DLBCL with amplification of JAK2/PD-L2 exhibits PMBCL-like CNA pattern and worse clinical outcome resembling those with MYD88 L265P mutation

Background Recently, copy number alteration (CNA) of 9p24.1 were demonstrated in 10% of diffuse large b-cell lymphoma (DLBCL), with gene expression and mutation profiles that were similar to those of primary mediastinal large B-cell lymphoma (PMBCL). However, their CNA-based profile and clinical impact still remain unclear. Methods Multiplex ligation-dependent probe amplification were employed to investigate the prevalence of JAK2/PD-L2 amplification in DLBCL and their CNA-based pattern of driver genes. The clinical outcome and characteristics were also analyzed. Results Using unsupervised hierarchical clustering, a small group of DLBCL (10.5%, 8/76) was clustered together with PMBCL as Cluster_2, demonstrating amplification of JAK2 (100%,8/8) and PD-L2 (75.0%,6/8). This subgroups of DLBCL demonstrated significant higher expression of PD-L1 than those with MYD88 L265P mutation(p = 0.024). And they exhibited dismal OS and PFS as compared with DLBCL_others(p = 0.003 and 0.001, respectively), which is similar to DLBCL with MYD88 L265P mutation. Conclusions DLBCL with amplification of JAK2/PD-L2 exhibits CNA pattern that is similar to PMBCL, and demonstrates unfavorable clinical outcome that resembles those with MYD88 L265P mutation. It is essential to identify this subgroup of DLBCL who may acquire more benefits from the JAK2 and PD-L1 signaling inhibition.

Therefore, in this study, we employed multiplex ligation-dependent probe amplification (MLPA) to investigate the prevalence of JAK2/PD-L2 amplification in DLBCL, and their CNA-based pattern of driver genes, including BCL2, CDKN2A and TP53 [6]. And we analyzed their long-term survival outcome after treatment of R-CHOP-like regime.

Case selection
We collected consecutive cases of DLBCL and PMBCL in our clinical FFPE archives of excisional biopsy database between Jan 2009 and Oct 2010. And 77 cases of DLBCL and 4 cases of PMBCL were found. After confirmation, one case of DLBCL was diagnosed as PMBCL. Thus, 76 cases of DLBCL and 5 cases of PMBCL were acquired finally (see Additional file 1). All patients were diagnosed at National Cancer Center/National Clinical Research Center for Cancer/Cancer Hospital, Chinese Academy of Medical Sciences and Peking Union Medical College according to the revised 4th edition of the WHO Classification of Tumours of Haematopoietic and Lymphoid Tissues [7]. The data regarding treatment and prognosis were acquired by means of medical record consultation and telephone conversation.

Multiplex ligation-dependent probe amplification (MLPA)
Genomic DNA were extracted from formalin-fixed, paraffin-embedded (FFPE) blocks using QIAamp DNA FFPE Tissue Kit (Qiagen, Valencia, CA). Then DNA copy number quantification and MYD88 L265P mutation were detected using MLPA kit (MRC-Holland, Netherlands). The PCR products were detected on an ABI 3500 Genetic Analyzer (Applied Biosystems, USA), and the final result were analyzed using Coffalyser 9.4 software. The relative peak ratio (PRR) of probe larger than 1.3 was defined as amplification, and less than 0.7 was defined as deletion (see Additional file 2). Genes which had two or more probes covering two different exomes were put into final analysis, including JAK2, PD-L2, MDM2, REL, PUS10, BCL2, NFATC1, SPIB, FOXP1,  NFKBIZ, BCL6, PRDM1, TNFAIP3, CDKN2A, PTEN,  ING1 and TP53 [6]. The details of MLPA probes of driver genes in DLBCL are shown in the online supporting material (see Additional file 3). True amplification of one gene was regarded only when all probes of this gene exhibited amplification, and vice versa (see Additional file 2).
MYD88 L265P mutation was identified when the probe had a high peak. MYD88 wildtype didn't show any peak (see Additional file 2).

Immunohistochemistry (IHC) staining of PD-L1(22C3)
IHC staining was performed on Dako Autostainer Link 48 (ASL48) platform. Each FFPE block were cut at a thickness of 4-μm, and then deparaffinized. Antigen retrieval were performed using the EnVision™ FLEX Target Retrieval Solution at low pH 6.0. Monoclonal PD-L1 (clone 22C3, Dako) were used as primary antibody, followed by incubation with EnVision™ FLEX+ Mouse LINKER, and then EnVision™ FLEX HRP reagent. Finally, the IHC was visualized by EnVision™ FLEX DAB. Each IHC slide contained a positive control (Lung carcinoma).
IHC score of PD-L1 were calculated by multiplying the percentage of positive cells with mean intensity (0, no staining, 1, weak staining, 2, moderate staining; 3, strong staining), which was reported in previous study [5]. The results were evaluated by an experienced hematopathologist (Xuemin).

Statistical analysis
The differences of clinicopathological characteristics among different groups were analyzed using Chi-square test, Fisher exact test or Kruskal-Wallis rank sum test. PD-L1 IHC score between different groups was analyzed using Wilcoxon test. Overall survival (OS) and progressfree survival (PFS) times were defined from the date of pathologic diagnosis to the date of the event or the last follow-up. The hazard ratio (HR) of each parameter was calculated by univariate Cox proportional regression analysis firstly, in which parameters with p < 0.05 were evaluated together using multivariate Cox proportional regression analysis. The survival curve were made according to Kaplan-Meier procedure. The day of last follow-up was March 1st 2019. All statistical analysis were two sided and p < 0.05 was defined as significance.
Unsupervised hierarchical clustering was carried out using Euclidean distance and complete method. Heatmap was plot using pheatmap package.
All above statistical analyses were run in R 3.4.1 statistic software.
As to survival, DLBCL in Cluster_2 demonstrated significant worse OS (p = 0.016) and PFS (p = 0.008) as compared with DLBCL in Cluster_1 (Fig. 1b). However, Cluster_1 and Cluster_3 didn't reveal significant difference in survival (Fig. 1b). We also analyzed the OS and PFS between DLBCL with and without JAK2/PD-L2 amplification, and got statistical significance (see Additional file 5).
Unlike DLBCL_MYD88_L265P and DLBCL_others, DLBCL_JAK2/PD-L2_amp showed a distinctive pattern similar to that of PMBCL, with high frequency of REL and NFKBIZ amplifications, but no amplification of BCL2 and NFATC1 and no deletion of PRDM1 was found (Fig. 2a).
JAK2/PD-L2 amplification identify a subgroup of DLBCL with unfavorable survival outcome similar to that of MYD88 L265P mutation Trying to explore the survival indication of JAK2/PD-L2 amplification and MYD88 L265P mutation, 49 cases of DLBCLs who received RCHOP-like regiment with or without surgical resection were enrolled to performed cox proportional regression analysis of OS and PFS. The median follow-up time was 108 months (range, 3-192 months).
In the univariate analysis, as compared with DLBCL_others, DLBCLs with MYD88 L265P mutation had significantly worse OS and PFS (p = 0.025 and 0.007, respectively), and the same to DLBCLs with JAK2/PD-L2 amplification (p = 0.003 and 0.001, respectively). Meanwhile, IPI risk category were significantly associated with OS and PFS (Fig. 2c, Tables 3  and 4).
In the multivariate analysis, IPI risk category and three subgroups of DLBCL were put into analysis. As compared with DLBCL_others, DLBCL with MYD88 L265P mutation still showed poor OS and PFS (p = 0.005 and 0.001, respectively), and the same to DLBCL with JAK2/PD-L2 amplification for PFS and OS (p = 0.006 and 0.001, respectively). Meanwhile, IPI risk category was still an independent risk predictors for OS and PFS (Fig. 2c, Tables 3 and 4).

Discussion
DLBCL presents with a wide spectrum of genetic aberration. Recently, Shi et al. study exhibited PD-L2 amplification in 75% PMBCL and 0% of DLBCL [11]. Chapuy et al. demonstrated 15% of 9p24.1 amplification in DLBCL [2]. Meanwhile, DLBCL with PD-L1 gene alterations was identified as a unique biological subgroup, having high risk features [3]. Y Wang et al. study demonstrated that 10% of DLBCL had CNA of 9p24.1, with gene expression and mutation profiles that were similar to those of PMBCL [5]. In our study, by using unsupervised hierarchical clustering, 10.5% (8/76) cases of DLBCL were clustered together with PMBCL as Clus-ter_2, indicating that they shared recurrent CNAs. They were enriched for JAK2 amplification and PD-L2 amplification (Fig. 1a). Using Hans model, most of DLBCL in Cluster_2 were non-GCB (75%, 6/8), and tend to be younger than other groups of DLBCL (Table 2), which was consistent with prior study [5]. Therefore, coupled with Y Wang et al. study, we confirmed that DLBCL with JAK2/PD-L2 amplification is a unique subgroup resembling the PMBCL with respect to CNA pattern.
With regard to survival, increasingly data exhibited that the suppression of immune surveillance in DLBCL was associated with poor survival. Godfrey J et al. study has demonstrated that DLBCL with PD-L1 gene alterations showed high risk features [3]. Meta-analysis also showed that PD-L1 expression was associated with poor OS and adverse clinicopathologic features in DLBCL [12].
In Y Wang et al. study, 10% of DLBCL harbored CNA of 9p24.1, of which 65% were gains and 35% were amplifications [5]. And, as compared with those who have no gain of 9p24.1, DLBCL with 9p24 amplification had a trend of better EFS, while patients with only gain tend to have worse prognosis [5]. Unfortunately, they didn't show any statistical significance [5]. In our study, 10.5% (8/76) of DLBCL were found that had CNA of JAK2. When JAK2 CNA was separated into gain (MLPA value between 1.7-2.0) and amplification (MLPA value > 2.0) as described [13], 50%(4/8) cases in DLBCL_JAK2/PD-L2_amp group were found that had JAK2 gain, which was slightly lower than that in Wang J et al. study [5] (as shown in Additional File 7). And both DLBCL with JAK2 gain and with amplification demonstrated significant poor prognosis as compared with rest of DLBCL (as shown in Additional File 7). More interesting, unlike Y Wang et al. study [5], 5 cases of PMBCL were included in our study as control, all of which demonstrating JAK2 gains, rather than amplifications (as shown in Additional File 7).
Objective response rates (ORR) of PD-1 blockade therapy was 10-36% in unselected patients with relapsed/refractory DLBCL [18,19]. The wide spectrum of ORR may be due to high heterogeneity of this subgroup. Ansell SM et al. study demonstrated 19% patients with 9p24.1 alteration in relapsed/refractory DLBCL [18]. In our cohort, the frequency of JAK2 and PD-L2 amplification in relapsed/refractory DLBCL were 20% and 13.3%, which were within the range of ORR in the prior studies [18,19]. While, 33.3% (5/15) patients were found that had MYD88 L265P mutation who may not be suitable for anti-PD-1 therapy. Thus, the genetic analysis in refractory/relapsed DLBCL is required for future therapy selection to increase the ORR of immune checkpoint inhibitors.
JAK2 amplification could augment the expression of itself and PD-1 ligands (PD-L1 and PD-L2), enhancing the HR_U hazard ratio by univariate analysis, HR_M hazard ratio by multivariate analysis * Because age was contained in the IPI, thus it wasn't put into multivariate analysis sensitivity to JAK2 kinase inhibitor [4]. Chemical JAK2 inhibition could reduce the RNA transcription and protein expression of PD-L1 [20]. Thus, selective inhibition of JAK2 would be a valuable complementary therapy for PD-L1 blockade.

Conclusions
DLBCL with amplification of JAK2/PD-L2 exhibits PMBCL-like CNAs pattern, and demonstrates unfavorable outcome resembling those with MYD88 L265P mutation. Thus, it is essential to identify this subgroup of DLBCL who may acquire more benefits from the JAK2 and PD-L1 signaling inhibition, and JAK2 amplification detection by MLPA would be feasible in routine practice. Meanwhile, the difference of survival outcome between our study and Wang J et al. study indicated that PMBCL-like DLBCL suggested by 9p24.1 CNA could be an intermixed subgroup, which required further exploration.