The characteristics of DLBCL patients and healthy donors
Healthy donors (n=130) and patients with DLBCL (n=349) confirmed by previous histopathological analysis were included in the study. A full list of clinical characteristics of healthy donors and DLBCL patients were listed in Supplementary Table 1. It showed that DLBCL patients and healthy donors had similar age, gender, white blood cell count (WBC), absolute neutrophil count (ANC), platelet count (PLT) and albumin/globulin ratio (AGR). However, the absolute monocyte count (AMC) and RDW in DLBCL patients were significantly higher than that in healthy controls; and the absolute lymphocyte count (ALC), hemoglobin (Hb), albumin (ALB) and globulin (GLB) in DLBCL patients were significantly lower than healthy donors. There were 174 patients (49.9%) treated with R-CHOP, and 175 patients (50.1%) treated with CHOP only. The subgroups of patients’ Ann Arbor tumor stage were 89 (25.5%) in stageⅠ, 86 (24.6%) in stage Ⅱ, 61 (17.5%) in stage Ⅲ and 113 (32.4%) in stage Ⅳ. There were no statistical differences in the age, gender, and other clinicopathological parameters between the training set and the testing set (Supplementary Table 2).
Cut-off values of RDW and PLT in DLBCL patients
RDW, PLT and Hb are three common parameters in routine blood test. Using ROC analysis and calculating the Youden index (specificity+sensitivity–1), the optimal cutoff values chosen for RDW and PLT were 14.35% and 126.5×109/L respectively in the training set (Supplementary Figure 1). However, there are gender differences in the definition of anemia. According to the guidelines of the World Health Organization, anemia in male patients is defined as hemoglobin (Hb) <13g/dL, and female patients have Hb <12g/dL. The cutoff values were applied to the whole cohort, DLBCL patients were then classified into high-level and low-level groups, where 93 (26.64%) patients fell in the high RDW group, 44 (14.43%) patients in low PLT group, and 187 patients with anemia.
Association of RDW, PLT and Hb with other clinical- pathological factors
Linear correlation analysis showed that higher RDW level was associated with higher NLR, lower ALB and lower Hb; while lower PLT correlated directly with lower WBC, but did not correlate with NLR, ALB or Hb (Supplementary Figure 2).
Further analysis showed that, the value of RDW>14.35% significantly correlated with a poorer ECOG-PS (P<0.001), more extranodal sites of disease (P=0.002), presence of B symptoms (P=0.011), bone marrow involvement (P=0.007), higher Ann Arbor stage (P<0.001), higher LDH level (P<0.001) and higher IPI score (P<0.001). However, we found no statistical significance between age and gender with RDW level. There were also significant correlations between patients with PLT≦126.5×109/L and higher Ann Arbor stage (P=0.003); more extranodal sites of disease (P=0.021); higher LDH level (P=0.013) and presence of B symptoms (P=0.033). There were no statistical correlations between low PLT with age, gender and bone marrow involvement. In addition, ECOG PS (P=0.096) and IPI score (P=0.061) had only borderline significance (Table 1). Because of the correlation between RDW and Hb, we further analyzed the correlation between Hb level and clinical parameters. Lower Hb level was significantly associated with higher NLR (r=0.253, P<0.001) and higher ALB (r=0.519, P<0.001), but not correlated with WBC or NLR (Supplementary Figure 3). Overall, Hb level was signiﬁcantly associated with age, gender, B symptoms, clinical disease stage, serum LDH level, ECOG-PS, extranodal sites of disease and IPI score, but it was not associated with bone barrow involvement (Table 1).
Levels of RDW, PLT, Hb at diagnosis and clinical outcomes
The median follow-up time for our study was 21.3 months (range: 0.80-126.93). During follow-up, a total of 134 (38.4%) patients presented with disease recurrence, disease progression or death, of which 79 (22.6%) died. In the training set, the survival rate was significantly worse in patients with higher RDW than in patients with lower RDW (5-year OS: 43%vs 69%; 5-year PFS: 29%vs 53%) (Supplementary Figure 4a,4b). Also, patients with lower PLT showed significantly worse PFS than the patients with higher levels (5-year PFS: 30% vs 49%) (Supplementary Figure 4d), but the overall survival was not significantly different (P=0.074) (Supplementary Figure 4c). Similar results were observed in the testing set and the whole cohort set (Supplementary Figure 4e-4l). In order to explore whether different chemotherapy regimens affect the evaluation efficacy of the level of RDW and PLT, we divided the patients into two groups, one group treated with R-CHOP regimen and the other group treated with CHOP regimen. Kaplan-Meier analysis showed poor OS and PFS in patients with high RDW (P=0.021 for OS and P=0.039 for PFS) and low PLT (P=0.001 for OS, P<0.001 for PFS) levels in the R-CHOP cohort. Patients with higher RDW and lower PLT in CHOP treated cohort had poorer OS (P=0.001 for RDW, P=0.045 for PLT), but the results of PFS were not statistically significant (Figure 1). Next, we analyzed the correlation between Hb level and other clinical-pathological parameters. We found that anaemic patients had poorer OS in the training set and CHOP cohort, and poorer OS and PFS in the overall set. (Supplementary Figure 5).
We further assessed the prognostic value of RDW, PLT and Hb in the IPI subgroup. The Kaplan-Meier analysis showed that the RDW, PLT and Hb levels may not distinguish those with favorable outcomes from those with adverse outcomes for patients with IPI score of 0-2 (data not shown). However, in patients with IPI scores 3-5, the RDW and PLT levels, but not Hb level (data not shown) were able to further risk-stratify patients into high-risk and low-risk groups. In R-CHOP cohort, the patients with lower PLT had significantly poorer OS (P=0.003) and PFS (P=0.013); and in higher level of RDW patients, OS (P=0.014) was significantly reduced (Figure 2); the whole cohort and CHOP cohort also showed similar results (Supplementary Figure 6).
High RDW, low PLT and Hb at diagnosis as poor prognostic factors
To investigate the association between RDW and PLT and Hb levels with patients’ clinical outcomes, we performed the Cox proportional risk model. Table 2 and Table 3 summarized the results of the univariate and multivariate analysis for factors influencing OS and PFS in all DLBCL patients. The univariate Cox proportional analysis revealed that old age, advanced Ann Arbor stage, poor ECOG PS, elevated LDH, B symptoms, more extranodal sites of disease, higher IPI score, bone marrow involvement, lower Hb level, higher RDW and lower PLT were all predictors of DLBCL patients for OS and PFS (Table 2). To explore whether RDW and PLT were independent prognostic factors of DLBCL patients, we performed a multivariate analysis, including age, advanced Ann Arbor stage, ECOG PS, LDH, extranodal sites, B symptoms, IPI score, bone marrow involvement, lower Hb level, RDW and PLT. Interestingly, our results showed that older age (P<0.001), advanced Ann Arbor stage (P=0.037), higher RDW (P=0.003) and lower PLT (P=0.046) were independent prognostic factors for OS. On the other hand, for PFS, only older age (P<0.001), advanced Ann Arbor stage (P=0.002) and lower PLT(P=0.002) were independent prognostic factors (Table 3). But the ECOG PS, LDH, extranodal sites, B symptoms, IPI, bone marrow involvement and lower Hb level were not independent prognostic factors for OS and PFS in our study for DLBCL patients.
We further performed univariate and multivariate analysis by applying the above indicators to the R-CHOP and CHOP cohorts. Bone marrow involvement in the univariate analysis was not statistically significant and the number of patients involved in bone marrow was small, hence, it was excluded from the multivariate analysis (Supplementary Table 3 and Table 4). Surprisingly, we found that elevated RDW was an independent prognostic factor (P=0.012) in CHOP cohort, and depressed PLT was an independent prognostic factor (P=0.003) in R-CHOP cohort for OS. However, RDW was not an independent prognostic factor for PFS either in R-CHOP cohort or in CHOP cohort, whereas PLT was an independent prognostic factor (P=0.003) in R-CHOP cohort but not in CHOP cohort (Table 4).
Development of a modified IPI by adding both RDW and PLT
From multivariate analysis, there were clearly four independent prognostic factors for OS in the whole cohort. We then used the four clinical parameters to construct a new adjusted IPI model, age>60 equaled to two points; RDW>14.35%, PLT≦126.5(×109/L) and Ann Arbor stage III/IV equaled to one point respectively . Three risk categories were generated: low (0-1 points), intermediate (2-3 points) and high (4-5 points).
Based on the risk stratification model, the results showed that patients assigned to the low-risk group had good outcomes (5-year OS: 83%, 5-year PFS: 62%) and high-risk patients had very poor outcomes (5-year OS: 9%, 5-year PFS: 0%, Figure 3a,b) in all patients cohort. Similar results were observed in the R-CHOP (n =174) cohort (Figure 3c, d) and CHOP cohort (n =175) (Figure 3e, f). To strengthen the results from the multivariate analysis, we conducted a Harrell’s C statistics analysis. The c-index of the IPI prognostic model for OS was 0.744 for patients treated with CHOP, 0.709 for patients treated with R-CHOP, 0.725 for all DLBCL patients, and 0.763, 0.718, 0,743 in NCCN-IPI prognostic model. When the factors of RDW and PLT values were added, the predictive power was increased in both IPI and NCCN-IPI prognostic model. And the c-index of the adjusted IPI in the three cohorts was 0.753, 0.732 and 0.748 (Table 5).