Diffuse large B-cell lymphoma (DLBCL) is the most common subtype of non-Hodgkin lymphoma (NHL), accounting for 30–40% of all NHL patients (1–3), which is considered to be a heterogeneous entity based on its biological characteristics and clinical outcomes (3–5). The survivals of DLBCL patients have notably improved since addition of rituximab to CHOP (rituximab, cyclophosphamide, doxorubicin, vincristine, and prednisone) chemotherapy (6,7). However, some DLBCL patients continue to present an inferior prognosis under standard R-CHOP therapy (1).

Ki-67, a nuclear nonhistone protein, is synthesized at the beginning of cell proliferation (8). Ki-67 expression has been widely used in clinical practice as an index to evaluate the proliferative activity of lymphoma. High Ki-67 expression was highly associated with worse OS for NHL (9). However, the relationship between Ki-67 expression and outcome with DLBCL are still contradictory and inconclusive in various studies (10–12).

BCL2 protein functions as an antiapoptotic protein inhibiting cells from programmed cell death (13). Both gene amplification and translocation are common mechanisms causing BCL2 protein overexpression in DLBCL. The clinical significance of BCL2 protein expression in DLBCL is still controversial. The impact of BCL2 overexpression on survival in DLBCL is still debatable in previous studies. Additionally, the prognostic value of BCL2 protein overexpression is also different between GCB and ABC subtypes (14,15).

Moreover, the predictive significance of some prognostic factors changed following the introduction of a CD20 monoclonal antibody, rituximab, underscores the necessity for revaluating the prognostic value of predictive factors after the introduction of rituximab (7,16).

In the present study, we intended to investigate the optimal prognosis cut off value of Ki-67 index in DLBCL patients, and to confirm the specific prognostic value of BCL2 and its association with cell of origin classification (COOC). Furthermore, we investigated whether the BCL2/Ki-67 index has a more significant importance on the outcome of DLBCL patients.

In the present study, we set out to evaluate the prognostic value of combining Ki-67 and BCL2 as an index which would be superior to the evaluation of the markers separately.

Ki-67 is an immunohistochemical marker of proliferating cells. Recent studies suggested that MYC and BCL2 protein co-expression is an independent indicator of poor prognosis in diffuse large B-cell lymphoma (19,20). Since MYC positive DLBCL usually manifest a high proliferation rate, some research suggested the proliferation fraction criterion to ≥90% improved the specificity for detection MYC⁺ double/triple translocations, which means Ki-67 ≥90% might predict poor outcome (21). Therefore, further investigation is necessary to clearly delineate the relationship between Ki-67 expression and prognosis in DLBCL. In our study, we confirmed the prognostic value of high Ki-67 index (≥90%) in GCB-DLBCL, which was in accordance with the prognosis of MYC gene rearrangement (22).

BCL2 is an anti-apoptotic protein which also has an antiproliferative effect influencing cell-cycle entry and is a powerful prognostic marker before rituximab. Studies showed that the addition of rituximab have eliminated the negative impact of the BCL2 expression (23,24). However, the prognostic value of BCL2 protein in DLBCL was still controversial (14,15). In our study, we showed BCL2 protein expression has a significant impact on OS and PFS in GCB-DLBCL, but not in NGC-DLBCL in the R-CHOP cohort, which was in accord with previous research (14).

Sustaining proliferative signaling and resisting cell death were two of the ten hallmarks of cancer, which play important role in cancer progression (25). In breast cancer, a BCL2/Ki-67 index based on IHC was highly prognostic in ER-positive patients. However, the prognostic value of BCL2/Ki-67 index has barely been investigated in DLBCL. With this purpose, we combined the high Ki-67 index (≥90%) and BCL2 positive patients together, named ‘BK’. We showed BK⁺ group had significantly poor outcome than BK⁻ group. Stratification analysis showed GCB-DLBCL but not NGC-DLBCL retained the prognostic value of BK⁺. In multivariate analysis by Cox proportional hazards regression, BK⁺ remained significantly prognostic factor of PFS in DLBCL. Since DLBCL is a heterogeneous entity, IHC test alone may not obtain the complete picture of this disease, hematologist need more effective indicators to tailor therapy. Nevertheless, the BCL2/Ki-67 index is a simple and convenient method to figure. out the patients' outcome of DLBCL, which would be a potential complement of current genetic heterogeneity.

Although most DLBCL patients are cured with 6–8 cycles of R-CHOP chemotherapy, about 10–15% ones have primary refractory disease and a further 20–30% relapse. There is an urgent need to improve outcome for these patients. In the precision medicine era, target CD20 alone might not be enough. Studies also suggest improvement in outcome on the use of rituximab with CHOP in ABC-DLBCL and the BCL2 negative subset of GCB-DLBCL. However, the BCL2 positive GCB-DLBCL has shown less improvement, and these cases may benefit from novel agents such as inhibitors of BCL2 function (14). Targeted inhibition of BCL2 with its highly selective inhibitor ABT-199 recently emerged as a promising treatment strategy for some B-cell malignancies such as CLL and MCL (26,27). ABT-199 was proved potentially effective in BCL2 positive DLBCL (28). In GCB-DLBCL with BCL2 positive might benefit from ABT-199 or other inhibitors of BCL2 function. Besides, to inhibition of MYC expression via BRD4 inhibitor, such as JQ1, might indirectly be another way to target high Ki-67 proliferation DLBCL (29).

In conclusion, we have described a method for the combinatorial assessment of BCL2 and Ki-67 as measured by IHC. The BCL2/Ki-67 index was a highly effective predictor of patients' outcome with DLBCL, especially in the GCB-DLBCL group. In multivariate analysis, BK⁺ remained significantly prognostic factor of PFS in DLBCL. In the precision medicine era, targeting BK⁺ therapies might be potentially promising ways to improve patients' outcome.