Genetic and Functional Drivers of Diffuse Large B Cell Lymphoma.

Diffuse large B cell lymphoma (DLBCL) is the most common form of blood cancer and is characterized by a striking degree of genetic and clinical heterogeneity. This heterogeneity poses a major barrier to understanding the genetic basis of the disease and its response to therapy. Here, we performed an integrative analysis of whole-exome sequencing and transcriptome sequencing in a cohort of 1,001 DLBCL patients to comprehensively define the landscape of 150 genetic drivers of the disease. We characterized the functional impact of these genes using an unbiased CRISPR screen of DLBCL cell lines to define oncogenes that promote cell growth. A prognostic model comprising these genetic alterations outperformed current established methods: cell of origin, the International Prognostic Index comprising clinical variables, and dual MYC and BCL2 expression. These results comprehensively define the genetic drivers and their functional roles in DLBCL to identify new therapeutic opportunities in the disease.

Using mouse liver cancer models based on somatic genome editing to predict immune checkpoint inhibitor responses.

BACKGROUND & AIMS: Tyrosine kinase inhibitors (TKIs) and immune checkpoint inhibitors (ICIs) are the only two classes of FDA-approved drugs for individuals with advanced hepatocellular carcinoma (HCC). While TKIs confer only modest survival benefits, ICIs have been associated with remarkable outcomes but only in the minority of patients who respond. Understanding the mechanisms that determine the efficacy of ICIs in HCC will help to stratify patients likely to respond to ICIs. This study aims to elucidate how genetic composition and specific oncogenic pathways regulate the immune composition of HCC, which directly affects response to ICIs. METHODS: A collection of mouse HCCs with genotypes that closely simulate the genetic composition found in human HCCs were established using genome-editing approaches involving the delivery of transposon and CRISPR-Cas9 systems by hydrodynamic tail vein injection. Mouse HCC tumors were analyzed by RNA-sequencing while tumor-infiltrating T cells were analyzed by flow cytometry and single-cell RNA-sequencing. RESULTS: Based on the CD8+ T cell-infiltration level, we characterized tumors with different genotypes into cold and hot tumors. Anti-PD-1 treatment had no effect in cold tumors but was greatly effective in hot tumors. As proof-of-concept, a cold tumor (Trp53KO/MYCOE) and a hot tumor (Keap1KO/MYCOE) were further characterized. Tumor-infiltrating CD8+ T cells from Keap1KO/MYCOE HCCs expressed higher levels of proinflammatory chemokines and exhibited enrichment of a progenitor exhausted CD8+ T-cell phenotype compared to those in Trp53KO/MYCOE HCCs. The TKI sorafenib sensitized Trp53KO/MYCOE HCCs to anti-PD-1 treatment. CONCLUSION: Single anti-PD-1 treatment appears to be effective in HCCs with genetic mutations driving hot tumors, while combined anti-PD-1 and sorafenib treatment may be more appropriate in HCCs with genetic mutations driving cold tumors. IMPACT AND IMPLICATIONS: Genetic alterations of different driver genes in mouse liver cancers are associated with tumor-infiltrating CD8+ T cells and anti-PD-1 response. Mouse HCCs with different genetic compositions can be grouped into hot and cold tumors based on the level of tumor-infiltrating CD8+ T cells. This study provides proof-of-concept evidence to show that hot tumors are responsive to anti-PD-1 treatment while cold tumors are more suitable for combined treatment with anti-PD-1 and sorafenib. Our study might help to guide the design of patient stratification systems for single or combined treatments involving anti-PD-1.

Immune pathway upregulation and lower genomic instability distinguish EBV-positive nodal T/NK-cell lymphoma from ENKTL and PTCL-NOS.

Primary Epstein-Barr virus (EBV)-positive nodal T/NK-cell lymphoma (PTCL-EBV) is a poorly understood disease which shows features resembling extranodal NK/T-cell lymphoma (ENKTL) and is currently not recognized as a distinct entity but categorized as a variant of primary T-cell lymphoma not otherwise specified (PTCL-NOS). Herein, we analyzed copynumber aberrations (n=77) with a focus on global measures of genomic instability and homologous recombination deficiency and performed gene expression (n=84) and EBV miRNA expression (n=24) profiling as well as targeted mutational analysis (n=16) to further characterize PTCL-EBV in relation to ENKTL and PTCL-NOS. Multivariate analysis revealed that patients with PTCL-EBV had a significantly worse outcome compared to patients with PTCL-NOS (P=0.002) but not to those with ENKTL. Remarkably, PTCL-EBV exhibited significantly lower genomic instability and homologous recombination deficiency scores compared to ENKTL and PTCL-NOS. Gene set enrichment analysis revealed that many immune-related pathways, interferon α/γ response, and IL6_JAK_STAT3 signaling were significantly upregulated in PTCLEBV and correlated with lower genomic instability scores. We also identified that NFκB-associated genes, BIRC3, NFKB1 (P50) and CD27, and their proteins are upregulated in PTCL-EBV. Most PTCL-EBV demonstrated a type 2 EBV latency pattern and, strikingly, exhibited downregulated expression of most EBV miRNA compared to ENKTL and their target genes were also enriched in immune-related pathways. PTCL-EBV also showed frequent mutations of TET2, PIK3CD and STAT3, and are characterized by microsatellite stability. Overall, poor outcome, low genomic instability, upregulation of immune pathways and downregulation of EBV miRNA are distinctive features of PTCL-EBV. Our data support the concept that PTCL-EBV could be considered as a distinct entity, provide novel insights into the pathogenesis of the disease and offer potential new therapeutic targets for this tumor.

Spectrum of B-cell neoplasms associated with immunoglobulin G4-related disease.

Immunoglobulin G4-related disease (IgG4-RD) has rarely been associated with lymphoid neoplasms, the spectrum of which remains unclear. B-cell lymphoid neoplasms (LN) associated with IgG4-RD diagnosed in a 4-year period were analysed. There were five men and three women at a median age of 76.5 (52-90) years; three with synchronous IgG4-RD and LN; three with IgG4-RD preceding LN by 2, 3, and 22 years; and two with LN preceding IgG4-RD by 2.5 and 7 years. All patients presented with disseminated lymphadenopathy. Monoclonal gammopathy of undetermined significance (MGUS)/smouldering multiple myeloma (SMM) was found in three patients, all with an IgGκ paraprotein. Levels of IgGκ and IgG4 correlated. Diffuse large B-cell lymphoma (DLBCL) was found in three patients, with one case showing co-existing lymphoma and IgG4-RD in the same lymph node biopsy. The remaining two cases were marginal zone lymphoma (MZL) developing in a lacrimal gland previously involved by IgG4-RD; and nodular lymphocyte predominant Hodgkin lymphoma (NLP-HL) diagnosed in a lymph node with concomitant IgG4-RD. Low-dose continuous prednisolone was given for MGUS/SMM, with both monoclonal IgGκ and IgG4 responding. Combination chemotherapy was given for DLBCL, with two patients achieving complete response and one patient dying from refractory lymphoma. The patient with MZL refused treatment, whereas the case of NLP-HL responded completely to chemotherapy. Our findings together with previous observations suggest that IgG4-RD has an increased risk of B-cell neoplasms. Patients with IgG4-RD presenting with lymphadenopathy require vigorous investigations to exclude lymphoid neoplasms.

The mutational landscape of Burkitt-like lymphoma with 11q aberration is distinct from that of Burkitt lymphoma.

The new recently described provisional lymphoma category Burkitt-like lymphoma with 11q aberration comprises cases similar to Burkitt lymphoma (BL) on morphological, immunophenotypic and gene-expression levels but lacking the IG-MYC translocation. They are characterized by a peculiar imbalance pattern on chromosome 11, but the landscape of mutations is not yet described. Thus, we investigated 15 MYC-negative Burkitt-like lymphoma with 11q aberration (mnBLL,11q,) cases by copy-number analysis and whole-exome sequencing. We refined the regions of 11q imbalance and identified the INO80 complex-associated gene NFRKB as a positional candidate in 11q24.3. Next to recurrent gains in 12q13.11-q24.32 and 7q34-qter as well as losses in 13q32.3-q34, we identified 47 genes recurrently affected by protein-changing mutations (each ≥3 of 15 cases). Strikingly, we did not detect recurrent mutations in genes of the ID3-TCF3 axis or the SWI/SNF complex that are frequently altered in BL, or in genes frequently mutated in germinal center-derived B-cell lymphomas like KMT2D or CREBBP An exception is GNA13, which was mutated in 7 of 15 cases. We conclude that the genomic landscape of mnBLL,11q, differs from that of BL both at the chromosomal and mutational levels. Our findings implicate that mnBLL,11q, is a lymphoma category distinct from BL at the molecular level.

The whole-genome landscape of Burkitt lymphoma subtypes.

Burkitt lymphoma (BL) is an aggressive, MYC-driven lymphoma comprising 3 distinct clinical subtypes: sporadic BLs that occur worldwide, endemic BLs that occur predominantly in sub-Saharan Africa, and immunodeficiency-associated BLs that occur primarily in the setting of HIV. In this study, we comprehensively delineated the genomic basis of BL through whole-genome sequencing (WGS) of 101 tumors representing all 3 subtypes of BL to identify 72 driver genes. These data were additionally informed by CRISPR screens in BL cell lines to functionally annotate the role of oncogenic drivers. Nearly every driver gene was found to have both coding and non-coding mutations, highlighting the importance of WGS for identifying driver events. Our data implicate coding and non-coding mutations in IGLL5, BACH2, SIN3A, and DNMT1. Epstein-Barr virus (EBV) infection was associated with higher mutation load, with type 1 EBV showing a higher mutational burden than type 2 EBV. Although sporadic and immunodeficiency-associated BLs had similar genetic profiles, endemic BLs manifested more frequent mutations in BCL7A and BCL6 and fewer genetic alterations in DNMT1, SNTB2, and CTCF. Silencing mutations in ID3 were a common feature of all 3 subtypes of BL. In vitro, mass spectrometry-based proteomics demonstrated that the ID3 protein binds primarily to TCF3 and TCF4. In vivo knockout of ID3 potentiated the effects of MYC, leading to rapid tumorigenesis and tumor phenotypes consistent with those observed in the human disease.

Molecular features of non-anaplastic peripheral T-cell lymphoma in children and adolescents.

Non-anaplasticperipheral T-cell lymphomas (PTCL) are rare tumors in children, adolescents, and young adults (CAYA) with poor prognosis and scarce genetic data. We analyzed lymphoma tissue from 36 patients up to 18 years old with PTCL, not otherwise specified (PTCL-NOS), hepatosplenic T-cell lymphoma, Epstein-Barr virus (EBV)-positive T-lymphoproliferative diseases, subcutaneous panniculitis-like T-cell lymphoma, and other PTCL types. Twenty-three patients (64%) had at least one genetic variant detectable, including TET2, KMT2C, PIK3D, and DMNT3A. TP53 and RHOA variants, commonly found in adults, were not identified. Eight of 20 (40%) CAYA PTCL-NOS had no detectable mutations. The genetic findings suggest that CAYA PTCL differ from adult cases.

Early Development of Colonic Adenocarcinoma With Minimal Polyposis in a Young Child With Metastatic Hepatoblastoma and Germline APC Mutation.

Germline adenomatous polyposis coli (APC) gene mutation is a cancer-predisposing condition commonly presenting as familial adenomatous polyposis. We describe a patient first diagnosed at the age of 3 years with metastatic hepatoblastoma. With a positive family history, germline testing confirmed maternally inherited APC mutation (p.Thr899Ansfs*13). The patient was subsequently diagnosed at 8 years with colonic adenocarcinoma in the absence of macroscopic polyposis. Total colectomy with adjuvant chemotherapy was delivered and the patient remained disease-free for 5 years since the second diagnosis. This report demonstrates the importance of considering germline APC mutation in children with hepatoblastoma, who may benefit from the early institution of colonoscopic surveillance.

Experience with provisional WHO-entities large B-cell lymphoma with IRF4-rearrangement and Burkitt-like lymphoma with 11q aberration in paediatric patients of the NHL-BFM group.

Large B-cell lymphoma with IRF4 rearrangement, and Burkitt-like lymphoma with 11q aberration are two provisional lymphoma entities in the 2017 revision of the WHO classification of lymphoid neoplasms. Despite being more frequent in young patients, knowledge regarding their true incidence and clinical features in unselected cohorts of paediatric and adolescent patients is limited. We screened for both entities among paediatric patients (<18 years of age) in the German NHL-BFM (Non-Hodgkin lymphoma Berlin-Frankfurt-Münster) group. Among follicular lymphomas and diffuse large B-cell lymphomas (DLBCL), 7/34 cases (21%) showed an IRF4 break-apart pattern by fluorescence in situ hybridisation (FISH) and are associated with stages I and II disease (P = 0·043). Among lymphomas morphologically resembling Burkitt lymphoma, DLBCL and high-grade B-cell lymphoma, unclassifiable, 13/102 cases (13%) lacked a MYC break-apart pattern but were positive for 11q proximal gain and telomeric loss by FISH. MYC-negative Burkitt-like lymphomas with the typical 11q gain-loss pattern by FISH were older (P = 0·004), showed less male predominance (P = 0·003), lower stage (P = 0·040), lower serum LDH level (P = 0·01) and less abdominal involvement (P = 0·008) compared to high grade B-cell lymphomas without 11q gain-loss pattern. Both entities showed excellent outcome with overall survival of 100% when managed according to NHL-BFM strategies and may provide candidates for future therapy de-escalation in clinical trials.

Middle East respiratory syndrome coronavirus and bat coronavirus HKU9 both can utilize GRP78 for attachment onto host cells.

Coronavirus tropism is predominantly determined by the interaction between coronavirus spikes and the host receptors. In this regard, coronaviruses have evolved a complicated receptor-recognition system through their spike proteins. Spikes from highly related coronaviruses can recognize distinct receptors, whereas spikes of distant coronaviruses can employ the same cell-surface molecule for entry. Moreover, coronavirus spikes can recognize a broad range of cell-surface molecules in addition to the receptors and thereby can augment coronavirus attachment or entry. The receptor of Middle East respiratory syndrome coronavirus (MERS-CoV) is dipeptidyl peptidase 4 (DPP4). In this study, we identified membrane-associated 78-kDa glucose-regulated protein (GRP78) as an additional binding target of the MERS-CoV spike. Further analyses indicated that GRP78 could not independently render nonpermissive cells susceptible to MERS-CoV infection but could facilitate MERS-CoV entry into permissive cells by augmenting virus attachment. More importantly, by exploring potential interactions between GRP78 and spikes of other coronaviruses, we discovered that the highly conserved human GRP78 could interact with the spike protein of bat coronavirus HKU9 (bCoV-HKU9) and facilitate its attachment to the host cell surface. Taken together, our study has identified GRP78 as a host factor that can interact with the spike proteins of two Betacoronaviruses, the lineage C MERS-CoV and the lineage D bCoV-HKU9. The capacity of GRP78 to facilitate surface attachment of both a human coronavirus and a phylogenetically related bat coronavirus exemplifies the need for continuous surveillance of the evolution of animal coronaviruses to monitor their potential for human adaptations.

Discovery and Sequence Analysis of Four Deltacoronaviruses from Birds in the Middle East Reveal Interspecies Jumping with Recombination as a Potential Mechanism for Avian-to-Avian and Avian-to-Mammalian Transmission.

The emergence of Middle East respiratory syndrome showed once again that coronaviruses (CoVs) in animals are potential source for epidemics in humans. To explore the diversity of deltacoronaviruses in animals in the Middle East, we tested fecal samples from 1,356 mammals and birds in Dubai, The United Arab Emirates. Four novel deltacoronaviruses were detected from eight birds of four species by reverse transcription-PCR (RT-PCR): FalCoV UAE-HKU27 from a falcon, HouCoV UAE-HKU28 from a houbara bustard, PiCoV UAE-HKU29 from a pigeon, and QuaCoV UAE-HKU30 from five quails. Complete genome sequencing showed that FalCoV UAE-HKU27, HouCoV UAE-HKU28, and PiCoV UAE-HKU29 belong to the same CoV species, suggesting recent interspecies transmission between falcons and their prey, houbara bustards and pigeons, possibly along the food chain. Western blotting detected specific anti-FalCoV UAE-HKU27 antibodies in 33 (75%) of 44 falcon serum samples, supporting genuine infection in falcons after virus acquisition. QuaCoV UAE-HKU30 belongs to the same CoV species as porcine coronavirus HKU15 (PorCoV HKU15) and sparrow coronavirus HKU17 (SpCoV HKU17), discovered previously from swine and tree sparrows, respectively, supporting avian-to-swine transmission. Recombination involving the spike protein is common among deltacoronaviruses, which may facilitate cross-species transmission. FalCoV UAE-HKU27, HouCoV UAE-HKU28, and PiCoV UAE-HKU29 originated from recombination between white-eye coronavirus HKU16 (WECoV HKU16) and magpie robin coronavirus HKU18 (MRCoV HKU18), QuaCoV UAE-HKU30 from recombination between PorCoV HKU15/SpCoV HKU17 and munia coronavirus HKU13 (MunCoV HKU13), and PorCoV HKU15 from recombination between SpCoV HKU17 and bulbul coronavirus HKU11 (BuCoV HKU11). Birds in the Middle East are hosts for diverse deltacoronaviruses with potential for interspecies transmission.IMPORTANCE During an attempt to explore the diversity of deltacoronaviruses among mammals and birds in Dubai, four novel deltacoronaviruses were detected in fecal samples from eight birds of four different species: FalCoV UAE-HKU27 from a falcon, HouCoV UAE-HKU28 from a houbara bustard, PiCoV UAE-HKU29 from a pigeon, and QuaCoV UAE-HKU30 from five quails. Genome analysis revealed evidence of recent interspecies transmission between falcons and their prey, houbara bustards and pigeons, possibly along the food chain, as well as avian-to-swine transmission. Recombination, which is known to occur frequently in some coronaviruses, was also common among these deltacoronaviruses and occurred predominantly at the spike region. Such recombination, involving the receptor binding protein, may contribute to the emergence of new viruses capable of infecting new hosts. Birds in the Middle East are hosts for diverse deltacoronaviruses with potential for interspecies transmission.

PD1 blockade with pembrolizumab is highly effective in relapsed or refractory NK/T-cell lymphoma failing l-asparaginase.

Natural killer (NK)/T-cell lymphomas failing L-asparaginse regimens have no known salvage and are almost invariably fatal. Seven male patients with NK/T-cell lymphoma (median age, 49 years; range, 31-68 years) for whom a median of 2 (range, 1-5) regimens (including l-asparaginase regimens and allogeneic hematopoietic stem-cell transplantation [HSCT] in 2 cases) failed were treated with the anti-programmed death 1 (PD1) antibody pembrolizumab. All patients responded, according to various clinical, radiologic (positron emission tomography), morphologic, and molecular (circulating Epstein-Barr virus [EBV] DNA) criteria. Two patients achieved complete response (CR) in all parameters. Three patients achieved clinical and radiologic CRs, with two having molecular remission (undetectable EBV DNA) but minimal EBV-encoded RNA-positive cells in lesions comprising predominantly CD3+CD4+ and CD3+CD8+ T cells (which ultimately disappeared, suggesting they represented pseudoprogression) and one having detectable EBV DNA despite morphologic CR. Two patients achieved partial response (PR). After a median of 7 (range, 2-13) cycles of pembrolizumab and a follow-up of a median of 6 (range, 2-10) months, all five CR patients were still in remission. The only adverse event was grade 2 skin graft-versus-host disease in one patient with previous allogeneic HSCT. Expression of the PD1 ligand was strong in 4 patients (3 achieving CR) and weak in 1 (achieving PR). PD1 blockade with pembrolizumab was a potent strategy for NK/T-cell lymphomas failing l-asparaginase regimens.

Rat Hepatitis E Virus as Cause of Persistent Hepatitis after Liver Transplant.

All hepatitis E virus (HEV) variants reported to infect humans belong to the species Orthohepevirus A (HEV-A). The zoonotic potential of the species Orthohepevirus C (HEV-C), which circulates in rats and is highly divergent from HEV-A, is unknown. We report a liver transplant recipient with hepatitis caused by HEV-C infection. We detected HEV-C RNA in multiple clinical samples and HEV-C antigen in the liver. The complete genome of the HEV-C isolate had 93.7% nt similarity to an HEV-C strain from Vietnam. The patient had preexisting HEV antibodies, which were not protective against HEV-C infection. Ribavirin was an effective treatment, resulting in resolution of hepatitis and clearance of HEV-C viremia. Testing for this zoonotic virus should be performed for immunocompromised and immunocompetent patients with unexplained hepatitis because routine hepatitis E diagnostic tests may miss HEV-C infection. HEV-C is also a potential threat to the blood product supply.

Mp1p homologues as virulence factors in Aspergillus fumigatus.

Recently, we showed that Mp1p is an important virulence factor of Talaromyces marneffei, a dimorphic fungus phylogenetically closely related to Aspergillus fumigatus. In this study, we investigated the virulence properties of the four Mp1p homologues (Afmp1p, Afmp2p, Afmp3p, and Afmp4p) in A. fumigatus using a mouse model. All mice died 7 days after challenge with wild-type A. fumigatus QC5096, AFMP1 knockdown mutant, AFMP2 knockdown mutant and AFMP3 knockdown mutant and 28 days after challenge with AFMP4 knockdown mutant (P<.0001). Only 11% of mice died 30 days after challenge with AFMP1-4 knockdown mutant (P<.0001). For mice challenge with AFMP1-4 knockdown mutant, lower abundance of fungal elements was observed in brains, kidneys, and spleens compared to mice challenge with QC5096 at day 4 post-infection. Fungal counts in brains of mice challenge with QC5096 or AFMP4 knockdown mutant were significantly higher than those challenge with AFMP1-4 knockdown mutant (P<.01 and P<.05). Fungal counts in kidneys of mice challenge with QC5096 or AFMP4 knockdown mutant were significantly higher than those challenge with AFMP1-4 knockdown mutant (P<.001 and P<.001) and those of mice challenge with QC5096 were significantly higher than those challenge with AFMP4 knockdown mutant (P<.05). There is no difference among the survival rates of wild-type A. fumigatus, AFMP4 knockdown mutant and AFMP1-4 knockdown mutant, suggesting that Mp1p homologues in A. fumigatus do not mediate its virulence via improving its survival in macrophage as in the case in T. marneffei. Afmp1p, Afmp2p, Afmp3p, and Afmp4p in combination are important virulence factors of A. fumigatus.

Receptor-type tyrosine-protein phosphatase κ directly targets STAT3 activation for tumor suppression in nasal NK/T-cell lymphoma.

Nasal-type natural killer/T-cell lymphoma (NKTCL) is an aggressive disease characterized by frequent deletions on 6q, and constitutive activation of signal transducer and activator of transcription 3 (STAT3). Phosphorylation at Tyr705 activates STAT3, inducing dimerization, nuclear translocation, and DNA binding. In this study, we investigated whether receptor-type tyrosine-protein phosphatase κ (PTPRK), the only protein tyrosine phosphatase at 6q that contains a STAT3-specifying motif, negatively regulates STAT3 activation in NKTCL. PTPRK was highly expressed in normal NK cells but was underexpressed in 4 of 5 (80%) NKTCL cell lines and 15 of 27 (55.6%) primary tumors. Significantly, PTPRK protein expression was inversely correlated with nuclear phospho-STAT3(Tyr705) expression in NKTCL cell lines (P = .025) and tumors (P = .040). PTPRK restoration decreased nuclear phospho-STAT3(Tyr705) levels, whereas knockdown of PTPRK increased such levels in NKTCL cells. Phosphatase substrate-trapping mutant assays demonstrated the binding of PTPRK to STAT3, and phosphatase assays showed that PTPRK directly dephosphorylated phospho-STAT3(Tyr705). Restoration of PTPRK inhibited tumor cell growth and reduced the migration and invasion ability of NKTCL cells. Monoallelic deletion and promoter hypermethylation caused underexpression of PTPRK messenger RNA in NKTCL, and methylation of the PTPRK promoter significantly correlated with inferior overall survival (P = .049) in NKTCL patients treated with the steroid-dexamethasone, methotrexate, ifosfamide, l-asparaginase, and etoposide regimen. Altogether, our findings show that PTPRK underexpression leads to STAT3 activation and contributes to NKTCL pathogenesis.