A B S T R A C T PurposeDiffuse large B-cell lymphoma (DLBCL) is curable in 60% of patients treated with rituximab plus cyclophosphamide, doxorubicin, vincristine, and prednisone (R-CHOP). MYC translocations, with or without BCL2 translocations, have been associated with inferior survival in DLBCL. We investigated whether expression of MYC protein, with or without BCL2 protein expression, could risk-stratify patients at diagnosis. Patients and MethodsWe determined the correlation between presence of MYC and BCL2 proteins by immunohistochemistry (IHC) with survival in two independent cohorts of patients with DLBCL treated with R-CHOP. We further determined if MYC protein expression correlated with high MYC mRNA and/or presence of MYC translocation. ResultsIn the training cohort (n ϭ 167), MYC and BCL2 proteins were detected in 29% and 44% of patients, respectively. Concurrent expression (MYC positive/BCL2 positive) was present in 21% of patients. MYC protein correlated with presence of high MYC mRNA and MYC translocation (both P Ͻ .001), but the latter was less frequent (both 11%). MYC protein expression was only associated with inferior overall and progression-free survival when BCL2 protein was coexpressed (P Ͻ .001). Importantly, the poor prognostic effect of MYC positive/BCL2 positive was validated in an independent cohort of 140 patients with DLBCL and remained significant (P Ͻ .05) after adjusting for presence of high-risk features in a multivariable model that included elevated international prognostic index score, activated B-cell molecular subtype, and presence of concurrent MYC and BCL2 translocations. ConclusionAssessment of MYC and BCL2 expression by IHC represents a robust, rapid, and inexpensive approach to risk-stratify patients with DLBCL at diagnosis.

Mantle cell lymphoma (MCL), an aggressive subtype of non-Hodgkin lymphoma, is characterized by the hallmark translocation t(11;14)(q13;q32) and the resulting overexpression of cyclin D1 (CCND1). Our current knowledge of this disease encompasses frequent secondary cytogenetic aberrations and the recurrent mutation of a handful of genes, such as TP53, ATM, and CCND1. However, these findings insufficiently explain the biologic underpinnings of MCL. Here, we performed whole transcriptome sequencing on a discovery cohort of 18 primary tissue MCL samples and 2 cell lines. We found recurrent mutations in NOTCH1, a finding that we confirmed in an extension cohort of 108 clinical samples and 8 cell lines. In total, 12% of clinical samples and 20% of cell lines harbored somatic NOTCH1 coding sequence mutations that clustered in the PEST domain and predominantly consisted of truncating mutations or small frame-shifting indels. NOTCH1 mutations were associated with poor overall survival (P ‫؍‬ .003). Furthermore, we showed that inhibition of the NOTCH pathway reduced proliferation and induced apoptosis in 2 MCL cell lines. In summary, we have identified recurrent NOTCH1 mutations that provide the preclinical rationale for therapeutic inhibition of the NOTCH pathway in a subset of patients with MCL.

BackgroundFollicular lymphoma (FL) is an indolent, yet incurable B cell malignancy. A subset of patients experience an increased mortality rate driven by two distinct clinical end points: histological transformation and early progression after immunochemotherapy. The nature of tumor clonal dynamics leading to these clinical end points is poorly understood, and previously determined genetic alterations do not explain the majority of transformed cases or accurately predict early progressive disease. We contend that detailed knowledge of the expansion patterns of specific cell populations plus their associated mutations would provide insight into therapeutic strategies and disease biology over the time course of FL clinical histories.Methods and FindingsUsing a combination of whole genome sequencing, targeted deep sequencing, and digital droplet PCR on matched diagnostic and relapse specimens, we deciphered the constituent clonal populations in 15 transformation cases and 6 progression cases, and measured the change in clonal population abundance over time. We observed widely divergent patterns of clonal dynamics in transformed cases relative to progressed cases. Transformation specimens were generally composed of clones that were rare or absent in diagnostic specimens, consistent with dramatic clonal expansions that came to dominate the transformation specimens. This pattern was independent of time to transformation and treatment modality. By contrast, early progression specimens were composed of clones that were already present in the diagnostic specimens and exhibited only moderate clonal dynamics, even in the presence of immunochemotherapy. Analysis of somatic mutations impacting 94 genes was undertaken in an extension cohort consisting of 395 samples from 277 patients in order to decipher disrupted biology in the two clinical end points. We found 12 genes that were more commonly mutated in transformed samples than in the preceding FL tumors, including TP53, B2M, CCND3, GNA13, S1PR2, and P2RY8. Moreover, ten genes were more commonly mutated in diagnostic specimens of patients with early progression, including TP53, BTG1, MKI67, and XBP1.ConclusionsOur results illuminate contrasting modes of evolution shaping the clinical histories of transformation and progression. They have implications for interpretation of evolutionary dynamics in the context of treatment-induced selective pressures, and indicate that transformation and progression will require different clinical management strategies.

Increased tumor-associated macrophages (TAMs) are reported to be associated with poor prognosis in classic Hodgkin lymphoma (CHL). We investigated the prognostic significance of TAMs in the E2496 Intergroup trial, a multicenter phase 3 randomized controlled trial comparing ABVD and Stanford V chemotherapy in locally extensive and advanced stage CHL. Tissue microarrays were constructed from formalinfixed, paraffin-embedded tumor tissue and included 287 patients. Patients were randomly assigned into training (n ‫؍‬ 143) and validation (n ‫؍‬ 144) cohorts. Immunohistochemistry for CD68 and CD163, and in situ hybridization for EBV-encoded RNA were performed. CD68 and CD163 IHC were analyzed by computer image analysis; optimum thresholds for overall survival (OS) were determined in the training cohort and tested in the independent validation cohort. Increased CD68 and CD163 expression was significantly associated with inferior failurefree survival and OS in the validation cohort.

Key Points• Programmed death ligands 1 and 2 are rearranged at a frequency of 20% in PMBCL.The pathogenesis of primary mediastinal large B-cell lymphoma (PMBCL) is incompletely understood. Recently, specific genotypic and phenotypic features have been linked to tumor cell immune escape mechanisms in PMBCL. We studied 571 B-cell lymphomas with a focus on PMBCL. Using fluorescence in situ hybridization here, we report that the programmed death ligand (PDL) locus (9p24.1) is frequently and specifically rearranged in PMBCL (20%) as compared with diffuse large B-cell lymphoma, follicular lymphoma, and Hodgkin lymphoma. Rearrangement was significantly correlated with overexpression of PDL transcripts. Utilizing high-throughput sequencing techniques, we characterized novel translocations and chimeric fusion transcripts involving PDLs at base-pair resolution. Our data suggest that recurrent genomic rearrangement events underlie an immune privilege phenotype in a subset of B-cell lymphomas. (Blood. 2014;123(13):2062-2065 IntroductionPrimary mediastinal large B-cell lymphoma (PMBCL) is an aggressive disease known to share certain genotypic and phenotypic features with classic Hodgkin lymphoma (CHL) and diffuse large B-cell lymphoma (DLBCL).1,2 However, the complete landscape of genetic alterations involved in PMBCL pathogenesis has yet to be fully elucidated.3 Among the most common chromosomal alterations in PMBCL are amplifications of chromosome 9p and translocations involving CIITA (16p13.13).4-7 These aberrations have been suggested to affect tumor-microenvironment interactions resulting in immune privilege. 7,8 Here, we demonstrate that rearrangements involving immune cell anergy-inducing programmed death ligand (PDL) 1 (CD274) and 2 (PDCD1LG2) are recurrent in and characteristic of PMBCL. Furthermore, we show such rearrangements are correlated with elevated transcript levels, and we characterize novel translocations identified using high-throughput sequencing. Study designWe studied 571 primary B-cell lymphoma samples in conjunction with 17 established B-cell-derived cell lines. Using in-house bacterial artificial chromosome probes, fluorescence in situ hybridization (FISH) or FISH combined with CD30 immunofluorescence (in the case of CHL specimens) was performed to characterize the PDL locus.7,9,10 These cases were also analyzed with Epstein-Barr virus (EBV)-encoded RNA in situ hybridization. Quantitative real-time polymerase chain reaction (qRT-PCR) was performed for CD274 and PDCD1LG2 transcript expression on a subset of cases from the FISH cohort (N 5 76). Surface PDCD1LG2 expression of the cell lines was determined via flow cytometry. To characterize the Hodgkin lymphoma cell lines found to be rearranged by FISH, 1 whole-transcriptome sequencing (RNA-seq) library (CHL-derived L-428) was reanalyzed, and 1 new wholegenome library (L-428) and 2 new RNA-seq libraries (CHL-derived L-1236 and nodular lymphocyte predominant Hodgkin lymphoma-derived DEV) were sequenced.7 This study was approved by the BC Cancer Agency ...