Comprehensive Genomic Profiling (CGP) of Angioimmunoblastic T-Cell Lymphoma (AITL) to Prospectively Inform Diagnosis and Clinical Management
Authors: Morley Samantha , He Jie , Intlekofer Andrew M. , Bailey Mark , Nahas Michelle , Lipson Doron , Yelensky Roman , Ross Jeffrey , Ali Siraj , Erlich Rachel , Stephens Phil , Miller Vincent A. , Vergilio Jo-Anne , Mughal Tariq I 
Published: October 2019

Abstract
Background: AITL is a rare mature T-cell lymphoma with an aggressive clinical course. Accurate diagnosis is confounded by low tumor content limiting molecular characterization, histologic resemblance to peripheral T-cell lymphoma (PTCL), and a scarcity of sensitive diagnostic tools for routine sample analysis. Also, to date, no confirmed standard of care exists; most patients receive combination chemotherapy, but this approach is largely ineffective. Several research studies report RhoAG17V as a genomic hallmark of AITL that often co-occurs with somatic mutations in epigenetic regulators (TET2, IDH2, or DNMT3A); however these findings have yet to be translated in the clinic. The detection of clinically-relevant genomic alterations in AITL has the potential to improve outcome for this patient population by identifying targeted therapeutic agents. CGP provides an opportunity to address these collective unmet needs; herein, we report on our experience using this methodology in AITL.
Methods: To assess RhoAG17V specificity as a diagnostic marker of AITL, RhoA mutation was explored in an unbiased fashion across the Foundation Medicine database. This led to inclusion in the study of 21 patient specimens with diagnoses of suspected AITL (4), confirmed AITL (12), PTCL (2), or other neoplasms (3). Prior autologous stem cell transplant was noted in 2 of 12 patients with confirmed AITL and in 1 with PTCL. Four patients with AITL were HHV-4 positive. Median age of the cohort was 64y (range 38-88y). Using the FoundationOne® Heme platform, DNA and RNA were extracted from samples submitted as either FFPE material (lymph nodes, skin, soft tissue) or blood. Adaptor ligated sequencing libraries were captured by solution hybridization using a custom bait set targeting 405 cancer-related genes, and 265 genes involved in cancer-related fusions. All captured libraries were sequenced in a CLIA-certified, CAP-accredited, NYS-approved laboratory (Foundation Medicine, Inc).
Results: RhoA G17V was found in 69% (11/16) of AITL samples; none harbored RhoA mutation at sites other than G17. In cases of suspected AITL, RhoAG17V was found in 3/4 samples, and in 8/12 of those confirmed. TET2 mutations, predominantly prematurely truncating and inactivating, were identified in 100% (16/16) of AITL cases, exceeding reported frequencies. Mutation of IDH2, affecting exclusively R172, was observed at a frequency of 31% (5/16), while DNMT3A was observed at a frequency of 25% (4/16) and was most commonly inactivating. In all 5 AITL samples with IDH2 mutation, RhoA and TET2 mutation also co-occurred. In PTCL samples lacking an AITL diagnosis, mutation in RhoA, TET2, IDH2, and DNMT3A was detected in 1/2, 2/2, 0/2, and 1/2 cases, respectively. An unbiased query of the Foundation Medicine database identified RhoAG17V mutation in 1 sample each of pleomorphic myxofibrosarcoma (MFS), marginal zone lymphoma (MZL), and cutaneous T-cell lymphoma (CTCL); in contrast to cases of AITL, these samples were devoid of TET2 and IDH2 mutations.
Conclusion: Using CGP, AITL samples harbored co-occurring RhoA/TET2 mutations at high frequency. Importantly, RhoAG17V has been proposed as a diagnostic marker for this aggressive PTCL subtype. Our findings are suggestive that assessing mutations in TET2 simultaneously with that of RhoA may have greater diagnostic utility than RhoA alone. All samples with concurrent RhoAG17V/TET2 mutation were from patients with suspected or confirmed AITL, with one exception. A single case was diagnosed as PTCL containing immunoblastic proliferations reminiscent of AITL morphology. In contrast, while RhoAG17V was detected in other malignancies (MFS, MZL, CTCL), these lacked TET2 and IDH2 mutation, suggesting the specificity of the co-occurring mutations for AITL. Co-occurrence of RhoAG17V/TET2 mutation, identified by CGP, may, therefore, enable genomic reclassification to AITL from other mature T-cell neoplasms when used in conjunction with other clinical criteria.
CGP may furthermore inform therapeutic approaches for AITL. Detection of frequent and co-occurring alterations in epigenetic regulators suggests DNA hypermethylation is common in this lymphoma and implicates hypomethylating agents as relevant. Studies are currently under way to examine clinical responses of AITL patients with IDH2 or TET2 mutations to IDH2 inhibitors and hypomethylating agents, respectively.

Disclosures
Morley: Foundation Medicine, Inc.: Employment, Equity Ownership. He:Foundation Medicine, Inc.: Employment, Equity Ownership. Bailey:Foundation Medicine, Inc.: Employment, Equity Ownership. Nahas:Foundation Medicine, Inc.: Employment, Equity Ownership. Lipson:Foundation Medicine, Inc.: Employment, Equity Ownership. Yelensky:Foundation Medicine, Inc.: Employment, Equity Ownership. Ross:Foundation Medicine, Inc.: Employment, Equity Ownership. Ali:Foundation Medicine, Inc.: Employment, Equity Ownership. Erlich:Foundation Medicine, Inc.: Employment, Equity Ownership. Stephens:Foundation Medicine, Inc.: Employment, Equity Ownership. Miller:Foundation Medicine, Inc.: Employment, Equity Ownership. Vergilio:Foundation Medicine, Inc.: Employment, Equity Ownership. Mughal:Foundation Medicine, Inc.: Employment, Equity Ownership.