Genomic and Targeted Mutational Analysis of T/NK-Cell Post-Transplant Lymphoproliferative Disorders Provides Insight into Disease Biology

Background

Post-transplant lymphoproliferative disorders (PTLDs) encompass heterogeneous entities with different histomorphology and behavior. T/NK-cell PTLDs (T/NK-PTLDs) are rare, accounting for 2-15% of all PTLD, which are often EBV-negative and associated with a poor prognosis. The pathogenesis of T/NK-PTLDs and the underlying molecular alterations are presently unknown.  In order to better understand their pathogenesis, we performed high resolution DNA profiling for copy number alterations and targeted mutational analyses of 18 T/NK-PTLDs.   

Methods

Cases of T/NK-PTLD were selected from the archives of two institutions.  Morphological features were assessed and immunohistochemistry (IHC) was performed to classify lymphomas according to the WHO 2008 criteria. DNA was extracted from formalin-fixed paraffin-embedded tissue. Copy number analysis (n=16; Affymetrix Oncoscan FFPE Assay) and targeted next-generation sequencing for 467 cancer-associated genes, after capture of all exons or select whole genes (n=17; Illumina HiSeq 2500), were performed.  Variants with allele frequency <10%, allele prevalence >1% in 1000 genomes project, read depth <10x, or quality score <10 were excluded and cross referenced with COSMIC, PROVEAN, and SIFT databases.  Mismatch repair deficiency was assessed using IHC and PCR analysis for a panel of mononucleotide repeat markers (Promega).  Cases exhibiting microsatellite instability (MSI) at ≥2 loci were classified as MSI-high.

Results

The T/NK-PTLDs represented virtually the entire spectrum of currently recognized T- and NK-cell lymphomas. Peripheral T-cell lymphoma (PTCL), NOS were the most frequent (n=6), extranodal location was common (n=14, 78%) and only a minority were EBV+ (n=2, 11%). Copy number alterations were observed in 13 cases (81%; complex =11, simple=2). In contrast to B-cell PTLDs, EBV+ T/NK-PTLDs showed more complex copy number changes than EBV- cases. Somatic mutations were detected in 17/18 cases (94%). Overall, 343 variants were identified: 277 missense (80%), 40 indels (12%) and 14 nonsense (4%). An average of 20.1 variants/case was observed with an average 594.8-fold coverage.

Mutations of epigenetic mediators and chromatin remodeling complex genes, including TET2 (n=5), MLL2 (n=4), MLL3 (n=4), DNMT3a (n=3), ARID1B (n=3) and ARID2 (n=2), were the most frequent alterations, seen in 11/17 (65%) cases. 

Disruption of TP53 was identified in 6/18 (33%) cases; biallelic inactivation in 3 cases with co-occurrence of missense (n=2) or nonsense (n=1) mutation and 17p LOH, monoallelic missense mutations in two cases, and loss of 17p encompassing the p53 locus in one case. 

Mutually exclusive activating STAT3 and STAT5B mutations were identified in 5/17 (29%) cases.  Mutations targeting the STAT3 SH2 domain were seen in one EBV- cutaneous anaplastic large cell lymphoma and two EBV+ PTLDs (PTCL, NOS and extranodal NK/T cell lymphoma (ENKTCL)). Both EBV+ cases also showed LOH of the STAT3 locus on 17q and 6q loss encompassing PTPRK, the phosphorylase responsible for downregulation of STAT3 signaling. A  JAK3 variant (S568P) adjacent a known hotspot was also identified in the ENKTCL.

Missense mutations in RHOA and FYN were detected in one angioimmunoblastic T-cell lymphoma (AITL). Mutations were also observed in genes not previously implicated in T/NK-cell lymphomas, including potentially damaging missense variants in MED12 (n=3) and TBX3 (n=3), and a novel missense MTOR mutation (W1456L) involving the FAT domain.

Recurrent copy number alterations comprised gains of 7q (n=4, including 2 hepatosplenic T-cell lymphomas [HSTCL]), 8q (n=3; MYC) and 9q (n=2; ABL1, SYK), and losses of 7p (n=4) and 6q (n=3; FOXO3, PRDM1).

Contrary to prior reports, MSI was uncommon in our series despite use of azathioprine therapy (n=9), with only 1 of 3 cases harboring mutations in mismatch repair genes was confirmed to be MSI-H.

Conclusion

Our analysis of a large series of T/NK-PTLDs represents the first survey of genomic and genetic changes in these lymphomas, and adds to the growing list of genes and pathways altered in T/NK-cell neoplasms. Our findings suggest shared genomic and molecular aberrations with T-cell lymphomas occurring in “immunocompetent” individuals. Functional studies are needed to assess if some of the observed genetic alterations could serve as therapeutic targets for these aggressive neoplasms.