Multi-omic profiling of follicular lymphoma reveals changes in tissue architecture and enhanced stromal remodeling in high-risk patients.

Follicular lymphoma (FL) is a generally incurable malignancy that evolves from developmentally blocked germinal center (GC) B cells. To promote survival and immune escape, tumor B cells undergo significant genetic changes and extensively remodel the lymphoid microenvironment. Dynamic interactions between tumor B cells and the tumor microenvironment (TME) are hypothesized to contribute to the broad spectrum of clinical behaviors observed among FL patients. Despite the urgent need, existing clinical tools do not reliably predict disease behavior. Using a multi-modal strategy, we examined cell-intrinsic and -extrinsic factors governing progression and therapeutic outcomes in FL patients enrolled onto a prospective clinical trial. By leveraging the strengths of each platform, we identify several tumor-specific features and microenvironmental patterns enriched in individuals who experience early relapse, the most high-risk FL patients. These features include stromal desmoplasia and changes to the follicular growth pattern present 20 months before first progression and first relapse.

Procrustes is a machine-learning approach that removes cross-platform batch effects from clinical RNA sequencing data.

With the increased use of gene expression profiling for personalized oncology, optimized RNA sequencing (RNA-seq) protocols and algorithms are necessary to provide comparable expression measurements between exome capture (EC)-based and poly-A RNA-seq. Here, we developed and optimized an EC-based protocol for processing formalin-fixed, paraffin-embedded samples and a machine-learning algorithm, Procrustes, to overcome batch effects across RNA-seq data obtained using different sample preparation protocols like EC-based or poly-A RNA-seq protocols. Applying Procrustes to samples processed using EC and poly-A RNA-seq protocols showed the expression of 61% of genes (N = 20,062) to correlate across both protocols (concordance correlation coefficient > 0.8, versus 26% before transformation by Procrustes), including 84% of cancer-specific and cancer microenvironment-related genes (versus 36% before applying Procrustes; N = 1,438). Benchmarking analyses also showed Procrustes to outperform other batch correction methods. Finally, we showed that Procrustes can project RNA-seq data for a single sample to a larger cohort of RNA-seq data. Future application of Procrustes will enable direct gene expression analysis for single tumor samples to support gene expression-based treatment decisions.

Integrative genomic analysis identifies unique immune environments associated with immunotherapy response in diffuse large B cell lymphoma.

Most diffuse large B-cell lymphoma (DLBCL) patients treated with bispecific antibodies (BsAb) or chimeric antigen receptor (CAR) T cells fail to achieve durable treatment responses, underscoring the need for a deeper understanding of mechanisms that regulate the immune environment and response to treatment. Here, an integrative, multi-omic approach was employed to characterize DLBCL immune environments, which effectively segregated DLBCLs into four quadrants - termed DLBCL-immune quadrants (IQ) - defined by cell-of-origin and immune-related gene set expression scores. Recurrent genomic alterations were enriched in each IQ, suggesting that lymphoma cell-intrinsic alterations contribute to orchestrating unique DLBCL immune environments. In relapsed/refractory DLBCL patients, DLBCL-IQ assignment correlated significantly with clinical benefit with the CD20 x CD3 BsAb, mosunetuzumab, but not with CD19-directed CAR T cells. DLBCL-IQ provides a new framework to conceptualize the DLBCL immune landscape and uncovers the differential impact of the endogenous immune environment on outcomes to BsAb and CAR T cell treatment.

CD5 Gene Signature Identifies Diffuse Large B-Cell Lymphomas Sensitive to Bruton's Tyrosine Kinase Inhibition.

PURPOSE: A genetic classifier termed LymphGen accurately identifies diffuse large B-cell lymphoma (DLBCL) subtypes vulnerable to Bruton's tyrosine kinase inhibitors (BTKis), but is challenging to implement in the clinic and fails to capture all DLBCLs that benefit from BTKi-based therapy. Here, we developed a novel CD5 gene expression signature as a biomarker of response to BTKi-based therapy in DLBCL. METHODS: CD5 immunohistochemistry (IHC) was performed on 404 DLBCLs to identify CD5 IHC+ and CD5 IHC- cases, which were subsequently characterized at the molecular level through mutational and transcriptional analyses. A 60-gene CD5 gene expression signature (CD5sig) was constructed using genes differentially expressed between CD5 IHC+ and CD5 IHC- non-germinal center B-cell-like (non-GCB DLBCL) DLBCLs. This CD5sig was applied to external DLBCL data sets, including pretreatment biopsies from patients enrolled in the PHOENIX study (n = 584) to define the extent to which the CD5sig could identify non-GCB DLBCLs that benefited from the addition of ibrutinib to rituximab, cyclophosphamide, doxorubicin, vincristine, and prednisone (R-CHOP). RESULTS: CD5 expression was observed in 12% of non-GCB DLBCLs. CD5+ DLBCLs displayed transcriptional features of B-cell receptor (BCR) activation and were enriched for BCR-activating mutations known to correlate with BTKi sensitivity. However, most CD5+ DLBCLs lacked canonical BCR-activating mutations or were LymphGen-unclassifiable (LymphGen-Other). The CD5sig recapitulated these findings in multiple independent data sets, indicating its utility in identifying DLBCLs with genetic and nongenetic bases for BCR dependence. Supporting this notion, CD5sig+ DLBCLs derived a selective survival advantage from the addition of ibrutinib to R-CHOP in the PHOENIX study, independent of LymphGen classification. CONCLUSION: CD5sig is a useful biomarker to identify DLBCLs vulnerable to BTKi-based therapies and complements current biomarker approaches by identifying DLBCLs with genetic and nongenetic bases for BTKi sensitivity.

XPO1 Enables Adaptive Regulation of mRNA Export Required for Genotoxic Stress Tolerance in Cancer Cells.

Exportin-1 (XPO1), the main soluble nuclear export receptor in eukaryotic cells, is frequently overexpressed in diffuse large B-cell lymphoma (DLBCL). A selective XPO1 inhibitor, selinexor, received approval as single agent for relapsed or refractory (R/R) DLBCL. Elucidating the mechanisms by which XPO1 overexpression supports cancer cells could facilitate further clinical development of XPO1 inhibitors. We uncovered here that XPO1 overexpression increases tolerance to genotoxic stress, leading to a poor response to chemoimmunotherapy. Upon DNA damage induced by MYC expression or exogenous compounds, XPO1 bound and exported EIF4E and THOC4 carrying DNA damage repair mRNAs, thereby increasing synthesis of DNA damage repair proteins under conditions of increased turnover. Consequently, XPO1 inhibition decreased the capacity of lymphoma cells to repair DNA damage and ultimately resulted in increased cytotoxicity. In a phase I clinical trial conducted in R/R DLBCL, the combination of selinexor with second-line chemoimmunotherapy was tolerated with early indication of efficacy. Overall, this study reveals that XPO1 overexpression plays a critical role in the increased tolerance of cancer cells to DNA damage while providing new insights to optimize the clinical development of XPO1 inhibitors. SIGNIFICANCE: XPO1 regulates the dynamic ribonucleoprotein nuclear export in response to genotoxic stress to support tolerance and can be targeted to enhance the sensitivity of cancer cells to endogenous and exogenous DNA damage. See related commentary by Knittel and Reinhardt, p. 3.

IL-8 correlates with nonresponse to neoadjuvant nivolumab in HPV positive HNSCC via a potential extracellular vesicle miR-146a mediated mechanism.

Therapy using anti-PD-1 immune checkpoint inhibitors (ICI) has revolutionized the treatment of many cancers including head and neck squamous cell carcinomas (HNSCC), but only a fraction of patients respond. To better understand the molecular mechanisms driving resistance, we performed extensive analysis of plasma and tumor tissues before and after a 4-week neoadjuvant trial in which HNSCC patients were treated with the anti-PD-1 inhibitor, nivolumab. Luminex cytokine analysis of patient plasma demonstrated that HPVpos nonresponders displayed high levels of the proinflammatory chemokine, interleukin-8 (IL-8), which decreased after ICI treatment, but remained higher than responders. miRNAseq analysis of tetraspanin-enriched small extracellular vesicles (sEV) purified from plasma of HPVpos nonresponders demonstrated significantly lower levels of seven miRNAs that target IL-8 including miR-146a. Levels of the pro-survival oncoprotein Dsg2, which has been to down-regulate miR-146a, are elevated with HPVpos tumors displaying higher levels than HPVneg tumors. Dsg2 levels decrease significantly following ICI in responders but not in nonresponders. In cultured HPVpos cells, restoration of miR-146a by forced expression or treatment with miR-146a-loaded sEV, reduced IL-8 level, blocked cell cycle progression, and promoted cell death. These findings identify Dsg2, miR-146a, and IL-8 as potential biomarkers for ICI response and suggest that the Dsg2/miR-146a/IL-8 signaling axis negatively impacts ICI treatment outcomes and could be targeted to improve ICI responsiveness in HPVpos HNSCC patients.

B-Cell Infiltrate in the Tumor Microenvironment Is Associated With Improved Survival in Resected Lung Adenocarcinoma.

Introduction: Relapse is common after resection of lung adenocarcinoma (LUAD). Features of the tumor microenvironment (TME) which influence postsurgical survival outcomes are poorly characterized. Here, we analyzed the TME of more than 1500 LUAD specimens to identify the relationship between B-cell infiltration and prognosis. Methods: Whole exome sequencing and bulk RNA sequencing were performed on LUADs and adjacent normal lung tissue. Relapse-free survival and overall survival (OS) were retrospectively correlated with characteristics of the tumor and TME in three data sets. Results: High B-cell content (defined as >10% B cells) was associated with improved OS in both a The Cancer Genome Atlas-resected LUAD data set (p = 0.01) and a separate institutional stage II LUAD data set (p = 0.04, median not reached versus 89.5 mo). A validation cohort consisting of pooled microarray data representing more than 1400 resected stage I to III LUADs confirmed the association between greater B-cell abundance, specifically higher B-cell expression, and longer postsurgical survival (median OS 90 versus 71 mo, p < 0.01). Relapse-free survival was longer for patients with adenocarcinomas with high B-cell content across data sets, but it did not reach statistical significance. Subcategorization of B-cell subsets indicated that high naive B-cell content was most predictive of survival. There was no correlation between programmed death-ligand 1 expression, lymphoid aggregates, or overall immune infiltrate density and survival outcomes across the cohorts. Conclusions: The growing adjuvant immunotherapy repertoire has increased the urgency for identifying prognostic and predictive biomarkers. Comprehensive profiling of more than 1500 LUADs suggests that high tumor-infiltrating B-cell content is a favorable prognostic marker.

B cell-dependent subtypes and treatment-based immune correlates to survival in stage 3 and 4 lung adenocarcinomas.

Lung cancer is the leading cause of cancer-related deaths worldwide. Surgery and chemoradiation are the standard of care in early stages of non-small cell lung cancer (NSCLC), while immunotherapy is the standard of care in late-stage NSCLC. The immune composition of the tumor microenvironment (TME) is recognized as an indicator for responsiveness to immunotherapy, although much remains unknown about its role in responsiveness to surgery or chemoradiation. In this pilot study, we characterized the NSCLC TME using mass cytometry (CyTOF) and bulk RNA sequencing (RNA-Seq) with deconvolution of RNA-Seq being performed by Kassandra, a recently published deconvolution tool. Stratification of patients based on the intratumoral abundance of B cells identified that the B-cell rich patient group had increased expression of CXCL13 and greater abundance of PD1+ CD8 T cells. The presence of B cells and PD1+ CD8 T cells correlated positively with the presence of intratumoral tertiary lymphoid structures (TLS). We then assessed the predictive and prognostic utility of these cell types and TLS within publicly available stage 3 and 4 lung adenocarcinoma (LUAD) RNA-Seq datasets. As previously described by others, pre-treatment expression of intratumoral 12-chemokine TLS gene signature is associated with progression free survival (PFS) in patients who receive treatment with immune checkpoint inhibitors (ICI). Notably and unexpectedly pre-treatment percentages of intratumoral B cells are associated with PFS in patients who receive surgery, chemotherapy, or radiation. Further studies to confirm these findings would allow for more effective patient selection for both ICI and non-ICI treatments.

Multicenter phase 2 study of oral azacitidine (CC-486) plus CHOP as initial treatment for PTCL.

Peripheral T-cell lymphomas (PTCL) with T-follicular helper phenotype (PTCL-TFH) has recurrent mutations affecting epigenetic regulators, which may contribute to aberrant DNA methylation and chemoresistance. This phase 2 study evaluated oral azacitidine (CC-486) plus cyclophosphamide, doxorubicin, vincristine, and prednisone (CHOP) as initial treatment for PTCL. CC-486 at 300 mg daily was administered for 7 days before C1 of CHOP, and for 14 days before CHOP C2-6. The primary end point was end-of-treatment complete response (CR). Secondary end points included safety and survival. Correlative studies assessed mutations, gene expression, and methylation in tumor samples. Grade 3 to 4 hematologic toxicities were mostly neutropenia (71%), with febrile neutropenia uncommon (14%). Nonhematologic toxicities included fatigue (14%) and gastrointestinal symptoms (5%). In 20 evaluable patients, CR was 75%, including 88.2% for PTCL-TFH (n = 17). The 2-year progression-free survival (PFS) was 65.8% for all and 69.2% for PTCL-TFH, whereas 2-year overall survival (OS) was 68.4% for all and 76.1% for PTCL-TFH. The frequencies of the TET2, RHOA, DNMT3A, and IDH2 mutations were 76.5%, 41.1%, 23.5%, and 23.5%, respectively, with TET2 mutations significantly associated with CR (P = .007), favorable PFS (P = .004) and OS (P = .015), and DNMT3A mutations associated with adverse PFS (P = .016). CC-486 priming contributed to the reprograming of the tumor microenvironment by upregulation of genes related to apoptosis (P < .01) and inflammation (P < .01). DNA methylation did not show significant shift. This safe and active regimen is being further evaluated in the ALLIANCE randomized study A051902 in CD30-negative PTCL. This trial was registered at www.clinicaltrials.gov as #NCT03542266.

Deciphering the origin and therapeutic targets of cancer of unknown primary: a case report that illustrates the power of integrative whole-exome and transcriptome sequencing analysis.

Cancer of unknown primary (CUP) represents a significant diagnostic and therapeutic challenge, being the third to fourth leading cause of cancer death, despite advances in diagnostic tools. This article presents a successful approach using a novel genomic analysis in the evaluation and treatment of a CUP patient, leveraging whole-exome sequencing (WES) and RNA sequencing (RNA-seq). The patient, with a history of multiple primary tumors including urothelial cancer, exhibited a history of rapid progression on empirical chemotherapy. The application of our approach identified a molecular target, characterized the tumor expression profile and the tumor microenvironment, and analyzed the origin of the tumor, leading to a tailored treatment. This resulted in a substantial radiological response across all metastatic sites and the predicted primary site of the tumor. We argue that a comprehensive genomic and molecular profiling approach, like the BostonGene© Tumor Portrait, can provide a more definitive, personalized treatment strategy, overcoming the limitations of current predictive assays. This approach offers a potential solution to an unmet clinical need for a standardized approach in identifying the tumor origin for the effective management of CUP.

Precise reconstruction of the TME using bulk RNA-seq and a machine learning algorithm trained on artificial transcriptomes.

Cellular deconvolution algorithms virtually reconstruct tissue composition by analyzing the gene expression of complex tissues. We present the decision tree machine learning algorithm, Kassandra, trained on a broad collection of >9,400 tissue and blood sorted cell RNA profiles incorporated into millions of artificial transcriptomes to accurately reconstruct the tumor microenvironment (TME). Bioinformatics correction for technical and biological variability, aberrant cancer cell expression inclusion, and accurate quantification and normalization of transcript expression increased Kassandra stability and robustness. Performance was validated on 4,000 H&E slides and 1,000 tissues by comparison with cytometric, immunohistochemical, or single-cell RNA-seq measurements. Kassandra accurately deconvolved TME elements, showing the role of these populations in tumor pathogenesis and other biological processes. Digital TME reconstruction revealed that the presence of PD-1-positive CD8+ T cells strongly correlated with immunotherapy response and increased the predictive potential of established biomarkers, indicating that Kassandra could potentially be utilized in future clinical applications.

Follicular Lymphoma Microenvironment Characteristics Associated with Tumor Cell Mutations and MHC Class II Expression.

Follicular lymphoma (FL) is a B-cell malignancy with a complex tumor microenvironment that is rich in nonmalignant immune cells. We applied single-cell RNA sequencing to characterize the diverse tumor and immune cell populations of FL and identified major phenotypic subsets of FL T cells, including a cytotoxic CD4 T-cell population. We characterized four major FL subtypes with differential representation or relative depletion of distinct T-cell subsets. By integrating exome sequencing, we observed that somatic mutations are associated with, but not definitive for, reduced MHC expression on FL cells. In turn, expression of MHCII genes by FL cells was associated with significant differences in the proportions and targetable immunophenotypic characteristics of T cells. This provides a classification framework of the FL microenvironment in association with FL genotypes and MHC expression, and informs different potential immunotherapeutic strategies based upon tumor cell MHCII expression. SIGNIFICANCE: We have characterized the FL-infiltrating T cells, identified cytotoxic CD4 T cells as an important component that is associated with tumor cell-intrinsic characteristics, and identified sets of targetable immune checkpoints on T cells that differed from FLs with normal versus low MHC expression. See related commentary by Melnick, p. 374. This article is highlighted in the In This Issue feature, p. 369.

Tadalafil Enhances Immune Signatures in Response to Neoadjuvant Nivolumab in Resectable Head and Neck Squamous Cell Carcinoma.

PURPOSE: We hypothesize that the addition of the phosphodiesterase-5 inhibitor tadalafil to the PD-1 inhibitor nivolumab, is safe and will augment immune-mediated antitumor responses in previously untreated squamous cell carcinoma of the head and neck (HNSCC). PATIENTS AND METHODS: We conducted a two-arm multi-institutional neoadjuvant randomized trial in any-stage resectable HNSCC (NCT03238365). Patients were stratified at randomization by human papillomavirus (HPV) status. Patients in both arms received nivolumab 240 mg intravenously on days 1 and 15 followed by surgery on day 28. Those in the combination therapy arm also received tadalafil 10 mg orally once daily for 4 weeks. Imaging, blood, and tumor were obtained pretreatment and posttreatment for correlative analysis. RESULTS: Neoadjuvant therapy was well-tolerated with no grade 3 to 5 adverse events and no surgical delays. Twenty-five of 46 (54%) evaluable patients had a pathologic treatment response of ≥20%, including three (7%) patients with a complete pathologic response. Regardless of HPV status, tumor proliferation rate was a negative predictor of response. A strong pretreatment T-cell signature in the HPV-negative cohort was a predictor of response. Tadalafil altered the immune microenvironment, as evidenced by transcriptome data identifying enriched B- and natural killer cell gene sets in the tumor and augmented effector T cells in the periphery. CONCLUSIONS: Preoperative nivolumab ± tadalafil is safe in HNSCC and results in more than 50% of the patients having a pathologic treatment response of at least 20% after 4 weeks of treatment. Pretreatment specimens identified HPV status-dependent signatures that predicted response to immunotherapy while posttreatment specimens showed augmentation of the immune microenvironment with the addition of tadalafil.

Oncogenic HSP90 Facilitates Metabolic Alterations in Aggressive B-cell Lymphomas.

HSP90 is critical for maintenance of the cellular proteostasis. In cancer cells, HSP90 also becomes a nucleating site for the stabilization of multiprotein complexes including signaling pathways and transcription complexes. Here we described the role of this HSP90 form, referred to as oncogenic HSP90, in the regulation of cytosolic metabolic pathways in proliferating B-cell lymphoma cells. Oncogenic HSP90 assisted in the organization of metabolic enzymes into non-membrane-bound functional compartments. Under experimental conditions that conserved cellular proteostasis, oncogenic HSP90 coordinated and sustained multiple metabolic pathways required for energy production and maintenance of cellular biomass as well as for secretion of extracellular metabolites. Conversely, inhibition of oncogenic HSP90, in absence of apparent client protein degradation, decreased the efficiency of MYC-driven metabolic reprogramming. This study reveals that oncogenic HSP90 supports metabolism in B-cell lymphoma cells and patients with diffuse large B-cell lymphoma, providing a novel mechanism of activity for HSP90 inhibitors. SIGNIFICANCE: The oncogenic form of HSP90 organizes and maintains functional multienzymatic metabolic hubs in cancer cells, suggesting the potential of repurposing oncogenic HSP90 selective inhibitors to disrupt metabolism in lymphoma cells.

Doublecortin-Like Kinase 1 (DCLK1) Is a Novel NOTCH Pathway Signaling Regulator in Head and Neck Squamous Cell Carcinoma.

Despite recent advancements, the 5 year survival of head and neck squamous cell carcinoma (HNSCC) hovers at 60%. DCLK1 has been shown to regulate epithelial-to-mesenchymal transition as well as serving as a cancer stem cell marker in colon, pancreatic and renal cancer. Although it was reported that DCLK1 is associated with poor prognosis in oropharyngeal cancers, very little is known about the molecular characterization of DCLK1 in HNSCC. In this study, we performed a comprehensive transcriptome-based computational analysis on hundreds of HNSCC patients from TCGA and GEO databases, and found that DCLK1 expression positively correlates with NOTCH signaling pathway activation. Since NOTCH signaling has a recognized role in HNSCC tumorigenesis, we next performed a series of in vitro experiments in a collection of HNSCC cell lines to investigate the role of DCLK1 in NOTCH pathway regulation. Our analyses revealed that DCLK1 inhibition, using either a pharmacological inhibitor or siRNA, resulted in substantially decreased proliferation, invasion, migration, and colony formation. Furthermore, these effects paralleled downregulation of active NOTCH1, and its downstream effectors, HEY1, HES1 and HES5, whereas overexpression of DCLK1 in normal keratinocytes, lead to an upregulation of NOTCH signaling associated with increased proliferation. Analysis of 233 primary and 40 recurrent HNSCC cancer biopsies revealed that high DCLK1 expression was associated with poor prognosis and showed a trend towards higher active NOTCH1 expression in tumors with elevated DCLK1. Our results demonstrate the novel role of DCLK1 as a regulator of NOTCH signaling network and suggest its potential as a therapeutic target in HNSCC.

Neoantigen vaccination induces clinical and immunologic responses in non-small cell lung cancer patients harboring EGFR mutations.

BACKGROUND: Neoantigen (NeoAg) peptides displayed at the tumor cell surface by human leukocyte antigen molecules show exquisite tumor specificity and can elicit T cell mediated tumor rejection. However, few NeoAgs are predicted to be shared between patients, and none to date have demonstrated therapeutic value in the context of vaccination. METHODS: We report here a phase I trial of personalized NeoAg peptide vaccination (PPV) of 24 stage III/IV non-small cell lung cancer (NSCLC) patients who had previously progressed following multiple conventional therapies, including surgery, radiation, chemotherapy, and tyrosine kinase inhibitors (TKIs). Primary endpoints of the trial evaluated feasibility, tolerability, and safety of the personalized vaccination approach, and secondary trial endpoints assessed tumor-specific immune reactivity and clinical responses. Of the 16 patients with epidermal growth factor receptor (EGFR) mutations, nine continued TKI therapy concurrent with PPV and seven patients received PPV alone. RESULTS: Out of 29 patients enrolled in the trial, 24 were immunized with personalized NeoAg peptides. Aside from transient rash, fatigue and/or fever observed in three patients, no other treatment-related adverse events were observed. Median progression-free survival and overall survival of the 24 vaccinated patients were 6.0 and 8.9 months, respectively. Within 3-4 months following initiation of PPV, seven RECIST-based objective clinical responses including one complete response were observed. Notably, all seven clinical responders had EGFR-mutated tumors, including four patients that had continued TKI therapy concurrently with PPV. Immune monitoring showed that five of the seven responding patients demonstrated vaccine-induced T cell responses against EGFR NeoAg peptides. Furthermore, two highly shared EGFR mutations (L858R and T790M) were shown to be immunogenic in four of the responding patients, all of whom demonstrated increases in peripheral blood neoantigen-specific CD8+ T cell frequencies during the course of PPV. CONCLUSIONS: These results show that personalized NeoAg vaccination is feasible and safe for advanced-stage NSCLC patients. The clinical and immune responses observed following PPV suggest that EGFR mutations constitute shared, immunogenic neoantigens with promising immunotherapeutic potential for large subsets of NSCLC patients. Furthermore, PPV with concurrent EGFR inhibitor therapy was well tolerated and may have contributed to the induction of PPV-induced T cell responses.

Conserved pan-cancer microenvironment subtypes predict response to immunotherapy.

The clinical use of molecular targeted therapy is rapidly evolving but has primarily focused on genomic alterations. Transcriptomic analysis offers an opportunity to dissect the complexity of tumors, including the tumor microenvironment (TME), a crucial mediator of cancer progression and therapeutic outcome. TME classification by transcriptomic analysis of >10,000 cancer patients identifies four distinct TME subtypes conserved across 20 different cancers. The TME subtypes correlate with patient response to immunotherapy in multiple cancers, with patients possessing immune-favorable TME subtypes benefiting the most from immunotherapy. Thus, the TME subtypes act as a generalized immunotherapy biomarker across many cancer types due to the inclusion of malignant and microenvironment components. A visual tool integrating transcriptomic and genomic data provides a global tumor portrait, describing the tumor framework, mutational load, immune composition, anti-tumor immunity, and immunosuppressive escape mechanisms. Integrative analyses plus visualization may aid in biomarker discovery and the personalization of therapeutic regimens.

Single-cell Spatial Proteomic Revelations on the Multiparametric MRI Heterogeneity of Clinically Significant Prostate Cancer.

PURPOSE: Multiparametric MRI (mpMRI) has become an indispensable radiographic tool in diagnosing prostate cancer. However, mpMRI fails to visualize approximately 15% of clinically significant prostate cancer (csPCa). The molecular, cellular, and spatial underpinnings of such radiographic heterogeneity in csPCa are unclear. EXPERIMENTAL DESIGN: We examined tumor tissues from clinically matched patients with mpMRI-invisible and mpMRI-visible csPCa who underwent radical prostatectomy. Multiplex immunofluorescence single-cell spatial imaging and gene expression profiling were performed. Artificial intelligence-based analytic algorithms were developed to examine the tumor ecosystem and integrate with corresponding transcriptomics. RESULTS: More complex and compact epithelial tumor architectures were found in mpMRI-visible than in mpMRI-invisible prostate cancer tumors. In contrast, similar stromal patterns were detected between mpMRI-invisible prostate cancer and normal prostate tissues. Furthermore, quantification of immune cell composition and tumor-immune interactions demonstrated a lack of immune cell infiltration in the malignant but not in the adjacent nonmalignant tissue compartments, irrespective of mpMRI visibility. No significant difference in immune profiles was detected between mpMRI-visible and mpMRI-invisible prostate cancer within our patient cohort, whereas expression profiling identified a 24-gene stromal signature enriched in mpMRI-invisible prostate cancer. Prostate cancer with strong stromal signature exhibited a favorable survival outcome within The Cancer Genome Atlas prostate cancer cohort. Notably, five recurrences in the 8 mpMRI-visible patients with csPCa and no recurrence in the 8 clinically matched patients with mpMRI-invisible csPCa occurred during the 5-year follow-up post-prostatectomy. CONCLUSIONS: Our study identified distinct molecular, cellular, and structural characteristics associated with mpMRI-visible csPCa, whereas mpMRI-invisible tumors were similar to normal prostate tissue, likely contributing to mpMRI invisibility.

Clinical and Biological Subtypes of B-cell Lymphoma Revealed by Microenvironmental Signatures.

Diffuse large B-cell lymphoma (DLBCL) is a biologically and clinically heterogeneous disease. Transcriptomic and genetic characterization of DLBCL has increased the understanding of its intrinsic pathogenesis and provided potential therapeutic targets. However, the role of the microenvironment in DLBCL biology remains less understood. Here, we performed a transcriptomic analysis of the microenvironment of 4,655 DLBCLs from multiple independent cohorts and described four major lymphoma microenvironment categories that associate with distinct biological aberrations and clinical behavior. We also found evidence of genetic and epigenetic mechanisms deployed by cancer cells to evade microenvironmental constraints of lymphoma growth, supporting the rationale for implementing DNA hypomethylating agents in selected patients with DLBCL. In addition, our work uncovered new therapeutic vulnerabilities in the biochemical composition of the extracellular matrix that were exploited to decrease DLBCL proliferation in preclinical models. This novel classification provides a road map for the biological characterization and therapeutic exploitation of the DLBCL microenvironment. SIGNIFICANCE: In a translational relevant transcriptomic-based classification, we characterized the microenvironment as a critical component of the B-cell lymphoma biology and associated it with the DLBCL clinical behavior establishing a novel opportunity for targeting therapies.This article is highlighted in the In This Issue feature, p. 1307.