Yip, Tameson

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 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 Introduction Primary mediastinal large B-cell lymphoma (PMLBCL) is recognized as a distinct lymphoid neoplasm. However, differentiation from diffuse large B-cell lymphoma (DLBCL) can be difficult due to the need for close clinicopathologic correlation and variability in morphology and immunophenotype. In addition, a recent study identified lymphomas that share molecular and morphological features with PMBCL, yet do not involve the mediastinum. Correct classification of PMBCL may impact therapeutic decision making, increasing the demand for more accurate diagnostic methods. Recent research identified a new gene expression signature that robustly differentiated PMLBCL from DLBCL in both training and independent validation cohorts using RNA isolated from routinely available formalin-fixed, paraffin-embedded (FFPE) tissues. Methods This assay, known as the “Lymph3Cx,” utilizes the nCounter platform (NanoString Technologies, Seattle, WA) and consists of probes for 58 target genes (including both discriminatory and housekeeping genes) that can distinguish PMLBCL cases from DLBCL as well as the “cell-of-origin” subtypes of DLBCL. In the current study reported here, the Molecular Diagnostics–Arizona Laboratory (MDAZL) performed a full validation of the Lymph3Cx assay for use in our clinical diagnostic laboratory. Results Assay performance included accuracy (92.9%), precision (100%), analytical sensitivity (100 ng RNA limit of detection), analytical specificity (measures are in place to prevent interfering substances), reproducibility between technologists and technical replicates (100%), specimen stability (18 months for unstained tissue sections), and RNA Isolation Kit Utility (two kits with equal performance) in order to become the first CLIA-certified, CAP-accredited clinical diagnostics laboratory to offer a molecular assay for distinction of PMLBCL from DLBCL. Conclusion The Lymph3Cx assay will be prospectively used for evaluation of Mayo Clinic patients and for use as an integrated biomarker in clinical trials.

Abstract Introduction Lymph2Cx lymphoma cell-of-origin assay (LM2CX) was developed by the Lymphoma/Leukemia Molecular Profiling Project (LLMPP) to better categorize diffuse large B-cell lymphoma (DLBCL). NanoString has licensed the assay and is currently pursuing FDA approval. In the meantime, the test is offered exclusively as a lab-developed test (LDT) by the Mayo Clinic Molecular Diagnostics–Arizona Lab (MDAZL) to Mayo Clinic Enterprise patients. In order to comply with these restrictions, the normal workflow for Mayo Clinic Rochester has been modified as cases are sent to Arizona. Rochester consultants order LM2CX using their local laboratory information system (LIS). Slides are prepared by Rochester histology and then shipped to Arizona, where they are entered into the Arizona LIS and processed. In July 2018, we discovered numerous cases that were ordered but not shipped. Because this step took place at the LIS transition between Arizona and Rochester, it was not detected immediately. Methods Allied Health Staff (AHS) colleagues in Rochester and Arizona had the unique opportunity to collaborate. After Arizona AHS identified the problem, they reached out to the Rochester pathology reporting specialists (PRSs) and began a joint improvement project. Together, we were able to measure the impact of the problem, with Arizona auditing digitally, while Rochester audited physical cases. We found 29% of the cases were handled improperly over a 6-month time period. In order to eliminate the gap, Rochester implemented several improvements, including training, tagging all LM2CX cases, and huddle discussions, while Arizona AHS monitored the process digitally. Results Since implementation of improvements, we have had zero defects. Modifications to AHS/consultant training in Rochester will ensure continued success. Conclusions This intervention illustrates the importance of strong collaborations in order to quickly respond to testing issues and provide the greatest value to patients.