Genomic and transcriptomic analyses of rare lymphomas

Post-transplant lymphoproliferative disorders (PTLDs) are heterogenous and potentially fatal disorders that arise in up to 20% of organ transplant recipients. According to World Health Organization (WHO), three major categories are distinguished: benign early lesions, polymorphic PTLD, and malignant monomorphic PTLD (M-PTLD). The latter category comprises hard-to-treat lymphoma entities of which diffuse large B-cell lymphoma (DLBCL) is the most common subtype. The molecular pathogenesis of posttransplant DLBCL (PT-DLBCL) is largely unknown. DLBCL is the most common non-Hodgkin’s lymphoma and typically has an aggressive behavior that if not treated would be immediately fatal. For most patients it is curable with immunotherapy but form some this therapy fails and they die. Less common M-PTLD include, among other, Burkitt lymphomas and the very rare Hepatosplenic T-cell lymphoma (HSTL). Burkitt Lymphoma (BL) is an aggressive, GCB-derived malignancy and one of its variants is the immunodeficiency-associated BL arising in patients with HIV infection or in those who underwent an organ transplant and immunosuppressive therapy. This tumor is biologically and molecularly hallmarked by IG-mediated  translocation t(8q24) resulting in upregulation of the oncogene MYC.  HSTL is also an aggressive lymphoma occurring predominantly in young male. Cytogenetically it is characterized by isochromosome 7q [i(7)(q10)], but the molecular consequences of this alteration remain unknown. Sporadically a ring chromosome 7 (r7) have been observed. The disease is also characterized by a rapid downhill progression and very poor responsiveness to treatment. Its clinical presentation consist of splenomegaly and hepatomegaly with no visible lymphadenopathy. The diagnostic is challenging because the symptoms can be shared by many other pathological conditions.

In this PhD work we performed an integrative genomic and transcriptomic analyses of these rare lymphomas using cytogenetics (classical and molecular), SNP-arrays, expression arrays, RNA-seq and a set of bioinformatics methodologies. The results are summarized below.

HSTL: We analyzed six i(7)(q10)-positive HSTL cases and three cases with ring 7 [r(7)]. Using aCGH, we profiled all cases and mapped the common deleted region (CDR) at 7p22.1p14.1 and the common gained region (CGR) at 7q22.11q31.1. The CGR spans a smaller 13 Mb region constantly amplified in cases with r(7). We found that TCRG (7p14.1) and TCRB (7q32) are involved in formation of r(7), probably a byproduct of somatic rearrangement of these loci. Transcriptomic analysis didn’t identified any CDR-related tumor suppressor gene, instead, loss of 7p22.1p14.1 was associated with an enhanced expression of CHN2 (7p14.1) and the encoded β2-chimerin. Gains of 7q22.11q31.1 associated with an increased expression of several cancer-related genes including ABCB1, a multidrug resistance gene. RNA-seq did not identify any disease-defining mutation neither gene fusions, leaving chromosome 7 imbalances as the only event driving the pathogenesis in this tumor. We hypothesize that the ∆7p22.1p14.1-associated enhanced expression of CHN2/β2-chimerin leads to downmodulation of the NFAT pathway and to a proliferative response, while upregulation of the CGR-related genes provides growth advantage and chemoresistance. Our study also identifies a gene signature comprising 24 genes distinguishing HSTL from other malignancies.

PT-DLBCL: we have shown previously that Epstein-Barr Virus-positive (EBV+) and -negative (EBV-) PT-DLBCL have distinct gene expression profiles, and the transcriptomic profile of EBV-PT-DLBCL is similar to that of DLBCL in immunocompetent individuals (IC-DLBCL).  To study if these differences/similarities between the 3 groups also hold at the genomic level, we performed aCGH on 21 EBV+ PT-DLBCL, 6 EBV- PT-DLBCL, and 11 control IC-DLBCL. The analysis showed EBV+ and EBV- PT-DLBCL have distinct genomic profiles and shared only one recurrent imbalance. EBV- PT-DLBCL, however, displayed at least 10 aberrations which were also present in IC-DLBCL including gain of 3/3q and 18q and losses of 6q23/TNFAIP3 as well as 9p21/CDKN2A. Gain/amplification of 9p24.1 targeting PDCD1LG2/PDL2 was the most prevalent aberration in EBV+ PT-DLBCL. Our study indicate that the oncogene FOXP1 and the tumor suppressor CDKNA2 may be implicated in EBV- DLBCL but do not play critical role in the pathogenesis of EBV+ PT-DLBCL. Altogether, genomic profiling of PT-/IC-DLBCL confirms that EBV- and EBV+ PT-DLBCL are distinct entities, while EBV- PT-DLBCL has features in common with IC-DLBCL. These findings support the hypothesis that EBV- PT-DLBCL are de novo lymphomas in transplant recipients

PT-BL: Recent studies of 59 molecular BL (mBL) identified a novel aberration manifested by a specific 11q-gain/loss pattern in two cases lacking MYC translocation. The aberration was subsequently detected in 15 MYC-negative high-grade B-cell lymphomas resembling BL and two high-grade B-cell lymphomas cell lines. Our study provide evidence that this 11q-gain/loss aberration is particularly frequent in BL in immunodeficient hosts, as it was identified in three out of seven patients with mBL  which underwent solid organ transplantation and  immunosuppressive therapy. The seven posttransplant BL cases reported here were analysed using conventional cytogenetics, aCGH, FISH, immunohistochemistry, gene expression profiling and bioinformatics. As controls, we included four cases of typical MYC-translocation-positive BL from immunocompetent hosts.  Altogether, we confirmed a recurrent occurrence of the 11q-gain/loss pattern in high grade  B-cell lymphoma and showed that this aberration is significantly more frequent in BL occurring in the setting of transplantation and immunosuppression than in immunocompetent patients,  suggesting that immunosuppression may favor its formation. As identification of patients with the 11q-gain/loss aberration is clinically important but cytogenetically challenging, we recommend the designed 11q-MGR/MLR FISH assay as a useful diagnostic tool to evaluate both, posttransplant- and immunocompetent BL patients.