Swerdlow, Steven H.

Introduction: We previously showed that HIV associated EBV negative (-) diffuse large B-cell lymphomas (DLBCL) of germinal center origins (GCB) have enhanced genomic stability and increased expression of DNA repair genes compared to their HIV negative counterparts (Maguire et al 2019). Given that immune deficiency can be induced virally or therapeutically, including the administration of immunosuppressive drugs post solid organ or hematopoietic stem cell transplant, we questioned whether these observations are specific to HIV or if they represent a more global immune impaired signature. Here we assessed the genomic stability of monomorphic DLBCL post-transplant lymphoproliferative disorders (PTLD) and the potential role of DNA repair. Methods: A total of 49 monomorphic DLBCL PTLDs (26 EBV(-), 23 EBV positive (+)) and 170 immune competent (IC) DLBCL (EBV status unknown) were studied. All were pathologically reviewed and samples with <60% tumor content were macro-dissected before RNA/DNA extraction. Cell of origin (COO) profiles were assessed using the DLBCL90 molecular assay which can distinguish primary mediastinal B-cell lymphoma (PMBL) from GCB, Activated B-cell (ABC) and unclassified (UNC) DLBCL (Ennishi et al 2019). NanoString gene expression profiling (GEP) was performed using the PanCancer Pathways panel and analyzed using NanoString nSolver Advanced Analysis. Copy number (CN) analysis was performed on a subset of samples (45 PTLD, 41 DLBCL) using the OncoScan CN assay and analyzed using Chromosome Analysis Suite (ChAS) v4.1. All p-values listed were Benjamini-Hochberg adjusted and Mann-Whitney tested for the GEP and CN data respectively. Results: In contrast to IC-DLBCL which are GCB enriched (50%), COO profiling revealed PTLD to be ABC enriched (43%). CN analyses revealed that the median CN/sample for PTLD (0.57, p<0.001), EBV(+)PTLD (0.25, p<0.001) and EBV(-)PTLD (0.80, p = 0.050) tumors were significantly less aberrant than IC-DLBCL (1.01). The data also showed that EBV(+)PTLD are less aberrant than EBV(-)PTLD tumors (0.25 vs 0.80, p<0.001). GEP pathway analysis identified DNA repair as downregulated in PTLD and EBV(+)PTLD but upregulated in EBV(-)PTLD compared IC-DLBCL. Nine DNA repair genes ( RAD51, CCNA2a, CHEK1b, POLR2Da, PCNAa, WHSC1a, GTF2H3a, ATM c, EP300 c) were differentially expressed (DE) in EBV(-)PTLD compared to IC-DLBCL (7 increased, 2 decreased, p ≤ 0.0425): where the most overexpressed gene RAD51, a critical mediator of Homologous Recombination (HR) and the Fanconi Anemia (FA) DNA repair pathways, was not DE in PTLD or EBV(+)PTLD compared to IC-DLBCL. Five a genes were increased in PTLD (p ≤ 0.0204) but not EBV(+)PTLD, 1 b was decreased in EBV(+)PTLD (p = 0.01) but not PTLD and 2 c were reduced in PTLD regardless of EBV status compared to IC-DLBCL (p ≤ 0.0024). Twenty-8 genes were DE in EBV(+)PTLD compared to IC-DLBCL. Of these, 23 were downregulated (p ≤ 0.0418) and included HR, FA and mismatch repair genes ( CHEK1, CHEK2, BRAC1/FANCS, FANCA, FANCB, FANCE, MSH2, MSH6). All but 2/23 were similarly expressed in PTLD compared to IC-DLBCL of which 11 were significant (p ≤ 0.0356). The five upregulated genes ( ZBTB32, DDB2, NBN, POLB, RBX1, p ≤ 0.0092) were also upregulated in PTLD (p ≤ 0.0042) but not EBV(-)PTLD compared to IC-DLBCL. Two additional genes ( SKP1, FBXW7) were also identified as increased in EBV(+)PTLD and PTLD (p ≤ 0.0204) compared to IC-DLBCL. In contrast, SKP1 was increased (p = 2.7E-4) and FBXW7 was decreased (p = 0.3610) in EBV(-)PTLD compared to IC-DLBCL. Together the protein products of RBX1, SKP1 and FBXW7 comprise the functional cores of a complete UPS complex, the SCF FBXW7 E3 ubiquitin ligase which have been reported to regulate the DNA damage response and promote genomic stability. Conclusions: Like HIV associated DLBCL, these results show that PTLD is more genomically stable than IC-DLBCL, and that genomic stability is further enhanced in EBV(+)PTLD which have increased expression of UPS related genes. In contrast, EBV(-)PTLD have increased expression of DNA damage repair genes, including RAD51. In summary, monomorphic DLBCL PTLD tumors have enhanced genomic stability that appears to be mediated by two distinct mechanisms stratified by EBV status. Further work is required to assess if this stratification of mechanisms is unique to PTLD or reflects a more global immune impaired EBV related signature.

Abstract We investigated the clinicopathologic features of 5 follicular lymphomas (FLs) that transformed (tFL) morphologically to diffuse large B-cell lymphomas (DLBCLs) and had a primary mediastinal large B-cell lymphoma (PMBL)–like gene expression profile (tFL-PMBLsig-pos). None of the tFL-PMBLsig-pos cases arose in the mediastinum, all cases tested had a germinal center B-cell phenotype, 20% were CD30+, 60% CD23+, 80% MAL+, 20% CD200+, and 0% CD273/PDL2+. Whole-exome sequencing detected alterations in genes associated with both FL/DLBCL (CREBBP, KMT2C, KMT2D, ARID1A, HIST1 members, and TNFRSF14) and PMBL (JAK-STAT pathway genes, B2M, and CD58). Copy number (CN) analysis detected gains/amplification of REL and STAT6 in 60%, gains of SOCS1 in 40%, and gains of chromosome 16, including IL4R, in 40% of the cases. CN gains/amplification of BCL6 and MYC and loss of TNFRSF14 and TNFAIP3 were identified in 20% of the cases. Three of 5 cases lacked a BCL2 rearrangement. Despite having some features that are less common in DLBCL (MAL and CD23 expression and JAK-STAT activation), these tFL-PMBLsig-pos cases lack the most characteristic CN alteration seen in PMBL (9p24.1 gain/amplification). This cohort expands the biologic heterogeneity of tFL, illustrating a subset with gene expression and some genetic features reminiscent of PMBL, with potential treatment implications that include the use of novel targeted therapies.

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.

Abstract Since the publication of the Revised European-American Classification of Lymphoid Neoplasms in 1994, subsequent updates of the classification of lymphoid neoplasms have been generated through iterative international efforts to achieve broad consensus among hematopathologists, geneticists, molecular scientists, and clinicians. Significant progress has recently been made in the characterization of malignancies of the immune system, with many new insights provided by genomic studies. They have led to this proposal. We have followed the same process that was successfully used for the third and fourth editions of the World Health Organization Classification of Hematologic Neoplasms. The definition, recommended studies, and criteria for the diagnosis of many entities have been extensively refined. Some categories considered provisional have now been upgraded to definite entities. Terminology for some diseases has been revised to adapt nomenclature to the current knowledge of their biology, but these modifications have been restricted to well-justified situations. Major findings from recent genomic studies have impacted the conceptual framework and diagnostic criteria for many disease entities. These changes will have an impact on optimal clinical management. The conclusions of this work are summarized in this report as the proposed International Consensus Classification of mature lymphoid, histiocytic, and dendritic cell tumors.

Abstract Objectives: We identified a case of follicular lymphoma (FL) that transformed into a morphologic diffuse large B-cell lymphoma (DLBCL), which by gene expression profiling showed a primary mediastinal (PMBL)-like gene expression profile (GEP) (Lymph3Cx; Blood 2018;132:2401-5). A search identified 4 additional transformed FL (tFL) cases with a PMBL-like GEP, which we further studied to determine how similar these tFLs were to classic cases of PMBL. Methods: The morphology and previously reported immunophenotype were reviewed, and CD30, CD23, MAL, CD273/PDL2, and CD200 immunohistochemical stains (IHC) were performed. Whole exome sequencing (WES) and copy number analysis (CNA) to evaluate genes typically altered in FL and PMBL were performed. Results: None of the tFLs arose in the mediastinum or had a previous history of mediastinal disease. All cases showed typical centroblastic DLBCL cytology, with fine sclerosis typical of PMBL. 3/3 were GCB by the Hans IHC algorithm, 1/3 were MYC+, 3/3 BCL2+, 1/5 CD30+, 3/5 CD23+, 4/5 MAL+, 0/5 CD273/PDL2+, 1/5 CD200+, and 0/2 EBER+. Rearrangements of MYC, BCL2, or BCL6 were identified by FISH in 0/3, 1/3, and 2/3 cases, respectively. WES demonstrated sequence variants in genes associated with both FL (CREBBP [60%], KMT2D [40%], and TNFRSF14 [40%]) and PMBL (JAK-STAT pathway genes [80%], B2M [20%], and CD58 [20%]). 2 of the mutations identified in the tFLs have previously been shown to result in JAK-STAT activation (STAT6 p.E372K [PNAS 2016;113:13015-20] and SOCS1 p.F101L [Oncogene 2002;21:4351-62] identified in 1/5 cases each). CNA showed gains/amplification of REL in 3/5 cases, gains/amplification of STAT6 in 2/5, gains of large sections of chromosome 16, including IL4R, in 2/5, and both deletions and gains of 11q in 1/5. See Figure demonstrating the 5 cases on the Y-axis and the chromosomes on the X-axis. Conclusions: The tFLs in this small series seem to represent PMBL-like DLBCLs, rather than classic PMBLs, and have a blended pattern of immunophenotypic and genomic features between FL/DLBCL and PMBL. Although the cases express some PMBL-associated markers (CD23 and MAL), there is less frequent staining for others (CD30, CD273/PDL2, and CD200). The cases harbor both FL-associated and PMBL-associated sequence variants, including 40% with mutations known to activate the JAK-STAT pathway. This frequency of mutations in JAK-STAT pathway genes is higher than that seen in typical FL/DLBCL, but perhaps lower than in classic PMBL (Blood 2019;134:802-13). PMBL also frequently has gains/amplifications of 9p24.1, which was not seen in our cohort. However, gains/amplification of REL/2p, which is seen in approximately 50% of PMBL, was identified in 60% of the tFLs. The 11q aberration identified in 1 case would be unusual for PMBL, and is instead more commonly associated with a subset of aggressive lymphomas with Burkitt-like features (Haematologica 2019;104:1822-9). Recently, lymphomas with similar blended features between DLBCL and PMBL, which were not arising in the setting of tFL, have been reported (Duns G, et al. Blood 2021). Our study extends the types of biological transformations, in addition to more classic DLBCL, that can be seen in FL. These tFLs with blended PMBL-DLBCL biology may have implications for therapeutic decision making including targeted therapies used in PMBL. Figure 1 Figure 1. Disclosures Rimsza: NanoString Technologies: Other: Fee-for-service contract. Steidl: Curis Inc.: Consultancy; Trillium Therapeutics: Research Funding; Bayer: Consultancy; Epizyme: Research Funding; Seattle Genetics: Consultancy; AbbVie: Consultancy; Bristol-Myers Squibb: Research Funding.

Abstract Objectives Primary cutaneous marginal zone lymphoma (PCMZL) is 1 of the 3 major subtypes of primary cutaneous B-cell lymphoma. The diagnosis of PCMZL may be challenging, as the differential diagnosis includes benign cutaneous lymphoproliferations as well as other primary or secondary cutaneous B-cell or T-cell lymphomas. This review describes our approach to the diagnosis of PCMZL. Methods Two cases are presented that illustrate how we diagnose each of the 2 subtypes of PCMZL. The clinicopathologic features of PCMZL and the ways in which these cases can be distinguished from both benign and other neoplastic entities are emphasized. Results A definitive diagnosis of PCMZL requires the incorporation of histologic and immunophenotypic features, molecular genetic studies in some cases, and just as importantly, clinical findings. Emerging data suggest that the heavy chain class-switched cases may be more like a clonal chronic lymphoproliferative disorder. Conclusions The 2 subtypes of PCMZL create different diagnostic challenges and require the use of a multiparameter approach. Although very indolent, it is important to distinguish PCMZLs from reactive proliferations, because they frequently recur and may require antineoplastic therapies. It is also critical to distinguish PCMZLs from other B- or T-cell lymphomas so that patients are properly evaluated and not overtreated.

Abstract Objectives Given the increased complexity of molecular and cytogenetic testing (MOL-CG), the Society for Hematopathology Education Committee (SH-EC) was interested in determining what the current expectations are for MOL-CG education in hematopathology (HP) fellowship training. Methods The SH-EC sent a questionnaire to HP fellowship program directors (HP-PDs) covering MOL-CG training curricula, test menus, faculty background, teaching, and sign-out roles. These findings were explored via a panel-based discussion at the 2018 SH-EC meeting for HP-PDs. Results HP fellows are expected to understand basic principles, nomenclature, and indications for and limitations of testing. Interpretation of common assays is within that scope, but not necessarily proficiency in technical troubleshooting of testing or analysis of complex raw data. Conclusions The consensus was that HP fellows should understand the components of MOL-CG testing necessary to incorporate those results into an accurate, clinically relevant, and integrated HP report.

Key Points ALK-negative ALCLs have chromosomal rearrangements of DUSP22 or TP63 in 30% and 8% of cases, respectively. DUSP22-rearranged cases have favorable outcomes similar to ALK-positive ALCLs, whereas other genetic subtypes have inferior outcomes.