Pittaluga, Stefania

Abstract Anaplastic large cell lymphomas (ALCLs) are CD30+ T-cell lymphomas that share pathologic features but differ in presentation, outcome, and genetics. Current classification incorporates clinical presentation and anaplastic lymphoma kinase (ALK) status but inadequately addresses molecular heterogeneity and therapeutic vulnerabilities. We studied 689 patients with ALCL in the LLMPP (Lymphoma/Leukemia Molecular Profiling Project) and performed expert consensus review, genetic subtyping (ALK, DUSP22, TP63, and triple negative), and immunohistochemistry for phosphorylated STAT3Tyr705. RNA sequencing with unsupervised gene expression profiling in 393 patients identified 2 main molecular types of ALCL that could be predicted with 91% accuracy based on the presence (type I) or absence (type II) of phosphorylated STAT3Y705 expression. Type I ALCLs included ALK+ ALCL and a subset of triple-negative ALCLs (TN-I); type II ALCLs included tumors with DUSP22 and/or TP63 rearrangements and the remaining triple-negative ALCLs (TN-II). Type I ALCLs were enriched for JAK-STAT3, whereas type II ALCLs were enriched for non–tyrosine kinase pathways, particularly epigenetic regulators such as EZH2. Immunohistochemistry showed overexpression of EZH2 and its trimethylated substrate H3K27. Prognosis in systemic ALCL was favorable for DUSP22-rearranged ALCL (5-year overall survival, 95%) and ALK+ ALCL (88%), intermediate for triple-negative ALCL (TN-I, 52% and TN-II, 37%), and poor for TP63-rearranged ALCL (0%). We introduce an integrated molecular classification that preserves currently diagnosed ALCL entities but identifies 4 molecularly distinct ALK− ALCL subtypes (DUSP22-rearranged, TP63-rearranged, TN-I, and TN-II). This classification can be easily implemented on paraffin tissue in routine practice or clinical trials, and stratifies ALCL into diagnostically, prognostically, biologically, and potentially therapeutically relevant subtypes.

Abstract Molecular characterization of high-grade B-cell lymphoma, not otherwise specified (HGBCL-NOS), is hindered by its rarity, evolving definition, and poor diagnostic reproducibility. To address this challenge, we analyzed 92 HGBCL-NOS tumors collected across Lymphoma/Leukemia Molecular Profiling Project sites. Leveraging comparison cohorts of diffuse large B-cell lymphoma, NOS (DLBCL-NOS) and Burkitt lymphoma (BL), and molecular frameworks described in these entities, our analysis revealed a heterogenous molecular landscape, reminiscent of DLBCL-NOS but with an enrichment of BL features. By cell-of-origin classification, 59% were germinal center B-cell–like (GCB), and 25% were activated B-cell–like (ABC). LymphGen, a genetic classifier for DLBCL-NOS, assigned a genetic subtype to 34% of HGBCL-NOS. Although classification rate was lower than in DLBCL-NOS (66%), assigned subtypes spanned the spectrum of LymphGen classes, including 31% of ABCs classified as MCD. Features differentiating HGBCL-NOS from DLBCL-NOS included MYC rearrangement (47% vs 6%); dark zone signature (DZsig) expression (45% vs 7%); and more frequent mutation of ID3, MYC, CCND3, and TP53, all common to BL. A genetic classifier that differentiates DLBCL-NOS from BL classified 53% of DZsig+ tumors as BL-like, and those classified as DLBCL-like were frequently BCL2-rearranged. Among DZsig− GCB tumors, 95% were DLBCL-like. Centralized pathology review reclassified almost half of tumors as DLBCL-NOS but did not identify a more homogenous HGBCL-NOS population, with no difference in features between confirmed and reclassified tumors. In conclusion, molecular testing enables a subset of HGBCL-NOS to be assigned to established categories. Based on rarity and diagnostic challenges, broader inclusion of HGBCL-NOS should be considered in biomarker-driven DLBCL trials.

Humans with monogenic inborn errors responsible for extreme disease phenotypes can reveal essential physiological pathways. We investigated germline mutations in GNAI2 , which encodes G αi2 , a key component in heterotrimeric G protein signal transduction usually thought to regulate adenylyl cyclase–mediated cyclic adenosine monophosphate (cAMP) production. Patients with activating G αi2 mutations had clinical presentations that included impaired immunity. Mutant G αi2 impaired cell migration and augmented responses to T cell receptor (TCR) stimulation. We found that mutant G αi2 influenced TCR signaling by sequestering the guanosine triphosphatase (GTPase)–activating protein RASA2, thereby promoting RAS activation and increasing downstream extracellular signal–regulated kinase (ERK)/mitogen-activated protein kinase (MAPK) and phosphatidylinositol 3-kinase (PI3K)–AKT S6 signaling to drive cellular growth and proliferation.

Abstract Rearrangements that place the oncogenes MYC, BCL2, or BCL6 adjacent to superenhancers are common in mature B-cell lymphomas. Lymphomas with diffuse large B-cell lymphoma (DLBCL) or high-grade morphology with both MYC and BCL2 rearrangements are classified as high-grade B-cell lymphoma with MYC and BCL2 rearrangements (“double hit”; HGBCL-DH-BCL2) and are associated with aggressive disease and poor outcomes. Although it is established that MYC rearrangements involving immunoglobulin (IG) loci are associated with inferior outcomes relative to those involving other non-IG superenhancers, the frequency of and mechanisms driving IG vs non-IG MYC rearrangements have not been elucidated. Here, we used custom targeted capture and/or whole-genome sequencing to characterize oncogene rearrangements across 883 mature B-cell lymphomas including Burkitt lymphoma, follicular lymphoma, DLBCL, and HGBCL-DH-BCL2 tumors. We demonstrate that, although BCL2 rearrangement topology is consistent across entities, HGBCL-DH-BCL2 have distinct MYC rearrangement architecture relative to tumors with single MYC rearrangements or with both MYC and BCL6 rearrangements (HGBCL-DH-BCL6), including both a higher frequency of non-IG rearrangements and different architecture of MYC::IGH rearrangements. The distinct MYC rearrangement patterns in HGBCL-DH-BCL2 occur on the background of high levels of somatic hypermutation across MYC partner loci in HGBCL-DH-BCL2, creating more opportunity to form these rearrangements. Furthermore, because 1 IGH allele is already disrupted by the existing BCL2 rearrangement, the MYC rearrangement architecture in HGBCL-DH-BCL2 likely reflects selective pressure to preserve both BCL2 and B-cell receptor expression. These data provide new mechanistic explanations for the distinct patterns of MYC rearrangements observed across different lymphoma entities.

Abstract With the introduction of large-scale molecular profiling methods and high-throughput sequencing technologies, the genomic features of most lymphoid neoplasms have been characterized at an unprecedented scale. Although the principles for the classification and diagnosis of these disorders, founded on a multidimensional definition of disease entities, have been consolidated over the past 25 years, novel genomic data have markedly enhanced our understanding of lymphomagenesis and enriched the description of disease entities at the molecular level. Yet, the current diagnosis of lymphoid tumors is largely based on morphological assessment and immunophenotyping, with only few entities being defined by genomic criteria. This paper, which accompanies the International Consensus Classification of mature lymphoid neoplasms, will address how established assays and newly developed technologies for molecular testing already complement clinical diagnoses and provide a novel lens on disease classification. More specifically, their contributions to diagnosis refinement, risk stratification, and therapy prediction will be considered for the main categories of lymphoid neoplasms. The potential of whole-genome sequencing, circulating tumor DNA analyses, single-cell analyses, and epigenetic profiling will be discussed because these will likely become important future tools for implementing precision medicine approaches in clinical decision making for patients with lymphoid malignancies.