The differential role of BCL-2 within molecular subtypes of DLBCL

Abstract

Gene expression profiling has divided diffuse large B-cell lymphoma (DLBCL) into distinct molecular subtypes with different outcomes following immunochemotherapy. Recently, there has been much interest in investigating the role of biomarkers, such as BCL-2, in predicting outcome in DLBCL, particularly in the context of these molecular subtypes.

Commentary

In this issue of Clinical Cancer Research, Iqbal and colleagues evaluate the prognostic significance of BCL-2 in patients with diffuse large B-cell lymphoma (DLBCL) who receive R-CHOP (rituximab, cyclophosphamide, doxorubicin, vincristine and prednisone)(1). Of interest, they find that BCL-2 protein over expression is a biomarker of poor outcome in the germinal center B-cell (GCB) but not in the activated B-cell (ABC) subtype of DLBCL. Their findings raise interesting questions about the diverse mechanisms of activation of BCL-2 in these tumors and the mode of action and differential activity of rituximab within the GCB and ABC subtypes of DLBCL.

The B-cell leukemia/lymphoma-2 gene (BCL-2) was discovered over 25 years ago through its association with the t(14:18) translocation in follicular lymphoma(2). This cytogenetic abnormality led to deregulated expression of the BCL-2 protein and was found in the majority of follicular lymphomas and in a variable number (10-40%) of de novo DLBCLs.(3) Then, with its identification, came the discovery of a family of structurally related proteins that function as important regulators of apoptosis(4). In DLBCL, the inhibitory action of BCL-2 on apoptosis was hypothesized as a cause of chemotherapy resistance and this notion was supported by several clinical studies in the pre-rituximab era demonstrating an inverse correlation between BCL-2 protein expression and survival(3, 5). Studies done in the post-rituximab era, however, raise the question of whether BCL-2 remains a biomarker of treatment failure and many studies have demonstrated that this is no longer the case(6, 7).

In that regard, looking at the role of BCL-2 within molecular subtypes of DLBCL is an interesting undertaking. Herein, Iqbal and colleagues, investigate the impact of BCL-2 protein and mRNA expression on survival within molecular subgroups of DLBCL treated with immunochemotherapy(1). The strengths of the study are, firstly, that the ‘gold standard’ - gene expression profiling -was performed to categorize tumors into GCB or ABC subtypes and this eliminates the potential inaccuracies of using immunohistochemical algorithms to predict cell of origin. Secondly, this was a homogeneous population in that all patients received R-CHOP and central expert pathology review was performed. While they and others had previously shown in the pre-rituximab era that BCL-2 protein over-expression is prognostic only in the ABC subtype, here they find the opposite where BCL-2 protein over expression is significantly associated with an inferior outcome in the GCB subtype(8). At the mRNA level, while high expression predicted lower event-free survival in DLBCL overall, there was only a trend towards inferior survival in the GCB subtype which may reflect the fact that mRNA expression may not translate to protein expression.

Their results raise interesting questions about the mechanisms of BCL-2 activation and how these may or may not be abrogated by the addition of rituximab to chemotherapy. In the ABC subtype, BCL-2 expression is associated with constitutive activation of the NF-kappa B pathway(9). While the mode of action of rituximab in the ABC subtype is poorly understood, in vitro studies have shown that it can down-regulate NF-kappa B by induction of the Raf-1 kinase inhibitor protein (a negative regulator of the pathway) and this mechanism may also occur in vivo and lead to reduced expression of BCL-2(10). It is also possible that rituximab modulates other pathways associated with BCL-2 in the ABC subtype.

In the GCB subtype, the presence of a t(14:18) translocation accounts for the over-expression of BCL-2 in most but not all cases. Interestingly, in this study, Iqbal and colleagues find that a t(14:18) translocation was not associated with the adverse outcome seen in the GCB subtype suggesting that GCB cases with t(14:18) likely have variable levels of BCL-2 protein and that some GCB cases without t(14:18) also express BCL-2 (figure 1). These latter cases are likely to have much higher expression of BCL-2 than translocated cases. The study’s findings suggest that rituximab does not overcome BCL-2 associated resistance in GCB tumors, raising a number of interesting questions with regard to the mechanisms of BCL-2 expression and other associated biological events. For example, the coexistence of BCL-2 translocations with other cytogenetic abnormalities, such as those involving MYC and/or BCL-6, may be important contributors to treatment resistance (11). Though we have limited clinical experience with these so-called ‘double-hit’ and ‘triple-hit’ tumors, the co-existence of two or more mutations are known to be clinically associated with a high rate of treatment failure, even in the immunochemotherapy era. In this regard, it would be interesting to know the frequency of these mutations in this series. The role of BCL-6 gene expression, which is generally high but variable in the GCB subtype, could be another co-variable within the BCL-2 positive cases (Figure 1).

Figure 1.

Shown here are potential mechanisms of BCL-2 mRNA expression in a series of unselected cases of germinal center B-like (GCB) and activated B-like (ABC) DLBCL. The array shows that most BCL-2 expression in GCB DLBCL is associated with t(14:18), although a subset of BCL-2 expressing cases do not harbor this translocation and other mechanisms of activation are at play. In ABC DLBCL, while activation of NF kappa B is associated with BCL-2 over-expression, it is likely that other mechanisms of activation are also important.

It is possible that BCL-2 expression is a surrogate biomarker for several other biologies. As the authors note, the BCL-2 negative cases were associated with a favorable microenvironment signature, termed the stromal-1 signature(12). This signature contains genes related to extracellular-matrix deposition and mesenchymal and histiocytic cell infiltration, and is found in varying proportions with the so called stromal-2 signature, which contains genes related to endothelial cells and important regulators of angiogenesis, and is associated with an adverse outcome. On the other hand, the BCL2-negative group was associated with higher proliferation, which has been identified as an adverse prognostic signature. In contrast, the BCL2-positive GCB tumors were associated with increased infiltrating follicular helper T-cells, which the authors speculate may protect the tumor cells against apoptosis, possibly through down regulation of BIM as described with follicular-dendritic cells. These varied biological associations in the BCL-2 positive and negative GCB tumors highlight the inherent drawbacks of such correlative studies and the difficult task of identifying the important mechanisms of drug resistance. These results point to BCL-2 as the obvious drugable target in GCB DLBCL, and the high affinity inhibitor of BH3-only proteins, navitoclax, as a prime candidate for testing.

In conclusion, this study highlights the importance of evaluating biomarkers in the context of molecular subtypes and demonstrates that distinct mechanisms of BCL-2 activation (even within a molecular subtype) are associated with different prognoses. The investigators’ findings suggest that some GCB tumors with BCL-2 over-expression may benefit from therapies that directly target the BCL-2 complex and overcome the inhibitory effects on apoptosis of BCL-2. Many of these agents are in development at present(13). While BCL-2 is a useful prognostic marker in subtypes of DLBCL, its expression likely reflects many different mechanisms that drive tumor survival and it is key to pair clinical investigation and tumor biology for the clinical development of new targeted agents.