Genetic and transcriptomic analyses of diffuse large B-cell lymphoma patients with poor outcomes within two years of diagnosis.

Despite the improvements in clinical outcomes for DLBCL, a significant proportion of patients still face challenges with refractory/relapsed (R/R) disease after receiving first-line R-CHOP treatment. To further elucidate the underlying mechanism of R/R disease and to develop methods for identifying patients at risk of early disease progression, we integrated clinical, genetic and transcriptomic data derived from 2805 R-CHOP-treated patients from seven independent cohorts. Among these, 887 patients exhibited R/R disease within two years (poor outcome), and 1918 patients remained in remission at two years (good outcome). Our analysis identified four preferentially mutated genes (TP53, MYD88, SPEN, MYC) in the untreated (diagnostic) tumor samples from patients with poor outcomes. Furthermore, transcriptomic analysis revealed a distinct gene expression pattern linked to poor outcomes, affecting pathways involved in cell adhesion/migration, T-cell activation/regulation, PI3K, and NF-κB signaling. Moreover, we developed and validated a 24-gene expression score as an independent prognostic predictor for treatment outcomes. This score also demonstrated efficacy in further stratifying high-risk patients when integrated with existing genetic or cell-of-origin subtypes, including the unclassified cases in these models. Finally, based on these findings, we developed an online analysis tool ( https://lymphprog.serve.scilifelab.se/app/lymphprog ) that can be used for prognostic prediction for DLBCL patients.

Integrated multi-omics profiling to dissect the spatiotemporal evolution of metastatic hepatocellular carcinoma.

Comprehensive molecular analyses of metastatic hepatocellular carcinoma (HCC) are lacking. Here, we generate multi-omic profiling of 257 primary and 176 metastatic regions from 182 HCC patients. Primary tumors rich in hypoxia signatures facilitated polyclonal dissemination. Genomic divergence between primary and metastatic HCC is high, and early dissemination is prevalent. The remarkable neoantigen intratumor heterogeneity observed in metastases is associated with decreased T cell reactivity, resulting from disruptions to neoantigen presentation. We identify somatic copy number alterations as highly selected events driving metastasis. Subclones without Wnt mutations show a stronger selective advantage for metastasis than those with Wnt mutations and are characterized by a microenvironment rich in activated fibroblasts favoring a pro-metastatic phenotype. Finally, metastases without Wnt mutations exhibit higher enrichment of immunosuppressive B cells that mediate terminal exhaustion of CD8+ T cells via HLA-E:CD94-NKG2A checkpoint axis. Collectively, our results provide a multi-dimensional dissection of the complex evolutionary process of metastasis.

ADAR1-mediated RNA editing promotes B cell lymphomagenesis.

Diffuse large B cell lymphoma (DLBCL) is one of the most common types of aggressive lymphoid malignancies. Here, we explore the contribution of RNA editing to DLBCL pathogenesis. We observed that DNA mutations and RNA editing events are often mutually exclusive, suggesting that tumors can modulate pathway outcomes by altering sequences at either the genomic or the transcriptomic level. RNA editing targets transcripts within known disease-driving pathways such as apoptosis, p53 and NF-κB signaling, as well as the RIG-I-like pathway. In this context, we show that ADAR1-mediated editing within MAVS transcript positively correlates with MAVS protein expression levels, associating with increased interferon/NF-κB signaling and T cell exhaustion. Finally, using targeted RNA base editing tools to restore editing within MAVS 3'UTR in ADAR1-deficient cells, we demonstrate that editing is likely to be causal to an increase in downstream signaling in the absence of activation by canonical nucleic acid receptor sensing.

The dual role of CD70 in B-cell lymphomagenesis.

BACKGROUND: CD70 is a costimulatory molecule that is transiently expressed on a small set of activated lymphocytes and is involved in T-cell-mediated immunity. However, the role of CD70 in B-cell malignancies remains controversial. METHODS: We investigated the clinical relevance of CD70 genetic alterations and its protein expression in two diffuse large B-cell lymphoma (DLBCL) cohorts with different ethnic backgrounds. We also performed transcriptomic analysis to explore the role of CD70 alterations in tumour microenvironment. We further tested the blockade of CD70 in combination with PD-L1 inhibitor in a murine lymphoma model. RESULTS: We showed that CD70 genetic aberrations occurred more frequently in the Chinese DLBCL cohort (56/233, 24.0%) than in the Swedish cohort (9/84, 10.8%), especially in those with concomitant hepatitis B virus (HBV) infection. The CD70 genetic changes in DLBCL resulted in a reduction/loss of protein expression and/or CD27 binding, which might impair T cell priming and were independently associated with poor overall survival. Paradoxically, we observed that over-expression of CD70 protein was also associated with a poor treatment response, as well as an advanced disease stage and EBV infection. More exhausted CD8+ T cells were furthermore identified in CD70 high-expression DLBCLs. Finally, in a murine lymphoma model, we demonstrated that blocking the CD70/CD27 and/or PD1/PD-L1 interactions could reduce CD70+ lymphoma growth in vivo, by directly impairing the tumour cell proliferation and rescuing the exhausted T cells. CONCLUSIONS: Our findings suggest that CD70 can play a role in either tumour suppression or oncogenesis in DLBCL, likely via distinct immune evasion mechanisms, that is, impairing T cell priming or inducing T cell exhaustion. Characterisation of specific dysfunction of CD70 in DLBCL may thus provide opportunities for the development of novel targeted immuno-therapeutic strategies.

Geospatial Immune Heterogeneity Reflects the Diverse Tumor-Immune Interactions in Intrahepatic Cholangiocarcinoma.

Intrahepatic cholangiocarcinoma (iCCA) exhibits extensive intratumoral heterogeneity and an extremely high mortality rate. Here, we performed whole-exome sequencing, RNA sequencing, T-cell receptor (TCR) sequencing, and multiplexed immunofluorescence on 207 tumor regions from 45 patients with iCCA. Over half of iCCA displayed intratumoral heterogeneity of immune infiltration, and iCCA were classified into sparsely, heterogeneously, and highly infiltrated subgroups with distinct immunogenomic characteristics. Sparsely infiltrated tumors displayed active copy-number loss of clonal neoantigens, and heterogeneous immune infiltration played an important role in the subclonal evolution across tumor subregions. Highly infiltrated tumors were characterized by extensive immune activation and a similar TCR repertoire across tumor subregions, but counteracted with T-cell exhaustion and pervasive antigen presentation defects. Notably, FGFR2 mutations and fusions correlated with low mutation burden and reduced immune infiltration. Our work delineated the dynamic tumor-immune interactions and developed a robust classification system to divide patients with iCCA into high and low immune evasion groups with different prognoses. SIGNIFICANCE: This study elucidates the impact of spatial immune heterogeneity upon tumor evolution of iCCA and reveals distinct immune evasion mechanisms developed in different immune microenvironments, which can be exploited for the development of personalized immunotherapy strategies. This article is highlighted in the In This Issue feature, p. 2221.

Genomic characterization of lymphomas in patients with inborn errors of immunity.

Patients with inborn errors of immunity (IEI) have a higher risk of developing cancer, especially lymphoma. However, the molecular basis for IEI-related lymphoma is complex and remains elusive. Here, we perform an in-depth analysis of lymphoma genomes derived from 23 IEI patients. We identified and validated disease-causing or -associated germline mutations in 14 of 23 patients involving ATM, BACH2, BLM, CD70, G6PD, NBN, PIK3CD, PTEN, and TNFRSF13B. Furthermore, we profiled somatic mutations in the lymphoma genome and identified 8 genes that were mutated at a significantly higher level in IEI-associated diffuse large B-cell lymphomas (DLBCLs) than in non-IEI DLBCLs, such as BRCA2, NCOR1, KLF2, FAS, CCND3, and BRWD3. The latter, BRWD3, is furthermore preferentially mutated in tumors of a subgroup of activated phosphoinositide 3-kinase δ syndrome patients. We also identified 5 genomic mutational signatures, including 2 DNA repair deficiency-related signatures, in IEI-associated lymphomas and a strikingly high number of inter- and intrachromosomal structural variants in the tumor genome of a Bloom syndrome patient. In summary, our comprehensive genomic characterization of lymphomas derived from patients with rare genetic disorders expands our understanding of lymphomagenesis and provides new insights for targeted therapy.

RAD51B Harbors Germline Mutations Associated With Pancreatic Ductal Adenocarcinoma.

PURPOSE: Genetic alterations in many components of the homologous recombination, DNA damage response, and repair (HR-DDR) pathway are involved in the hereditary cancer syndromes, including familial pancreatic cancer. HR-DDR genes beyond BRCA1, BRCA2, ATM, and PALB2 may also mutate and confer the HR-DDR deficiency in pancreatic ductal adenocarcinoma (PDAC). METHODS: We conducted a study to examine the genetic alterations using a companion diagnostic 15-gene HR-DDR panel in PDACs. HR-DDR gene mutations were identified and characterized by whole-exome sequencing and whole-genome sequencing. Different HR-DDR gene mutations are associated with variable homologous recombination deficiency (HRD) scores. RESULTS: Eight of 50 PDACs with at least one HR-DDR gene mutation were identified. One tumor with BRCA2 mutations is associated with a high HRD score. However, another tumor with a CHEK2 mutation is associated with a zero HRD score. Notably, four of eight PDACs in this study harbor a RAD51B gene mutation. All four RAD51B gene mutations were germline mutations. However, currently, RAD51B is not the gene panel for germline tests. CONCLUSION: The finding in this study thus supports including RAD51B in the germline test of HR-DDR pathway genes.

In situ areal mass measurement of laser shock-loaded aluminum using point-projection x-ray backlighting.

X-ray backlighting is been widely used today in dynamic phenomena observation. By applying proper synchronizing techniques, the in-situ data of the intensity distribution of the fragments in laser-driven shock-loaded aluminum were obtained for a particular moment using x-ray backlighting imaging. The image resolution was better than 40 µm in this context by introducing a pinhole. In order to obtain the areal mass of the fragments, a set of reference Al step wedges with certain thicknesses was employed. Furthermore, a novel, to the best of our knowledge, calibration method is introduced to calibrate the x-ray intensity distribution. It was effective to decrease the non-uniformity influence of the x-ray intensity with this calibration method by simulating a light field. After calibration, the standard deviation of 30 regions of interest reduced to 4.17%. In consequence, the areal mass distribution of the fragments is well quantified. It should be noted that the uncertainty in the areal mass conversion mainly comes from the non-uniformity of the x-ray intensity distribution with about 5% and the measurement uncertainty of the step thicknesses with less than 10%.

Distinct clinical and genetic features of hepatitis B virus-associated follicular lymphoma in Chinese patients.

Hepatitis B virus (HBV) infection has been associated with an increased risk for B-cell lymphomas. We previously showed that 20% of diffuse large B-cell lymphoma (DLBCL) patients from China, an endemic area of HBV infection, have chronic HBV infection (surface antigen-positive, HBsAg+) and are characterized by distinct clinical and genetic features. Here, we showed that 24% of follicular lymphoma (FL) Chinese patients are HBsAg+. Compared with the HBsAg- FL patients, HBsAg+ patients are younger, have a higher histological grade at diagnosis, and have a higher incidence of disease progression within 24 months. Moreover, by sequencing the genomes of 109 FL tumors, we observed enhanced mutagenesis and distinct genetic profile in HBsAg+ FLs, with a unique set of preferentially mutated genes (TNFAIP3, FAS, HIST1H1C, KLF2, TP53, PIM1, TMSB4X, DUSP2, TAGAP, LYN, and SETD2) but lack of the hallmark of HBsAg- FLs (ie, IGH/BCL2 translocations and CREBBP mutations). Transcriptomic analyses further showed that HBsAg+ FLs displayed gene-expression signatures resembling the activated B-cell-like subtype of diffuse large B-cell lymphoma, involving IRF4-targeted genes and NF-κB/MYD88 signaling pathways. Finally, we identified an increased infiltration of CD8+ memory T cells, CD4+ Th1 cells, and M1 macrophages and higher T-cell exhaustion gene signature in HBsAg+ FL samples. Taken together, we present new genetic/epigenetic evidence that links chronic HBV infection to B-cell lymphomagenesis, and HBV-associated FL is likely to have a distinct cell-of-origin and represent as a separate subtype of FL. Targetable genetic/epigenetic alterations identified in tumors and their associated tumor microenvironment may provide potential novel therapeutic approaches for this subgroup of patients.

Genome-wide CRISPR screens reveal synthetic lethal interaction between CREBBP and EP300 in diffuse large B-cell lymphoma.

Diffuse large B-cell lymphoma (DLBCL) is the most common type of aggressive lymphoid malignancy and a highly heterogeneous disease. In this study, we performed whole-genome and transcriptome sequencing, and a genome-wide CRISPR-Cas9-knockout screen to study an activated B-cell-like DLBCL cell line (RC-K8). We identified a distinct pattern of genetic essentialities in RC-K8, including a dependency on CREBBP and MDM2. The dependency on CREBBP is associated with a balanced translocation involving EP300, which results in a truncated form of the protein that lacks the critical histone acetyltransferase (HAT) domain. The synthetic lethal interaction between CREBBP and EP300 genes, two frequently mutated epigenetic modulators in B-cell lymphoma, was further validated in the previously published CRISPR-Cas9 screens and inhibitor assays. Our study suggests that integration of the unbiased functional screen results with genomic and transcriptomic data can identify both common and unique druggable vulnerabilities in DLBCL and histone acetyltransferases inhibition could be a therapeutic option for CREBBP or EP300 mutated cases.

Genome-wide mutational signatures revealed distinct developmental paths for human B cell lymphomas.

Both somatic hypermutation (SHM) and class switch recombination (CSR) are initiated by activation-induced cytidine deaminase (AID). Dysregulation of these processes has been linked to B cell lymphomagenesis. Here we performed an in-depth analysis of diffuse large B cell lymphoma (DLBCL) and follicular lymphoma (FL) genomes. We characterized seven genomic mutational signatures, including two B cell tumor-specific signatures, one of which is novel and associated with aberrant SHM. We further identified two major mutational signatures (K1 and K2) of clustered mutations (kataegis) resulting from the activities of AID or error-prone DNA polymerase η, respectively. K1 was associated with the immunoglobulin (Ig) switch region mutations/translocations and the ABC subtype of DLBCL, whereas K2 was related to the Ig variable region mutations and the GCB subtype of DLBCL and FL. Similar patterns were also observed in chronic lymphocytic leukemia subtypes. Thus, alterations associated with aberrant CSR and SHM activities can be linked to distinct developmental paths for different subtypes of B cell lymphomas.

ADAR1-Dependent RNA Editing Promotes MET and iPSC Reprogramming by Alleviating ER Stress.

RNA editing of adenosine to inosine (A to I) is catalyzed by ADAR1 and dramatically alters the cellular transcriptome, although its functional roles in somatic cell reprogramming are largely unexplored. Here, we show that loss of ADAR1-mediated A-to-I editing disrupts mesenchymal-to-epithelial transition (MET) during induced pluripotent stem cell (iPSC) reprogramming and impedes acquisition of induced pluripotency. Using chemical and genetic approaches, we show that absence of ADAR1-dependent RNA editing induces aberrant innate immune responses through the double-stranded RNA (dsRNA) sensor MDA5, unleashing endoplasmic reticulum (ER) stress and hindering epithelial fate acquisition. We found that A-to-I editing impedes MDA5 sensing and sequestration of dsRNAs encoding membrane proteins, which promote ER homeostasis by activating the PERK-dependent unfolded protein response pathway to consequently facilitate MET. This study therefore establishes a critical role for ADAR1 and its A-to-I editing activity during cell fate transitions and delineates a key regulatory layer underlying MET to control efficient reprogramming.

Genomic basis for RNA alterations in cancer.

Transcript alterations often result from somatic changes in cancer genomes1. Various forms of RNA alterations have been described in cancer, including overexpression2, altered splicing3 and gene fusions4; however, it is difficult to attribute these to underlying genomic changes owing to heterogeneity among patients and tumour types, and the relatively small cohorts of patients for whom samples have been analysed by both transcriptome and whole-genome sequencing. Here we present, to our knowledge, the most comprehensive catalogue of cancer-associated gene alterations to date, obtained by characterizing tumour transcriptomes from 1,188 donors of the Pan-Cancer Analysis of Whole Genomes (PCAWG) Consortium of the International Cancer Genome Consortium (ICGC) and The Cancer Genome Atlas (TCGA)5. Using matched whole-genome sequencing data, we associated several categories of RNA alterations with germline and somatic DNA alterations, and identified probable genetic mechanisms. Somatic copy-number alterations were the major drivers of variations in total gene and allele-specific expression. We identified 649 associations of somatic single-nucleotide variants with gene expression in cis, of which 68.4% involved associations with flanking non-coding regions of the gene. We found 1,900 splicing alterations associated with somatic mutations, including the formation of exons within introns in proximity to Alu elements. In addition, 82% of gene fusions were associated with structural variants, including 75 of a new class, termed 'bridged' fusions, in which a third genomic location bridges two genes. We observed transcriptomic alteration signatures that differ between cancer types and have associations with variations in DNA mutational signatures. This compendium of RNA alterations in the genomic context provides a rich resource for identifying genes and mechanisms that are functionally implicated in cancer.

Heterogeneous immunogenomic features and distinct escape mechanisms in multifocal hepatocellular carcinoma.

BACKGROUND & AIMS: The presence of multifocal tumors, developed either from intrahepatic metastasis (IM) or multicentric occurrence (MO), is a distinct feature of hepatocellular carcinoma (HCC). Immunogenomic characterization of multifocal HCC is important for understanding immune escape in different lesions and developing immunotherapy. METHODS: We combined whole-exome/transcriptome sequencing, multiplex immunostaining, immunopeptidomes, T cell receptor (TCR) sequencing and bioinformatic analyses of 47 tumors from 15 patients with HCC and multifocal lesions. RESULTS: IM and MO demonstrated distinct clonal architecture, mutational spectrum and genetic susceptibility. The immune microenvironment also displayed spatiotemporal heterogeneity, such as less T cell and more M2 macrophage infiltration in IM and higher expression of inhibitory immune checkpoints in MO. Similar to mutational profiles, shared neoantigens and TCR repertoires among tumors from the same patients were abundant in IM but scarce in MO. Combining neoantigen prediction and immunopeptidomes identified T cell-specific neoepitopes and achieved a high verification rate in vitro. Immunoediting mainly occurred in MO but not IM, due to the relatively low immune infiltration. Loss of heterozygosity of human leukocyte antigen (HLA) alleles, identified in 17% of multifocal HCC, hampered the ability of major histocompatibility complex to present neoantigens, especially in IM. An integrated analysis of Immunoscore, immunoediting, TCR clonality and HLA loss of heterozygosity in each tumor could stratify patients into 2 groups based on whether they have a high or low risk of recurrence (p = 0.038). CONCLUSION: Our study comprehensively characterized the genetic structure, neoepitope landscape, T cell profile and immunoediting status that collectively shape tumor evolution and could be used to optimize personalized immunotherapies for multifocal HCC. LAY SUMMARY: Immunogenomic features of multifocal hepatocellular carcinoma (HCC) are important for understanding immune-escape mechanisms and developing more effective immunotherapy. Herein, comprehensive immunogenomic characterization showed that diverse genomic structures within multifocal HCC would leave footprints on the immune landscape. Only a few tumors were under the control of immunosurveillance, while others evaded the immune system through multiple mechanisms that led to poor prognosis. Our study revealed heterogeneous immunogenomic landscapes and immune-constrained tumor evolution, the understanding of which could be used to optimize personalized immunotherapies for multifocal HCC.

The genomic landscape of Epstein-Barr virus-associated pulmonary lymphoepithelioma-like carcinoma.

Pulmonary lymphoepithelioma-like carcinoma (LELC) is a rare and distinct subtype of primary lung cancer characterized by Epstein-Barr virus (EBV) infection. Herein, we reported the mutational landscape of pulmonary LELC using whole-exome sequencing, targeted deep sequencing and single-nucleotide polymorphism arrays. We identify a low degree of somatic mutation but widespread existence of copy number variations. We reveal predominant signature 2 mutations and frequent loss of type I interferon genes that are involved in the host-virus counteraction. Integrated analysis shows enrichment of genetic lesions affecting several critical pathways, including NF-κB, JAK/STAT, and cell cycle. Notably, multi-dimensional comparison unveils that pulmonary LELC resemble NPC but are clearly different from other lung cancers, natural killer/T-cell lymphoma or EBV-related gastric cancer in terms of genetic features. In all, our study illustrates a distinct genomic landscape of pulmonary LELC and provides a road map to facilitate genome-guided personalized treatment.

Studies on chain shuttling polymerization reaction of nonbridged half-titanocene and bis(phenoxy-imine) Zr binary catalyst system.

In this contribution, olefin block copolymers were produced via chain shuttling polymerization (CSP), using a new combination of catalysts and a chain shuttling agent (CSA) diethylzinc (ZnEt2). The binary catalyst system included nonbridged half-titanocene catalyst, Cp*TiCl2(O-2,6-iPr2C6H3) (Cat A) and bis(phenoxy-imine) zirconium, {η 2-1-[C(H)=NC6H11]-2-O-3-tBu-C6H3}2ZrCl2 (Cat B), as well as co-catalyst methylaluminoxane (MAO). In contrast to dual-catalyst system in the absence of CSA, the blocky structure was obtained in the presence of CSA and rationalized from rheological studies. The binary catalyst system could cause the CSP reaction to occur in the presence of CSA ZnEt2, which yielded broad distribution ethylene/1-octene copolymers (M w/M n: 35.86) containing block polymer chains with high M w. The presented dual-catalytic system was applied for the first time in CSP and has a potential to be extended to produce a library of olefin block copolymers that can be used as advanced additives for thermoplastics.

Characterization and validation of somatic mutation spectrum to reveal heterogeneity in gastric cancer by single cell sequencing.

Gastric cancer (GC) is a highly heterogeneous disease with multiple cellular types and poor prognosis. However, the cellular evolution and molecular basis of GC at the individual intra-tumor level has not been well demonstrated. We performed single-cell whole exome sequencing to detect somatic single-nucleotide variants (SNVs) and significantly mutated genes (SMGs) among 34 tumor cells and 9 normal cells from a patient with GC. The Complete Prediction for Protein Conformation (CPPC) approach directly predicting the folding conformation of the protein 3D structure with Protein Folding Shape Code, combined with functional experiments were used to confirm the characterization of mutated SMGs in GC cells. We identified 201 somatic SNVs, including 117 non-synonymous mutations in GC cells. Further analysis identified 24 significant mutated genes (SMGs) in single cells, for which a single amino acid change might affect protein conformation. Among them, two genes (CDC27 and FLG) that were mutated only in single cells but not in the corresponding tumor tissue, were recurrently present in another GC tissue cohort, and may play a potential role to promote carcinogenesis, as confirmed by functional characterization. Our findings showed a mutational landscape of GC at intra-tumor level for the first time and provided opportunities for understanding the heterogeneity and individualized target therapy for this disease.