Expression but Not Promoter Hypermethylation of the Tyrosine Phosphatase PTPN6 Is Associated with Activated STAT3 and Inferior Prognosis in Diffuse Large B Cell Lymphoma Molecular Subtypes.

Abstract 2655

Diffuse large B cell lymphoma (DLBCL) has been classified into two distinct molecular subtypes: germinal center B cell–like (GCB), non-germinal centre-like (non-GCB). To improve outcomes for DLBCL patients, it is necessary to identify additional novel targets within GCB and non-GCB classifications to further stratify patients for prognosis and assist in choosing therapy for the individual patient. We have recently demonstrated that STAT3 activation is frequent in the DLBCL tumors, however the exact molecular mechanism(s) of STAT3 activation in DLBCL tumors are not known. Molecular mechanisms such as epigenetic silencing of negative regulators of the STAT3 pathway such as protein tyrosine phosphatase 6 (PTPN6) may contribute to STAT3 activation. We aimed to learn whether PTPN6 was expressed in GCB and non-GCB DLBCL, and if so, how that expression correlated with STAT3 activation and prognosis.

We first performed epigenetic studies of PTPN6 in 38 DLBCL tumors and 6 DLBCL cell lines by methylation specific (MSP) PCR and high resolution melting array (HRM) methods. Surprisingly, PTPN6 promoter hypermethylation (0/38) was not detected in patient sample and cell lines by both the methods. Since the MSP PCR technique yields qualitative rather than quantitative data, we next performed pyrosequencing on the 38 DLBCL samples, and found results consistent with the MSP PCR analysis. Our data conclusively demonstrate that PTPN6 hypermethylation is absent in DLBCL tumors. We next sequenced the 600 bp upstream of the transcription initiation site of PTPN6 promoter 2 and 1 in 10 DLBCL tumors. We did not detect any point mutations in the promoter 2 and 1 core regions. Since PTPN6 promoter hypermethylation and mutations were absent in DLBCL tumors, we determined the expression level of the PTPN6 protein in 40 DLBCL tumors by molecular subtype. Formalin fixed paraffin-embedded DLBCL tumor samples were stained by immunohistochemistry (IHC) for the determination of molecular subtype using the Hans algorithm and the detection of PTPN6 expression. Using a threshold of ≥30%, 75% (30/40) of cases were PTPN6 positive with various levels of expression: 11 cases had 30–80% of tumor cells staining positive and 19 had >80% of cells PTPN6 positive. PTPN6 expression by IHC was only correlated with higher IPI scores and a trend towards a shorter event free survival (EFS) (p=0.07). Within the 29 GCB tumors 69% (20/29) were PTPN6 positive; 100% (10/10) of non-GCB cases were PTPN6 positive. These data clearly suggest that PTPN6 expression is found in both GCB and non-GCB with the latter being uniformly positive (p=0.03) and PTPN6 negative cases being uniformly GCB. PTPN6 mRNA and protein was detected in all three ABC lines (LY3, Ly10, DHL2). Within the GCB lines (DHL6, Ly1 and Ly19) DHL6 was weakly positive and Ly1 and Ly19 were negative for PTPN6 mRNA and protein. Furthermore, within the GCB group PTPN6 positive cases had inferior EFS as compared to PTPN6 negative cases. In the non-GCB group all cases were PTPN6 positive with an EFS similar to PTPN6 positive GCB cases. The role of PTPN6 in the persistent activation of the STAT3 pathway in DLBCL patients has not been investigated. We hypothesized that tumors with activated STAT3 would have loss of PTPN6. Interestingly, this hypothesis was disproven. Within the 15-pSTAT3 positive cases 12 (80%) were PTPN6 positive. Conversely, 26% (6/23) of the pSTAT3 negative cases were PTPN6 negative. The distribution of pSTAT3 and PTPN6 by IHC in samples was evaluated in both GCB and non-GCB groups. Within the PTPN6+/pSTAT3+ group out of the 12 cases most were non-GCB (8/12; 66%). However, within the PTPN6-/pSTAT3- group all the 5 cases were GCB (5/5; 100%). Survival analysis revealed that the groups with the best EFS were those with PTPN6-/pSTAT3- tumors (n=5); those with PTPN6+/pSTAT3+ group (n=12) had the shortest EFS; and those with PTPN6+/pSTAT3- tumors (n=17) being intermediate between the other groups.

In summary, we have demonstrated for the first time that PTPN6 is highly expressed in DLBCL tumors, and a common abnormality in non-GCB subtypes, which is positively correlated with activated STAT3. PTPN6 expression in the DLBCLs is not regulated through SHP1 promoter hypermethylation or point mutations. The finding that SHP1 loss was found only in GCB cases and was especially favorable should be explored further and may provide an important new stratification factor for future DLBCL studies.