Unsupervised hierarchical clustering of CNAs of driver genes and its survival analysis in DLBCL and PMBCL patients.
Based on array CGH, Lenz G et al. study previously identified speciﬁc CNAs in PMBCL which were different from ABC and GCB of DLBCL. ABC DLBCLs often have CNAs in FOXP1, NFKBIZ, CDKN2A, CDKN2B, INF4a, BCL2, NFATC1 and SPIB, while GCB DLBCLs frequently harbor CNAs in REL, PTEN, MDM2, MIHG1 and ING1. PMBCL often demonstrate CNAs of JAK2 and PD-L2. Using unsupervised hierarchical clustering, we explored the CNA-based pattern of these genes in DLBCL and PMBCL. The result showed that a small group of DLBCL (10.5%, 8/76) was clustered together with PMBCL as Cluster_2, with amplification of JAK2 (100%,8/8) and PD-L2 (75.0%%,6/8)(Fig. 1a). This subgroup of DLBCL occurred at the site of cervical lymph node (3 cases), gastrointestinal tract (3 cases), nasal cavity (1 case) and spleen(1 cases) (Fig. 1a, Table 1)( Additional File 4). The frequency of JAK2 and PD-L2 amplification in the whole cohort of DLBCL were 10.5% (8/76) and 7.9% (6/76), while both of them were 100% in PMBCL(Fig. 1a) (see Additional file 1). Meanwhile, all cases in Cluster_3 harbored amplification of NFKBIZ which is essential for NF-κB activation in ABC DLBCL, but no amplification of NFKBIZ was found in Cluster_1.
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 or without JAK2/PD-L2 amplification, and got statistical significance(see Additional file 5).
Of note, we found that DLBCL in Cluster_2 enriched for JAK2/PD-L2 amplification had less frequency of MYD88_L265P mutation (12.5%, 1/8) (Fig. 1a, Table 4), which was supported by the Cancer Genome Atlas data (TCGA, PanCancer Atlas) from cBioPortal[9, 10] (Fig. 1c).
JAK2/PD-L2 amplification identify a distinctive CNA-based pattern of DLBCL similar to that of PMBCL
Since DLBCL with JAK2/PD-L2 amplification had less frequency of MYD88 L265P mutation, our study separated DLBCL patients into three subgroups: DLBCL with JAK2/PD-L2 amplification (DLBCL_JAK2/PD-L2_amp), DLBCL with MYD88 L265P mutation (DLBCL_MYD88_L265P), and DLBCL without JAK2/PD-L2 amplification nor MYD88_L265P mutation (DLBCL_others) (Fig. 2a). Based on the unsupervised cluster result(Fig. 1a), one patient who had both JAK2/PD-L2 amplification and MYD88 L265P mutation was clustered into Cluster_2. Therefore, this patient was put into DLBCL_JAK2/PD-L2_amp subgroup accordingly. We also analyzed the data when this case was included in DLBCL_MYD88_L265P subgroup, and got the similar result(see Additional file 6).
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).
With respect to clinicopathologic characteristics, DLBCL_JAK2/PD-L2_amp tend to be younger than DLBCL_MYD88_L265P (p=0.003) (Table 2). While, Hans model, international prognostic index (IPI) risk category and MYC break-apart didn’t show any significant differences (Table 2).
PD-L1 expression in DLBCL with JAK2/PD-L2 amplification was significantly higher than that in DLBCL with MYD88 L265P mutation
Totally, 32 cases were performed PD-L1 (22C3) IHC detection, including DLBCL_MYD88_L265P (14 cases), DLBCL_JAK2/PD-L2_amp (8 cases), DLBCL_others (5 cases) and PMBCL (5 cases). The result showed that PD-L1 expression in DLBCL_JAK2/PD-L2_amp was significantly higher than that in DLBCL_MYD88_L265P (p=0.024) and DLBCL_others (p=0.037) (Fig. 2b and 2d). While no significant difference was found between DLBCL_JAK2/PD-L2_amp and PMBCL (p=0.768) (Fig. 2b).
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 for patients alive 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).
Either JAK2/PD-L2 amplification or MYD88 L265P mutation are frequently seen in relapse/refractory DLBCL with PFS less than 2 years.
DLBCL with PFS less than 2 years was defined as primary relapse/refractory cases. Among these cases who treated by RCHOP-like regime, the frequency of JAK2 and PD-L2 amplification were 20%(3/15) and 13.3% (2/15). Meanwhile, the frequency of MYD88 L265P mutation were 33.3% (5/15). DLBCL with either JAK2/PD-L2 amplification or MYD88 L265P accounted for 46.7% (7/15).