeterminate potential. To elucidate the functional roles of endogenous levels of a DNMT3A R882 mutant, we generated a novel Dnmt3a R878C conditional knock-in mouse model. In contrast to viable heterozygotes, mice homozygous for the Dnmt3a R878C mutation in the hematopoietic system were not viable (Dnmt3a R878C is homologous to human DNMT3A R882C). Hematopoietic cell-specific heterozygous expression of Dnmt3a R878C led to significant expansion of adult quiescent hematopoietic stem cells (HSCs); however, these mice had no hematological malignancies. The expanding HSC population in heterozygous Dnmt3a R878C knock-in mice had an accumulation of G0-phase cells. In contrast to aberrantly enhanced self-renewal capacity in vitro, heterozygous Dnmt3a R878C knock-in HSCs had no competitive repopulating advantage in vivo over wild-type HSCs. Considering the capacity of the heterozygous Dnmt3a R878C mutant for HSC pool expansion, our Dnmt3a R878C knock-in mouse line is a useful platform on which to dissect the pathophysiology of clonal hematopoiesis. This mouse line can also help to elucidate the biological and molecular actions of DNMT3A mutations in the malignant transformation of normal HSCs.

Whole-genome landscape of adult T-cell leukemia/lymphoma.

Adult T-cell leukemia/lymphoma (ATL) is an aggressive neoplasm immunophenotypically resembling regulatory T cells, associated with human T-cell leukemia virus type-1. Here, we performed whole-genome sequencing (WGS) of 150 ATL cases to reveal the overarching landscape of genetic alterations in ATL. We discovered frequent (33%) loss-of-function alterations preferentially targeting the CIC long isoform, which were overlooked by previous exome-centric studies of various cancer types. Long but not short isoform-specific inactivation of Cic selectively increased CD4+CD25+Foxp3+ T cells in vivo. We also found recurrent (13%) 3'-truncations of REL, which induce transcriptional upregulation and generate gain-of-function proteins. More importantly, REL truncations are also common in diffuse large B-cell lymphoma, especially in germinal center B-cell-like subtype (12%). In the non-coding genome, we identified recurrent mutations in regulatory elements, particularly splice sites, of several driver genes. In addition, we characterized the different mutational processes operative in clustered hypermutation sites within and outside immunoglobulin/T-cell receptor genes and identified the mutational enrichment at the binding sites of host and viral transcription factors, suggesting their activities in ATL. By combining the analyses for coding and noncoding mutations, structural variations, and copy number alterations, we discovered 56 recurrently altered driver genes, including 11 novel ones. Finally, ATL cases were classified into 2 molecular groups with distinct clinical and genetic characteristics based on the driver alteration profile. Our findings not only help to improve diagnostic and therapeutic strategies in ATL, but also provide insights into T-cell biology and have implications for genome-wide cancer driver discovery.

Single-Cell Analysis of the Multicellular Ecosystem in Viral Carcinogenesis by HTLV-1.

Premalignant clonal expansion of human T-cell leukemia virus type-1 (HTLV-1)-infected cells occurs before viral carcinogenesis. Here we characterize premalignant cells and the multicellular ecosystem in HTLV-1 infection with and without adult T-cell leukemia/lymphoma (ATL) by genome sequencing and single-cell simultaneous transcriptome and T/B-cell receptor sequencing with surface protein analysis. We distinguish malignant phenotypes caused by HTLV-1 infection and leukemogenesis and dissect clonal evolution of malignant cells with different clinical behavior. Within HTLV-1-infected cells, a regulatory T-cell phenotype associates with premalignant clonal expansion. We also delineate differences between virus- and tumor-related changes in the nonmalignant hematopoietic pool, including tumor-specific myeloid propagation. In a newly generated conditional knockout mouse model recapitulating T-cell-restricted CD274 (encoding PD-L1) gene lesions found in ATL, we demonstrate that PD-L1 overexpressed by T cells is transferred to surrounding cells, leading to their PD-L1 upregulation. Our findings provide insights into clonal evolution and immune landscape of multistep virus carcinogenesis. SIGNIFICANCE: Our multimodal single-cell analyses comprehensively dissect the cellular and molecular alterations of the peripheral blood in HTLV-1 infection, with and without progression to leukemia. This study not only sheds light on premalignant clonal expansion in viral carcinogenesis, but also helps to devise novel diagnostic and therapeutic strategies for HTLV-1-related disorders.

Efficient production of human neutrophils from iPSCs that prevent murine lethal infection with immune cell recruitment.

Neutrophils play an essential role in innate immune responses to bacterial and fungal infections, and loss of neutrophil function can increase the risk of acquiring lethal infections in clinical settings. Here, we show that engineered neutrophil-primed progenitors derived from human induced pluripotent stem cells can produce functional neutrophil-like cells at a clinically applicable scale that can act rapidly in vivo against lethal bacterial infections. Using 5 different mouse models, we systematically demonstrated that these neutrophil-like cells migrate to sites of inflammation and infection and increase survival against bacterial infection. In addition, we found that these human neutrophil-like cells can recruit murine immune cells. This system potentially provides a straight-forward solution for patients with neutrophil deficiency: an off-the-shelf neutrophil transfusion. This platform should facilitate the administration of human neutrophils for a broad spectrum of physiological and pathological conditions.

Evi1 upregulates Fbp1 and supports progression of acute myeloid leukemia through pentose phosphate pathway activation.

Evi1 is a transcription factor essential for the development as well as progression of acute myeloid leukemia (AML) and high Evi1 AML is associated with extremely poor clinical outcome. Since targeting metabolic vulnerability is the emerging therapeutic strategy of cancer, we herein investigated a novel therapeutic target of Evi1 by analyzing transcriptomic, epigenetic, and metabolomic profiling of mouse high Evi1 leukemia cells. We revealed that Evi1 overexpression and Evi1-driven leukemic transformation upregulate transcription of gluconeogenesis enzyme Fbp1 and other pentose phosphate enzymes with interaction between Evi1 and the enhancer region of these genes. Metabolome analysis using Evi1-overexpressing leukemia cells uncovered pentose phosphate pathway upregulation by Evi1 overexpression. Suppression of Fbp1 as well as pentose phosphate pathway enzymes by shRNA-mediated knockdown selectively decreased Evi1-driven leukemogenesis in vitro. Moreover, pharmacological or shRNA-mediated Fbp1 inhibition in secondarily transplanted Evi1-overexpressing leukemia mouse significantly decreased leukemia cell burden. Collectively, targeting FBP1 is a promising therapeutic strategy of high Evi1 AML.

Loss-of-function mutations in BCOR contribute to chemotherapy resistance in acute myeloid leukemia.

Primary refractory acute myeloid leukemia (AML) is unresponsive to conventional chemotherapy and has a poor prognosis. Despite the recent identification of novel driver mutations and advances in the understanding of the molecular pathogenesis, little is known about the relationship between genetic abnormalities and chemoresistance in AML. In this study, we subjected 39 samples from patients with primary refractory AML to whole-exome and targeted sequencing analyses to identify somatic mutations contributing to chemoresistance in AML. First, we identified 49 genes that might contribute to chemotherapy resistance through the whole-exome sequencing of samples from 6 patients with primary refractory AML. We then identified a significantly higher frequency of mutations in the gene encoding BCL-6 co-repressor (BCOR) in patients with primary refractory AML through the targeted sequencing of all coding sequence of 49 genes. Notably, the presence of BCOR mutations appeared to have a negative impact on prognosis in our cohort and previous larger studies. Subsequently, to investigate the biological effect of BCOR mutations on sensitivity to anticancer drugs, we established BCOR knockout human leukemic cell lines using the CRISPR/Cas9 system. Here, BCOR knockout cell lines exhibited statistically significant reductions in sensitivity to anticancer drugs, compared with the wild-type controls both in vitro and in vivo in xenograft mouse models. In conclusion, loss-of-function BCOR mutations appear to contribute to chemotherapy resistance and may be a promising therapeutic target in primary refractory AML.

Multiple mutations within individual oncogenes.

Recent studies of the cancer genome have identified numerous patients harboring multiple mutations (MM) within individual oncogenes. These MM (de novo MM) in cis synergistically activate the mutated oncogene and promote tumorigenesis, indicating a positive epistatic interaction between mutations. The relatively frequent de novo MM suggest that intramolecular positive epistasis is widespread in oncogenes. Studies also suggest that negative and higher-order epistasis affects de novo MM. Comparison of de novo MM and MM associated with drug-resistant secondary mutations (secondary MM) revealed several similarities with respect to allelic configuration, mutational selection and functionality of individual mutations. Conversely, they have several differences, most notably the difference in drug sensitivities. Secondary MM usually confer resistance to molecularly targeted therapies, whereas several de novo MM are associated with increased sensitivity, implying that both can be useful as therapeutic biomarkers. Unlike secondary MM in which specific secondary resistant mutations are selected, minor (infrequent) functionally weak mutations are convergently selected in de novo MM, which may provide an explanation as to why such mutations accumulate in cancer. The third type of MM is MM from different subclones. This type of MM is associated with parallel evolution, which may contribute to relapse and treatment failure. Collectively, MM within individual oncogenes are diverse, but all types of MM are associated with cancer evolution and therapeutic response. Further evaluation of oncogenic MM is warranted to gain a deeper understanding of cancer genetics and evolution.

Clinical application of genomic aberrations in adult T-cell leukemia/lymphoma.

Adult T-cell leukemia/lymphoma (ATL) is an aggressive peripheral T-cell malignancy with a markedly poor prognosis. The low prevalence of ATL among human T-cell leukemia virus type-1 (HTLV-1) carriers and the long latency period before ATL onset suggest that additional genetic lesions are required for ATL leukemogenesis. Recently, a large-scale genetic analysis clarified the entire picture of genetic alterations, identified a number of novel driver genes, and delineated their characteristics. Frequent alterations are observed in the molecules belonging to T-cell receptor/NF-κB signaling and other T-cell-related pathways. A notable feature of the ATL genome is the predominance of gain-of-function alterations, including activating mutations in PLCG1, PRKCB, and CARD11. As many as one-fourth of all ATL cases harbor structural variations disrupting the 3'-untranslated region of the PD-L1 gene, leading to immune evasion of tumor cells. The frequency and pattern of these somatic alterations differ among clinical subtypes. Aggressive subtypes are associated with an increased burden of genetic alterations, and higher frequencies of TP53 and IRF4 mutations, PD-L1 amplifications, and CDKN2A deletions than indolent subtypes. In contrast, STAT3 mutations are more characteristic of indolent ATL. Furthermore, these subtypes are further classified into molecularly distinct subsets with a different prognosis by genetic alterations. We present an overview of the current understanding of somatic alterations in ATL, with specific focus on their utility in clinical settings. Furthermore, we highlight their genetic features by exploring their similarities and differences among peripheral T-cell lymphomas.

Landscape and function of multiple mutations within individual oncogenes.

Sporadic reports have described cancer cases in which multiple driver mutations (MMs) occur in the same oncogene1,2. However, the overall landscape and relevance of MMs remain elusive. Here we carried out a pan-cancer analysis of 60,954 cancer samples, and identified 14 pan-cancer and 6 cancer-type-specific oncogenes in which MMs occur more frequently than expected: 9% of samples with at least one mutation in these genes harboured MMs. In various oncogenes, MMs are preferentially present in cis and show markedly different mutational patterns compared with single mutations in terms of type (missense mutations versus in-frame indels), position and amino-acid substitution, suggesting a cis-acting effect on mutational selection. MMs show an overrepresentation of functionally weak, infrequent mutations, which confer enhanced oncogenicity in combination. Cells with MMs in the PIK3CA and NOTCH1 genes exhibit stronger dependencies on the mutated genes themselves, enhanced downstream signalling activation and/or greater sensitivity to inhibitory drugs than those with single mutations. Together oncogenic MMs are a relatively common driver event, providing the underlying mechanism for clonal selection of suboptimal mutations that are individually rare but collectively account for a substantial proportion of oncogenic mutations.

Nationwide epidemiological survey of familial myelodysplastic syndromes/acute myeloid leukemia in Japan: a multicenter retrospective study.

Although several pedigrees of familial myelodysplastic syndromes/acute myeloid leukemia (fMDS/AML) have been reported, the epidemiology and clinical features has been poorly understood. To explore the epidemiology of this entity, we performed a retrospective nationwide epidemiological survey in Japan using questionnaire sheets. The questionnaire was sent to 561 institutions or hospitals certified by Japanese Society of Hematology, unearthing the existence of 41 pedigrees of fMDS/AML. Among them, we obtained the clinical information of 31 patients in 20 pedigrees. The median age of the initial diagnosis was 51 years (range 9-88 years) and the WHO classification 2008 ranged from refractory anemia (RA) to AML. Focusing on the familial MDS patients, refractory anemia with excess blasts (RAEB)-2 was the largest group (27.3%). The median overall survival (OS) of fMDS and fAML in this study were 71.6 and 12.4 months, and the five-year OS were 61.3 and 50%, respectively.

TP53 and PTEN mutations were shared in concurrent germ cell tumor and acute megakaryoblastic leukemia.

BACKGROUND: The occurrence of a mediastinal germ cell tumor (GCT) and hematological malignancy in the same patient is very rare. Due to its rarity, there have been only two reports of the concurrent cases undergoing detailed genetic analysis with whole-exome sequencing (WES), and the possible clonal relationship between the both tumors remained not fully elucidated. METHODS: We performed whole-exome sequencing analysis of mediastinal GCT and acute myeloid leukemia (AML) samples obtained from one young Japanese male adult patient with concurrent both tumors, and investigated the possible clonal relationship between them. RESULTS: Sixteen somatic mutations were detected in the mediastinal GCT sample and 18 somatic mutations in the AML sample. Mutations in nine genes, including TP53 and PTEN both known as tumor suppressor genes, were shared in both tumors. CONCLUSIONS: All in our case and in the previous two cases with concurrent mediastinal GCT and AML undergoing with whole-exome sequencing analysis, TP53 and PTEN mutations were commonly shared in both tumors. These data not only suggest that these tumors share a common founding clone, but also indicate that associated mediastinal GCT and AML harboring TP53 and PTEN mutations represent a unique biological entity.