Genomic Landscape of Young ATLL Patients Identifies Frequent Targetable CD28 Fusions
Authors: Yoshida Noriaki , Shigemori Kay , Ng Samuel Y. , Nirmal Ajit , Donaldson Nicolas , Cordero Nicolas , Stevenson Kristen E. , Bu Xia , Takeuchi Mai , Ohshima Koichi , Yoda Akinori , Weinstock David M 
Published: November 2019

Introduction
Adult T-cell leukemia/lymphoma (ATLL) is a T-cell neoplasm induced by human T-cell leukemia virus type 1 (HTLV-1). In endemic areas, HTLV-1 infection typically occurs during breastfeeding but the median age of ATLL presentation in Japan is >70. Approximately 5% of ATLL patients in Japan present at age <50. Here, we hypothesized that ATLL in younger patients will have distinct genetic alterations.
Methods
DNA and RNA samples were extracted from frozen lymph nodes. Targeted capture sequencing for 98 genes and analysis of RNA sequencing (RNAseq) were performed in 8 samples of young patients with ATL (defined as ≤ 50 years in the current study)
Results and discussion
The most frequently mutated genes were CCR4 and CARD11, each in 3 of the analyzed cases (37.5%). Mutations of PLCG1, PRKCB and STAT3 were frequent in a previous genomic analysis (Kataoka et al. Nat Genet. 2015) that were not age-selected, but were not identified in our 8 cases.
Three (37.5%) cases harbored concurrent CTLA4-CD28 and ICOS-CD28 fusions. This contrasts with previous reports, in which cases with both fusions were found in <1% of PTCL and ATLL patients. We confirmed the presence of both fusions in all 3 cases by RT-PCR. The structure of the CTLA4-CD28 fusion suggests that the extracellular portion of CTLA4 is expressed on the cell surface where it can interact with CD80 and CD86 to activate signaling through the intracellular CD28 portion. In contrast to CTLA4-CD28, the ICOS-CD28 fusion links the N-terminal signal peptide of ICOS with the extracellular and intracellular portions of CD28. This fusion should simply result in overexpression of CD28 and haploinsufficiency of ICOS.
To evaluate the function of CTLA4-CD28, we used Ba/F3 cells, which are strictly dependent on exogenous IL-3 for proliferation. We transduced Ba/F3 cells with CTLA4-CD28 or a CTLA4-CD28 mutant (mut) with three amino acid substitutions that abrogate CD28 signaling. We then cultured these cells in the presence or absence of Raji cells, which express CD80 and CD86. In the absence of Raji cells, neither CTLA4-CD28 nor CTLA4-CD28mut expression conferred IL-3-independent growth. However, Ba/F3 cells expressing CTLA4-CD28 cultured in the presence of Raji cells achieved IL-3 independence. This was not the case for Ba/F3 cells expressing CTLA4-CD28mut, indicating a requirement on functional CTLA4-CD28 signaling. Western-blot analysis identified activation of CD28 signal in Ba/F3 cells with CTLA4-CD28 co-cultured with Raji cells. These data indicate that ligand-bound CTLA4-CD28 can promote downstream signaling in the absence of an endogenous TCR complex. Gene set enrichment analysis (GSEA) comparing the 3 ATLL cases with CD28 fusions to the 5 cases that lacked fusions demonstrated enrichment of previously defined gene signatures associated with AKT and RAF signaling, both of which are downstream of CD28 activation. GSEA also identified enrichments of T-cell function-related signatures, including pathways involved in interferon responses, in cases with CD28 fusions
Cases with CTLA4-CD28 and ICOS-CD28 fusions become haploinsufficient for CTLA4 and ICOS. As expected, these cases had lower expression of both genes by RNAseq. Interestingly, these cases also had higher expression of CD80. By immunohistochemistry, ATLL cells with CTLA4-CD28 and ICOS-CD28 fusions expressed CD80 and macrophages in the tumor microenvironment expressed CD86. Thus, both intra- and intercellular interactions could drive CTLA4-CD28 and ICOS-CD28 signaling in these cases.
Treatment of Ba/F3 cells expressing CTLA4-CD28 and cultured in the presence of Raji cells with a CTLA4 blocking antibody suppressed proliferation in a dose-dependent fashion. A previous case report (Mol Genet Genomic Med, 2015) described a patient with Sezary Syndrome and CTLA4-CD28 fusion who had a deep but transient response to the anti-CTLA4 antibody ipilimumab. This study did not clarify the extent to which response resulted from blocking of cell-autonomous CTLA4-CD28 signaling versus activation of a cell non-autonomous immune response. Nonetheless, it strongly supports the testing of CTLA4 blockade in additional cases of T-cell malignancies, including ATLL, that harbor CTLA4-CD28 fusions.

Disclosures
Stevenson: Celgene: Research Funding. Ohshima:Kyowa Kirin Co., Ltd.: Honoraria, Research Funding; Chugai Pharmaceutical Co., Ltd.: Honoraria, Research Funding; Celgene Corp.: Honoraria, Research Funding; NEC Corp.: Research Funding; SRL, Inc.: Consultancy. Weinstock:Verastem Oncology: Research Funding; Celgene: Research Funding.