20.    Refractory and relapsed predictive biomarkers in Hodgkin Lymphoma and                          potential directed-therapy targets

Hodgkin lymphoma (HL) is a lymphatic system malignancy. The neoplastic cell, named as Hodgkin-Reed Sternberg (HRS), derive from a late B-cell maturation stage through their transit in germinal center. Despite 90% of early and 60% of late stage chemosensitive patients can achieve a long disease free survival (DFS) with
current therapy, refractory and relapsed HL patients remain a challenge. There are no predictive molecular biomarkers to identify refractory or at risk of relapse patients at diagnosis nor alternative treatment beyond high risk toxicity-related rescue chemo schemes. We have previously reported that HL relies on the constitutive
non-canonical NFkB pathway activation mediated by Rel-B and NIK. This leads to high BCL2 levels and sustained survival. Our ChIP-Seq data showed a Rel-B hierarchy DNA-binding network in HL. ChIP-Seq data merged with expression arrays results following Rel-B depletion uncovered an extensive set of Rel-B target genes including BCL2. Our molecular studies suggest the constitutive non-canonical pathway signaling could at least partially explain the lack of response in refractory/relapsed HL patients. We aimed to analyze if mediators of this pathway
could be useful as predictive biomarkers to identify refractory and at risk of relapse HL patients at primary diagnosis, as well as potential targetable factors. We analyzed the citoplasmic NIK and BCL2 expression in HRS cells of lymph node biopsies in 96 HL patients by immunohistochemistry [50 female Md age (range) 59 (6-82) and 46 male 42 (9-78)]. The univariate analysis showed no correlation among NIK or BCL2 expression and the prognosis clinical and pathological parameters. As previously determined by our in vitro experiments a direct correlation was found between NIK and BCL2 expression in the biopsies studied (p=0.01). The analysis of
survival, applying the Kaplan-Meier Curves, showed that >60% of NIK positive HRS cells was associated with shorter DFS [Log Rank Test p=0.008]. >60% of BCL2 positive HRS cells correlated with poor prognosis in terms of overall survival [Log Rank Test p=0.002]. The statistical significance was maintained in the multivariate
analysis as shown by the Cox Regression and the Logistic Regression analysis (p=0.01). NIK and BCL2 are useful predictive biomarkers to identify refractory and at risk of relapse HL patients at diagnosis. Furthermore, they represent attractive therapeutic targets. We have already demonstrated that 1,3 (2H, 4H)- isoquinolinedione, a NIK inhibitor, induced cell death in human HL cell lines. Besides, this non-canonical pathway also offers as targetable BCL2, one of its main downstream targets. Furthermore, BCL2 inhibitors are currently being used in other onco-hematological diseases. We do believe non-canonical NF-kB signaling disruption could contribute to overcome the lack of response of refractory and relapsed HL patients to current conventional therapies.