Genomic Landscape of Young ATLL Patients Identifies Frequent Targetable CD28 Fusions

Abstract

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.

Clinicopathological significance of CD28 overexpression in adult T‐cell leukemia/lymphoma

“…Second, it was difficult to estimate which of the CD28 genes, the altered type or wild type, contributed more to the CD28 overexpression. Third, consistent with earlier reports, 22,23 we definitively detected the presence of tumor cells ectopically overexpressing CD80/CD86 in some ATLL cases.…”

“…In addition, there have been recent reports of ectopic expression of CD80 and CD86 on tumor cells in some ATLL cases, although these molecules were originally expressed on APCs and not on T cells. 22,23 These observations suggest that intracellular and intercellular interactions via the CD80/CD86-CD28 pathway may exist and contribute to ATLL pathogenesis.…”

Detection of Gene Fusion Transcripts in Peripheral T-Cell Lymphoma Using a Multiplexed Targeted Sequencing Assay

“…52,53 In agreement with previous studies, 9,10,21 ICO-Se1_CD28e2 fusion was found in 9 of 117 (7.7%) of PTCLs of TFH derivation (8 AITLs and 1 TFH-PTCL), but in only 1 of 46 PTCL-NOS and 1 of 8 ATLLs. The young age of patients with a CD28 fusion recently reported in ATLL patients 54 was not observed in this study (mean age, 64 years), and the patient harboring the double fusion ICO-Se1_CD28e2 associated with CTLA4e3_CD28e4 was 75 years old. The current study further confirms the low frequency of CTLA4e3_CD28e4donly one casedamong TFH-PTCL.…”

HTLV-1-related adult T-cell leukemia/lymphoma: insights in early detection and management

“…Similar mutational patterns to those found in the Japanese cohorts were identified in patients of African and Caribbean ancestry [25 ▪ ], with a smaller study noting a high frequency of mutation in epigenetic and histone-modifying genes, notably EP300 [26]. Fusions in CD28 are not infrequent, and these may be more prevalent in patients under the age of 50 [27 ▪▪ ]. More recently, a whole genome sequencing approach has identified new variants in ATL, including CIC (31% of cases) and ITGB1 (5%) [28].…”