Cortelezzi, Agostino

Abstract Background: Over the last two decades several phenotypic, molecular, and chromosomal markers have been identified that are significantly associated with the prognosis of CLL patients. Therefore, clinicians managing CLL patients would benefit from a simplified prognostic index. Methods: We analyzed prospectively collected data from 337 Binet A CLL patients enrolled in Italian the O-CLL1-GISL protocol with the aim of developing scores capable of predicting treatment free survival (TFS). Factors independently associated with TFS were included in the prognostic indexes. To account for differences in the magnitude of the association between the individual independent factors and TFS, we assigned a weighted risk score to each factor based on ranges of their corresponding hazard ratios (HRs) (i.e., 1 point for HR 1.1-1.9; 2 points for HR 2.0-2.9, etc.). The total risk score was then calculated by the sum of the ratings of each factor on individual basis. Risk groups were identified combining risk categories with a non-statistically different TFS. Results: We developed two scores based on weighted multivariable models: the first included clinical and laboratory parameters [clinical score (c-score)], while the second was based on biological markers [biological score (b-score)] (Table 1). The c-score allowed to predict the TFS of patients through the combination of Rai stage, b2-microglobulin and absolute lymphocyte count (ALC), while the b-score predicted TFS by IGHV mutational status and CD38 expression. The c-score showed a C-statistic of 0.72, while the b-score was 0.67, although cases stratified according to the b-score showed a more specific mRNA/microRNA profile. When the two scores were forced in a multivariate analysis, both showed an independent predictive value on TFS with a similar HR, demonstrating their complementarity. Thus, we attempted to integrate the two scores performing a further multivariate analysis in which all parameters, significantly associated with TFS at univariate analysis, were tested (Table 1). ALC, Rai stage, b2-microglobulin together with IGHV mutational status, resulted independently associated with TFS. We constructed a weighted score [comprehensive score (co-score)], including all the above 4 variables, which allowed the identification of 3 different risk groups with significantly different TFS (Figure 1). The C-statistic of the g-score was 0.75, showing a better concordance than the other two scores. Moreover, its validity was externally validated in a series of 297 newly diagnosed Binet A CLL patients from the Mayo Clinic, USA. Conclusions: Using this multistep process and external validation, we developed a score with high discriminatory power and predictive significance on the individual patient level. Table 1. Univariate and multivariate Cox proportional Hazards Models Variable Univariate analysis Multivariate analysis Clinical model Biological model Comprehensive model HR (95% CI) P HR (95% CI) P score HR (95% CI) P score HR (95% CI) P score Age (years) <60/>60 1.12 (0.73-1.74) 0.59 - - - - - - - - - Sex Male/Female 0.93 (0.6-1.44) 0.93 - - - - - - - - - Rai stage 0/I-II 2.30 (1.47-3.50) <0.0001 2.13 (1.24-3.03) 0.004 0/2 - - - 1.76 (1.11-2.78) 0.015 0/1 ALC (109/L) <10/>10 3.43 (1.99-5.92) <0.0001 2.91 (1.85-5.20) <0.0001 0/2 - - - 2.70 (1.54-4.72) 0.001 0/2 b-2 microglobulin normal/elevated 3.04 (1.96-4.70) <0.0001 2.78 (1.79-4.30) <0.0001 0/2 - - - 2.65 (1.66-4.21) <0.0001 0/2 LDH normal/elevated 1.25 (0.57-2.71) 0.57 - - - - - - - - - CD38 negative/positive 3.22 (2.06-5.02) <0.0001 - - - 2.11 (1.15-3.16) 0.02 0/2 1.40 (0.80-2.42) 0.24 - ZAP-70 negative/positive 2.34 (1.51-3.61) <0.0001 - - - 1.21 (0.70-2.16) 0.485 - 1.0 (0.98-1.01) 0.72 - IGHV mutated/unmutated 3.57 (2.32-5.50) <0.0001 - - - 2.10 (1.12-3.90) 0.019 0/2 2.39 (1.27-4.50) 0.007 0/2 FISH risk low+int/high 2.93 (1.46-5.90) 0.002 - - - 1.65 (0.76-3.34) 0.216 - 1.80 (0.84-3.88) 0.13 - Abbreviations: ALC: absolute lymphocyte count; CI: confidential interval; HR: hazard ratio. Figure 1. TFS according to comprehensive progression risk score. Figure 1. TFS according to comprehensive progression risk score. Disclosures Shanafelt: Cephalon: Research Funding; Glaxo-Smith-Kline: Research Funding; Genentech: Research Funding; Hospira: Research Funding; Celgene: Research Funding; Jannsen: Research Funding; Polyphenon E International: Research Funding; Pharmacyclics: Research Funding. Kay:Genentech: Research Funding; Celgene Corporation: Membership on an entity's Board of Directors or advisory committees, Research Funding; Gilead: Membership on an entity's Board of Directors or advisory committees, Research Funding; Hospira: Research Funding; Tolero Pharma: Research Funding; Pharmacyclics: Research Funding.

Abstract Abstract 2883 CLL-like monoclonal B-cell lymphocytosis (MBL) shares common immunophenotypic features and cytogenetic abnormalities with CLL and is generally perceived as its premalignant state. The World Health Organization has set a consensus cut-off of 5×109/L circulating B cells to discriminate between what constitutes ‘disease’ and what not. However, the clonal size within MBL is extremely variable. High-count (HC), clinical MBL is associated with absolute lymphocytosis and progresses to CLL requiring treatment at a rate of 1–2% per year, whereas low-count (LC) MBL is found in the general population through high-sensitivity techniques and carries a risk of progression that is limited if any. Given the high frequency of CLL-like MBL in the general population, it is important to understand the underlying mechanisms and also identify biological markers endowing malignant potential that may distinguish between the different forms. To this end, we performed a detailed immunogenetic profiling of 334 CLL-like MBL cases (78 LC and 256 HC) for a total of 355 productive VDJ rearrangements (including double rearrangements), 91 from LC MBL and 264 from HC MBL. We also compared the immunoglobulin (IG) gene repertoires of MBL to 544 CLL Rai Stage 0 (CLL-0) that were part of an IG sequence dataset of 7424 CLL cases previously analyzed by our group. LC and HC MBL had distinct IG gene repertoires, with over-representation of the IGHV1–69 and IGHV4–34 genes in HC and the IGHV4–59/61 genes in LC MBL, respectively (p<0.001). The HC MBL repertoire exhibited clear similarities to CLL-0 in terms of IGHV gene usage (similar frequencies of IGHV1–69 and IGHV4–34). Regarding somatic hypermutation, no differences were identified between LC MBL versus HC MBL versus CLL-0, in that the frequency of mutated rearrangements (<98% identity to the germline) was overall similar (LC MBL: 72.5%, HC MBL: 76.1%, CLL-0: 75%). In this respect, all the aforementioned subgroups differed significantly (p<0.001) from the frequency previously reported by us in CLL where only 55% of rearrangements carried mutated IGHV genes. We finally analyzed the expression of stereotyped B cell receptor (BcR) IGs, identified through a cluster analysis of the MBL sequences together with all CLL sequences from our cohort and 5494 non-CLL IG sequences retrieved from public databases. Overall, only 6/91 (5.5%) LC MBL rearrangements could be clustered with other sequences in subsets with stereotyped BcRs. Two of these six LC MBL cases were clustered together with IG sequences from various entities, including CLL, other lymphomas and autoimmune diseases; thus, they were considered to carry ‘public’ BcR stereotypes. In contrast, HC MBL showed a significantly (p=0.0002) higher frequency of ‘CLL-specific’ BcR stereotypes versus LC MBL, with 60/264 (22.7%) HC MBL cases carrying stereotyped VH CDR3s. This frequency was comparable to the one observed in CLL-0 (20.2%). Notably, a gradation was observed in the frequency of BcR IG stereotypy depending on the absolute count of CLL-like cells, starting with 5.5% in LC MBL, raising to 22.7–20.2% in in HC MBL/CLL-0 and, finally, peaking at 30.4% in the entire CLL cohort as previously reported. Altogether, these findings suggest that rather than a true premalignant condition, LC MBL may merely reflect immune senescence or result from persistent antigen stimulation. On the other hand, HC MBL appears to be a continuum with Rai stage 0 in the evolution to clinically overt CLL, being maybe one step behind where it requires either additional genetic hits or simply extra time to cross the numerical border that discriminates it from CLL. Hence, the identification of molecular genetic markers that may predict progression of HC MBL/CLL-0 into full-fledged CLL is strongly warranted. Disclosures: Shanafelt: Genentech: Research Funding; GlaxoSmith Klein: Research Funding; Teva/Cephalon: Research Funding; Celgene: Research Funding.

Key Points Low-count and high-count monoclonal B-cell lymphocytosis (MBL) have distinct immunogenetic signatures, with only the latter resembling CLL. Rather than a true premalignant condition, low-count MBL may merely reflect immune senescence or result from persistent antigen stimulation.