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MAPK and JAK-STAT pathways dysregulation in plasmablastic lymphoma

Plasmablastic lymphoma (PBL) is an aggressive B-cell lymphoma with an immunoblastic/large-cell morphology and terminal B-cell differentiation. The differential diagnosis from Burkitt lymphoma, plasma cell myeloma and some variants of diffuse large B-cell lymphoma may be challenging because of the overlapping morphological, genetic and immunophenotypic features. Furthermore, the genomic landscape in PBL is not well known. To characterize the genetic and molecular heterogeneity of these tumors, we investigated 34 cases of PBL using an integrated approach, including fluorescence in situ hybridization, targeted sequencing of 94 B-cell lymphoma-related genes, and copy-number arrays. PBL were characterized by high genetic complexity including MYC translocations (87%), gains of 1q21.1-q44, trisomy 7, 8q23.2- q24.21, 11p13-p11.2, 11q14.2-q25, 12p and 19p13.3-p13.13, losses of 1p33, 1p31.1-p22.3, 13q and 17p13.3-p11.2, and recurrent mutations of STAT3 (37%), NRAS and TP53 (33%), MYC and EP300 (19%) and CARD11, SOCS1 and TET2 (11%). Pathway enrichment analysis suggested a cooperative action between MYC alterations and MAPK (49%) and JAK-STAT (40%) signaling pathways. Of note, Epstein-Barr virus (EBV)-negative PBL cases had higher mutational and copy-number load and more frequent TP53, CARD11 and MYC mutations, whereas EBVpositive PBL tended to have more mutations affecting the JAK-STAT pathway. In conclusion, these findings further unravel the distinctive molecular heterogeneity of PBL identifying novel molecular targets and the different genetic profile of these tumors in relation to EBV infection.


Introduction
Plasmablastic lymphoma (PBL) is an aggressive lymphoid neoplasm characterized by a diffuse proliferation of large neoplastic cells with an immunoblastic or plasmablastic morphology but expressing a plasma cell phenotype including positivity for BLIMP1/PRDM1, XBP1, CD138, CD38, Vs38c and MUM1/IRF4 and negativity or weak positivity for CD45 and markers of mature B cells such as CD20 and PAX5. CD79a is positive in approximately 50-85% of the cases. 1,2 This tumor was initially described as a subtype of diffuse large B-cell lymphoma (DLBCL) present in the oral cavity of patients positive for human immunodeficiency virus (HIV). Subsequent studies expanded the spectrum of presentation of this lymphoma to other extranodal sites and immunodeficient conditions such as iatrogenic immunosuppression associated with post-transplant therapy or chronic treatments for autoimmune diseases and aging. 1,3,4 Epstein-Barr virus (EBV) is known to be an important element in the development of PBL, especially in HIV-positive patients, and can be detected in 60-75% of PBL cases by in situ hybridization. 1,4,5 The role of EBV infection has been related to the anti-apoptotic effect in B cells through several mechanisms related to EBV antigens. 4 Around 50% of PBL carry immunoglobulin (IG)/MYC rearrangements, a hallmark of Burkitt lymphoma (BL). This rearrangement seems to be observed more frequently in EBV-positive than in EBV-negative PBL (74% vs. 43%). 4,6,7 Few studies have investigated the genetic landscape of PBL. Copy number profiling revealed that recurrent gains of 1p36, 1p36-p34, 1q21-q23, 7q11, 11q12-q13 and 22q12-q13 are common features of PBL. 8 More recently a 34-gene targeted next-generation sequencing study highlighted PRDM1 and STAT3 as key genes in the pathogenesis of PBL in addition to MYC. 9 Furthemore, during the preparation of this manuscript, a genomic analysis of HIV-positive PBL patients from South Africa identified recurrent mutations in the JAK-STAT and RAS-MAPK signaling pathways and recurrent copy number alterations including large chromosomal gains of 1q and chr7 and focal gains in 6p22.1, 1q21.3 and 11p13 but the possible differential representation of these alterations in relationship to EBV infection could not be defined. 10 A gene expression profiling study has shown that, compared to DLBCL, PBL downregulates B-cell receptor signaling genes including transcriptionally activated targets of nuclear factor κB (NFκB) signaling and upregulation of MYB and MYC target genes. 11 Some PBL share morphological and immunophenotypic features with other aggressive lymphoid neoplasias such as BL, plasmablastic transformation of plasma cell myeloma (PCM) and some variants of DLBCL with immunoblastic features and/or activated B-cell like (ABC) cell of origin. 2,12,13 Of note, plasmablastic PCM has been associated with MYC translocations and worse prognosis, 4,14 which adds difficulty to the already challenging differential diagnosis. The management of patients with PBL is still not standardized and most patients are treated with cyclophosphamide, doxorubicin, vincristine, and prednisone (CHOP) or CHOP-like regimens. However, responses are relatively poor with a median overall survival of about 1 year. 1,3,15 More intensive regimens, such as infusional EPOCH, Hyper-CVAD, or CODOX-M/IVAC, have also been used without improving the prognosis. 16 On the other hand, bortezomib, a proteosome inhibitor used in the treatment of PCM, has shown potential efficacy in PBL with promising early results. 17 A better understanding of the genetic profiling of PBL may substantiate its distinction from other entities and may contribute to the design of novel therapeutic strategies. We performed a high-resolution genetic analysis of PBL to discover the hallmarks of this disease which may enhance greater diagnostic accuracy and provide insight into the clinical behavior of this lymphoma.

Immunohistochemistry and fluorescence in situ hybridization
Immunohistochemical studies were performed using standard protocols. (Online Supplementary Table S1). Fluorescence in situ hybridization (FISH) analyses for the detection of BCL2, BCL6, MYC and IGH translocations were performed using standard techniques and commercial Dual Color Break-Apart Rearrangement Probes (Vysis, Abbott Molecular, Wiesbaden, Germany).

Copy number analysis
Copy number alterations were examined in 33 PBL samples using an Oncoscan FFPE Assay Kit (Thermo Fisher Scientific, Waltham, MA, USA) according to standard protocols. Gains and losses and copy number neutral loss of heterozygosity (CNN-LOH) regions were evaluated using Nexus Biodiscovery 9.0 software (Biodiscovery, Hawthorne, CA, USA). The proportion of tumor cells or cancer cell fraction carrying each copy number alteration (CCFCNA) was estimated from the B-allele frequency and corrected for the tumor cell content or purity of the sample obtained from ASCAT (Online Supplementary Methods). 18 Copy number alterations were considered clonal if their CCFCNA was ≥85%. 19 Previously published data from 35 BL, 41 PCM and 49 ABC-DLBCL were used for comparisons. [20][21][22][23]

Mutational analysis
Twenty-seven PBL were examined for the mutational status of 94 B-cell lymphoma-related genes (Online Supplementary Table S2) using a SureSelectXT Target Enrichment System Capture NGS strategy library (Agilent Technologies, Santa Clara, CA, USA) before sequencing with MiSeq equipment (Illumina, San Diego, CA, USA). The bioinformatic pipeline included a filtering process excluding intronic, synonymous and single nucleotide polymorphic variants and a selection of driver mutations with potential functional effect (Online Supplementary Methods, Online Supplementary Figure S1). The cancer cell fraction carrying each specific mutation (CCFmut) was calculated as previously described. 18 As applied to copy number alterations, mutations were classified Genetic landscape of plasmablastic lymphoma haematologica | 2021; 106 (10) as clonal or subclonal if their cancer cell fractions were ≥85% or <85%, respectively. 19 Previously published mutational data from 28 BL, 203 PCM and 295 ABC-DLBCL were used for comparisons. [24][25][26] Gene expression analysis A total of 12 PBL were analyzed using the Nanostring PanCancer Pathways Panel (NanoString, Seattle, WA, USA) to investigate gene expression differences between different subsets of PBL (Online Supplementary Methods, Online Supplementary Table  S3).

Statistical analyses
R software version 3.6.2 was used for statistical analyses. Differences in the distribution of individual parameters among subsets of patients were analyzed by the Fisher exact test for categorical variables, and the Student t-test for continuous variables. Nonparametric Wilcoxon tests were applied when necessary. Pvalues for multiple comparisons were adjusted using the Benjamini-Hochberg correction (false discovery rate). A P value <0.05 was considered statistically significant unless otherwise indicated.

Pathological features and fluorescence in situ hybridization
Twenty-four patients were male and ten female with a median age of 52 years (range, 11-87 years). HIV infection was confirmed in 14 out of 16 cases with available data (88%) and one patient (PBL6) had a post-transplant associated immunodeficiency. Seven patients for whom HIV information was not available were older than 60 years (Table 1). Most cases had an extranodal presentation (84%), mainly affecting the head and neck region (44%) and gastrointestinal tract (30%). As expected, virtually all cases were negative for B-cell markers, and only two cases showed weak expression of PAX5. Conversely, plasma cell markers such as CD138 and MUM1/IRF4 were positive in 68% and 100% of the cases, respectively. CD79a was expressed in 60% (18/30) of the tumors, usually with a focal pattern, and CD56 was positive in 21% (5/24) ( Table 1). EBV (as determined by EBV-encoded small RNA; EBER) was positive in 18 out of 30 (60%) cases whereas human herpes virus-8 was negative in all analyzed cases (Table 1; Figure  1A-H). MYC rearrangements were detected in 26 of the 30 (87%) analyzed cases, most of them with IGH as translocation partner (8/9 cases evaluated). Neither BCL2 nor BCL6 translocations were observed (Table 1, Figure 1I, J).
All STAT3 mutations except one were nonsynonymous variants located in the SH2 domain (positions 585 to 688). Two different hotspots were found within the SH2 domain including D661Y/V and Y640F (3 cases each), which have been previously described to upregulate STAT3 downstream target genes. 29 To assess the STAT3 activation status p-STAT3 was studied by immunhistochemistry in six cases (2 mutated and 4 wild-type). RNA expression of genes related to the JAK-STAT pathway (JAK-STAT pathway score), was also analyzed in 12 cases using the NanoString platform (Online Supplementary Methods). p-STAT3 was positive in four cases (67%), including the two mutated cases but also two wild-type cases. The four plasmablastic PCM studied were negative for p-STAT3 (Online Supplementary Figure S4, Online Supplementary Table S6). STAT3 mRNA expression was similar in mutated and unmutated cases but it showed a tendency to be higher in EBV-positive thannegative tumors (Wilcoxon test, P=0.07). (Online Supplementary Results, Online Supplementary Figures S4 and  S5). Additionally, there were no significant differences in the expression of JAK-STAT pathway-related genes between STAT3-mutated and wild-type cases (Wilcoxon test; P=0.15), although a tendency to higher expression was observed in EBV-positive cases (Wilcoxon test; P=0.1) (Online Supplementary Figure S6).
All 12 TP53 mutations were located in the DNA binding domain and three of them were stop-gain mutations ( Figure  2D). Bi-allelic inactivation of TP53 was observed in six cases, all negative for EBV expression, in which the 17p13/TP53 locus was also affected by losses or CNN-LOH. NRAS mutations mainly affected the known hotspots Q61 (4 cases) and G13 (3 cases). Finally, all but one exonic MYC mutations were located on the transactivation domain, which has been previously demonstrated to induce MYC stability and inhibition of apoptosis ( Figure  2D). 30 Interestingly, multiple mutations (considering >10 variants and including intronic and synonymous ones) were found in the five cases with MYC driver mutations and in one additional case with only intronic mutations, all six with concomitant MYC rearrangements. Additionally, a high rate of these mutations involved activated-induced cytidine deaminase (AID)-motifs (corresponding to sequences WA/TW/WRCY/RGYW/WGCW) 31 (65% vs. 39% expected; P<0.05) suggesting that those mutations fulfill a somatic hypermutation pattern (Online Supplementary  Table S7).

Clonal evolution in plasmablastic lymphomas
To analyze the hierarchy of genetic events in PBL, the cancer cell fraction was determined for 357 alterations (including 250 copy number alterations and 107 mutations) from 24 PBL cases, of which 31% were clonal (cancer cell fraction >85%) (Online Supplementary Tables S4 and S5). The majority of alterations showed a wide spectrum of cancer cell fractions, except for TP53 mutations, 17 losses and 13q deletions which were mainly clonal (Shapiro test; P<0.05) (Online Supplementary Figure S7). Interestingly, clonal and subclonal mutations targeting recurrently mutated genes (STAT3, NRAS, TP53 and MYC) affected the same protein domains, suggesting that those subclonal driver mutations confer similar advantage to the cell (Online Supplementary Figure S8).

E D
CNN-LOH. Our cases showed recurrent regions of amplification with some of them in the context of chromothripsislike patterns. Amplifications of 1q21 had already been described as an important prognostic marker in PCM. 34 We also identified IL6R, ADAR and CKS1B genes included in the 1q21.3-q23.2 amplified region. 27,28 These genes have been identified to drive disease aggressiveness in 1q21amplified PCM cases, and consequently, could be considered possible targets of the 1q21 amplification. 27,28 In detail, 1q21 amplification and overexpression of CKS1B have been described to confer poor prognosis in PCM, as a result of enhanced degradation of p27/Kip1. 28 Besides, IL6R and ADAR genes cooperate, inducing hyperactivation of the JAK-STAT pathway, leading to the transcription of pro-survival and anti-apoptotic genes in PCM. 27 Of note, the copy number profile identified in PBL differed from that in other lymphoma entities such as BL, ABC-DLBCL and PCM. PBL were genetically more complex than BL, and lacked common aberrations of ABC-DLBCL such as 9p21.3 losses and 18q gains or typical trisomies of the hyperdiploid profile found in PCM. On the other hand, PBL had specific alterations including gains of 12p (including KRAS) and 16q11.2-q24.3 CNN-LOH.
Regarding the mutational profile of PBL, the most frequently mutated genes were STAT3, NRAS and TP53, followed by MYC, EP300, CARD11, SOCS1 and TET2. These mutations mainly affect the MAPK pathway, epigenome/chromatin modifier genes, the JAK-STAT pathway and cell cycle genes. Altough some of these results have already been described (Online Supplementary Figure   S12), 9,10 we also demonstrated a previously undetected association between clonal TP53 alterations and EBV negativity. Interestingly, several of those genes could be targets for novel therapies (Online Supplementary Table S8). The identification of recurrent STAT3 and NRAS mutations is in line with recent results in HIV-positive PBL in South Africa. 10 Moreover, Garcia-Reyero et al. also found recurrent missense PRDM1 mutations, 9 which we were not able to identify in our cases. Several of those described PRDM1 mutations were predicted in the study as neutral, in contrast to the truncating PRDM1 mutations associated with ABC-DLBCL. 35 In our study we identifed recurrently mutated pathways that have an important role in the pathogenesis of PBL. In this sense, the MAPK pathway was the most frequently mutated pathway, with mutations in NRAS, KRAS, BRAF and MAP2K1 being found in 49% of the cases. This finding is similar to the recent observation in South-African patients and highlights its relevance in PBL. 10 KRAS and NRAS mutations were mainly located in the hotspot amino acid positions 13 and 61, previously described to impair intrinsic GTPase activity, leading to constitutive activation of the MAPK pathway. 36 Two BRAF mutations affecting the G469V/A position and located on the p-loop of the kinase domain were also recognized. These mutations have been demonstrated in lung cancer to induce a higher kinase activity compared to non-mutated BRAF. 37 These MAPK pathway mutations were also frequently found in PCM (46-59% of cases), leading to MEK/ERK activation and resulting in increased proliferation, growth and survival. 26,38 Genetic landscape of plasmablastic lymphoma haematologica | 2021; 106(10) 2689   26 including the most frequently mutated genes interrogated in both series (>10%). Asteriskes indicate differentially mutated genes between groups (false discovery rate <0.05).

A B C
The JAK-STAT pathway was also mutated in 40% of our cases. We found recurrent activating STAT3 variants located in the SH2 domain, in addition to inactivating mutations in SOCS1, a negative regulator of the JAK-STAT pathway. Activating STAT3 mutations and expression of the activated protein are frequently seen in T-cell and NK-cell malignancies but are rare in B-cell lymphomas, being previously described only in high-grade B-cell lymphoma, a subset of CD30 + DLBCL and ALK + large B-cell lymphoma, which also has a plasmablastic phenotype. 29,39,40 Moreover, these STAT3 mutations have been suggested to confer hydrophobicity, facilitating the activation of STAT3 through Y705 phosphorylation and the consequent upregulation of STAT3 downstream target genes. 29 Immunohistochemical staining for p-STAT3 demonstrated positivity in 67% of the cases independently of the STAT3 mutational status. The same results were obtained by Liu et al. In addition, no differences in the expression of JAK-STAT pathways were observed in STAT3-mutated and wild-type tumors. Although the number of cases is limited, these results suggest that PBL may have alternative mechanisms of STAT pathway activation. 10 Interestingly, plasmablastic PCM were negative for p-STAT3. The potential diagnostic value of this difference deserves further confirmation.
Independently of these activating mutations, the JAK-STAT pathway is also deregulated in PCM and ABC-DLBCL, promoting cell survival and proliferation. 41,42 In addition, the interaction of IL6 with its receptor IL6R has been described to induce STAT3 activation in PCM cell lines. 43 Interestingly, a newly established PBL cell line has been demonstrated to be dependent on IL6 for proliferation and survival. 44 All these findings suggest the importance of IL6/JAK/STAT3 signaling in the pathogenesis of PBL disease.
On the other hand, in addition to MYC rearrangements, genes governing the cell cycle were also affected in PBL by other mechanisms including bi-allelic inactivation of important regulator genes such as TP53 and CDKN2A. Of note, these TP53 alterations, including mutations and deletions, were restricted to EBV-negative PBL, without other associated clinical, morphological or molecular features. The low frequencies of TP53 mutations identified in previous studies is probably due to the small number of EBV-negative cases in those series (Online Supplementary Figure S12). 8,10 Interestingly, the mutational landscape of PBL differed from that of BL since it lacked variants affecting genes related to BL pathogenesis such as ID3, SMARCA4 and TCF3. Moreover, compared with ABC-DLBCL, PBL lacked typical MYD88-L265P and CD79B mutations, affecting NFκB pathway. Finally, PBL resembles PCM for the presence of mutations affecting the MAPK pathway. 26 Mutations in this pathway were detected as clonal or subclonal in different cases. In contrast, TP53 alterations and NRAS mutations were frequently clonal, suggesting that they are early events in PBL in addition to MYC rearrangements.
As previously observed in other lymphoma subtypes, our results suggest that EBV-positivity defines a specific PBL phenotype with particular molecular properties such as lower levels of genetic complexity, and distinct pathogenic mechanisms. 9,45-48 Specifically, EBV-positive PBL showed more frequent STAT3 mutations whereas inactivating TP53 alterations (mutations, deletions and CNN-LOH) were sig-nificantly enriched in EBV-negative tumors, which have lower expression of p53 signaling pathway-related genes. In BL, EBV positivity has also been associated with fewer driver mutations, especially among apoptosis-related genes such as TP53. 45 Additionally, similar to PBL, EBV-positive DLBCL had relatively fewer genomic alterations than EBVnegative DLBCL, whereas EBV-negative cases had more 17p deletions. 48 This suggests two different mechanisms to avoid apoptosis in which TP53 depletion substitutes for the anti-apoptotic effect that EBV infection exerts in the cell. In addition, EBV-negative PBL have a higher frequency of mutations affecting epigenome/chromatin modifiers and NFκB signaling pathways. In ABC-DLBCL, genetic alterations of BCR-TLR pathways lead to high NFκB activity which induces production of IL-6 and IL-10. 49,50 These observations suggest that an autocrine action of IL-6 could also occur in EBV-negative PBL, activating the JAK-STAT pathway as an alternative mechanism to STAT3 mutations present in EBV-positive cases.
In summary, we used an integrative approach of FISH, copy number and next-generation sequencing mutational analyses in a large series of PBL. Our results revealed a specific PBL genetic landscape which, differing from that of other related lymphoma entities, is characterized by recurrent alterations in the MAPK and JAK-STAT pathways in addition to previously known MYC rearrangements. Moreover, we identified a distinct mutation profile for EBVpositive and EBV-negative cases, a finding observed in other forms of aggressive B-cell lymphoma. The detection of recurrently altered MAPK and JAK-STAT pathways in PBL opens new perspectives in the biology of this disease, identifying possible new targets for therapy.

Disclosures
No conflicts of interest to disclose.