Gimenez, J.

Background: Anaplastic large cell lymphomas represent a group of T-cell lymphomas that have long been broadly classified by the presence or absence of ALK fusion proteins (ALK+ or ALK−, respectively). However, recent discovery of extensive additional molecular heterogeneity has both promoted refinement of disease classification and uncovered new opportunities for biomarker-guided targeted therapy. In addition to recognition of a new genetic subtype, ALK− ALCL with DUSP22 rearrangement (R), a new molecular classification has been proposed based on the recent discovery of 2 overarching molecular types of ALCL, defined by the presence (Type I) or absence (Type II) of a gene expression signature highly enriched for JAK-STAT3 activation. BCL2 is a mitochondrial membrane protein that functions to inhibit the actions of pro-apoptotic proteins such as BAX and BAK and can be targeted clinically by BCL2 small molecule inhibitors. BCL2 has been reported to be expressed more frequently in ALK− ALCL than in ALK+ ALCL, but its expression in the context of current molecular findings is unknown. Methods: ALCLs were centrally reviewed and classified by current WHO/ICC criteria. BCL2 was performed using IHC and scored as positive if ≥30% of tumor cells showed staining. DUSP22-R was assessed by fluorescence in situ hybridization (FISH) using a breakapart probe set and a dual-color, dual-fusion (D) probe set for the most common DUSP22 translocation, t(6;7)(p25.3;q32.3). pSTAT3Y705 IHC, which is >90% accurate for Type I/II assignment based on gene expression, was performed and scored based on a previously validated cut-off of ≥30%. Results: We evaluated 217 ALCLs (148M/69F; age: mean, 57 y; range, 5-93 y), including 55 systemic ALK+ ALCLs, 127 systemic ALK− ALCLs, and 35 primary cutaneous (pc) ALCLs. Overall, 86/217 cases were positive for BCL2 (40%). BCL2 was positive in 67/127 ALK− ALCLs (53%) and 15/35 pcALCLs (43%; P=NS), but in only 4/51 ALK+ (7%; P<0.0001). DUSP22-R was present in 90 cases (63 ALK− ALCL and 27 pcALCL). BCL2 was positive in 57/90 cases with DUSP22-R (63%) compared to 29/127 without DUSP22-R (23%; P<0.0001). Among 85 cases with DUSP22-R and informative D-FISH testing, BCL2 was positive in 24/36 cases with t(6;7)(p25.3;q32.3) (67%) and 31/49 cases without t(6;7)(p25.3;q32.3) (63%; P=NS). Among 213 cases with informative pSTAT3Y705 staining, BCL2 was positive in 13/88 pSTAT3+ cases (Type I, 15%) and 70/125 pSTAT3− cases (Type II, 56%; P<0.0001). Conclusions: We identified a complex pattern of BCL2 expression across ALCLs that strongly correlates with a recently introduced molecular classification based on gene expression profiling. While our findings are in keeping with the previously reported low expression in ALK+ ALCL, we found that the higher expression in both systemic ALK− ALCL and pcALCL is particularly associated with DUSP22-R and the absence of pSTAT3Y705 expression (Type II ALCLs). The discovery of 2 overarching types of ALCL associated with the presence or absence JAK-STAT3 pathway activation represents a new conceptual framework for classifying ALCL and understanding its therapeutic vulnerabilities. Of note, previous data demonstrated that Type II ALCLs are enriched for epigenetic alterations and particularly overexpress the histone methyltransferase EZH2. Correlation between BCL2 expression and EZH2 expression also is observed in other lymphomas such as diffuse large B-cell lymphoma, where dual targeting with EZH2 and BCL2 inhibitors has been proposed. Thus, our findings provide further rationale for stratifying Type I and Type II ALCLs and identify BCL2 as a differentially expressed oncoprotein that might represent a target for precision therapy approaches.

Background: Anaplastic large cell lymphomas (ALCLs) represent a heterogeneous group of T-cell lymphomas that currently are classified by the presence or absence of ALK tyrosine kinase (TK) fusion genes (ALK+ or ALK−) and clinical presentation (systemic, cutaneous, or breast implant-associated). Two overarching molecular types of ALCL recently were discovered, defined by the presence (Type I) or absence (Type II) of a gene expression signature highly enriched for JAK-STAT3 activation. Fusions involving non-ALK TK genes occur in some ALK− cases, but the fusion landscape of ALCL remains incompletely characterized. Methods: Expert consensus pathology review was conducted in the Lymphoma/Leukemia Molecular Profiling Project (LLMPP). RNAseq was performed and fusions were identified using FusionCatcher. Here, we focused on recurrent in-frame coding fusions. Previously unreported fusions were validated by RT-PCR. Type I/II was assigned using a previously validated gene expression-based model. Genes with adjusted (adj) P<0.05 were considered differentially expressed. Overall survival (OS) was assessed for systemic ALCL, when available. Results: We evaluated 379 ALCLs (229M/150F; mean age, 56 y). Of 199 candidate fusions (excluding reciprocal events), 28 were recurrent and passed quality metrics. At least 1 of these 28 fusions was present in 150 cases (40%). ALK fusions were present in 106 ALK+ ALCLs (all Type I; P<0.0001). Of these, NPM1::ALK was seen in 68/106 (64%). Alternate partners (X::ALK) included ATIC (N=24) and CTLC, COL1A2, MSN, MYH9, RNF213, SATB1, TFH, TPM3, and TRAF1 (1-3 cases each). X::ALK was associated with older age (mean, 52 y) than NPM1::ALK (32 y; P<0.0001) and showed relative overexpression of 49 genes, including multiple activators of small GTPases such as CGNL1 (FC, 5.6; Padj=2.4×10-6), SRGAP1, ALS2, DOCK1, and NCKAP1. ALK expression was similar in cases with NPM1::ALK and X::ALK and there was no significant difference in OS. Non-ALK TK fusions were seen in 17/273 ALK− cases (6%), including TYK2 (N=9); JAK2 (N=6); and ROS1 (N=2). All were Type I ALCLs (P<0.0001). ALK− cases with non-ALK TK fusions overexpressed 38 genes compared to ALK− cases without TK fusions, including cytokines involved in the IL17 signaling pathway such as CXCL6 (FC, 12.5; Padj=4.2×10-3), CXCL1, and CSF3. We then assessed the relationship between ALK− cases with non-ALK TK fusions and ALK+ cases by separately comparing each subset to ALK− cases without TK fusions. FC values for ALK− cases with non-ALK TK fusions were significantly correlated with FC values for ALK+ ALCLs over the set of expressed genes (R=0.54; P<0.0001). At a median follow-up of 32 months, 0/4 systemic ALCL patients with non-ALK TK fusions had died, while 30% of those with ALK− ALCL without TK fusions had died, but this was not statistically significant (P=0.26). The other 13 ALK− cases with non-ALK TK fusions were either localized ALCLs or did not have outcome data available. Of 20 TP63 fusions, 17 (85%) were TBL1XR1::TP63 and 3 were X::TP63. As previously reported, TP63 fusions were associated with poor OS (median OS, 10 months; not reached for other ALK− ALCLs; P=0.007). Four ALCLs had a novel NCL::UBTF fusion involving genes encoding the nucleolar proteins nucleolin and nucleolar (upstream binding) transcription factor-1. Four cases each had PARG::BMS1 and TNK1::GPS2 (the latter seen only in Type I ALCLs). Conclusions: This large consortium-based analysis of coding fusions further elucidates the molecular distinction between Type I and Type II ALCLs. Both ALK and non-ALK TK fusions were seen exclusively in Type I ALCLs, which we have shown previously are enriched for JAK-STAT3 pathway genes and correlate strongly with pSTAT3Y705 positivity by immunohistochemistry. These fusions were not identified in Type II ALCLs, which previous data suggest are associated with epigenetic alterations. ALK+ ALCLs with NPM1::ALK and X::ALK showed differences in gene expression, suggesting biological differences. Furthermore, ALCLs with non-ALK TK fusions shared gene expression features with ALK+ ALCL. Clinical fusion detection could enhance molecular classification of Type I and Type II ALCLs and may guide precision therapy.