Impact Of Clinical and Biological Factors On Outcome In a Cohort Of Unselected Chronic Lymphocytic Leukemia (CLL) Patients
Authors: Piras Giovanna , Murru Roberta , Palmas Angelo D. , Asproni Rosanna , Uras Antonella , Monne Maria , Murineddu Marco , Pira Giovanna , Calvisi Anna , Angelucci Emanuele , Latte Giancarlo 
Published: October 2019

Abstract

Introduction
Chronic Lymphocytic Leukemia (CLL) is a heterogeneous disease with a variable clinical course. Therapeutic decisions are based mainly on clinical grounds. Several prognostic markers based on genetic, phenotypic and molecular lesions have emerged in the past decade as relevant, alone or in combination with each other, in order to predict the clinical course and the best treatment option for CLL patients. Among these, NOTCH1 and TP53 mutation, have been described as new prognostic markers and predictive of survival in CLL. In this study, a real-life cohort of 165 CLL patients from Sardinia (Italy), was retrospectively analyzed for clinical, laboratory data (including NOTCH-1, TP53 mutation), first line treatment and followed up for survival.

Methods
Patients (99 males, 66 females, age ranged 31-89 y, 24% <55 y, Binet A 60%, Binet B/C 40%) were diagnosed with CLL between 1988-2012 by standard criteria. First-line treatment: 34% patients received no therapy, 30% fludarabine based regimens (+/-Rituximab), 32% alkylating agents based regimens (+/- Rituximab), 4% other regimens. Cytogenetic analysis was performed on peripheral blood cultured in the presence of the immunostimulatory CpG-oligonucleotide DSP30/Interleukin-2. FISH analysis on interphase nuclei was done using conventional 4-probe panel. The presence of NOTCH1 c.7544_7545delCT was investigated by ARMS PCR approach. The TP53 exons 4-11 were amplified in 33 patients and direct sequenced. Data from chromosome banding analysis (n=73), CD38 expression (n=133), IGHV mutation status (n=110) were also available. Categorical variables were compared by chi-squared test and Fisher exact test when appropriate. Statistical significance was defined as P value= 0,05. Kaplan-Meier curves and log rank test were used to determine overall survival and Cox regression analysis to calculate hazardous ratios using the R statistical program.

Results
Survival curve for the entire cohort ofpatients showed a median survival time of 195 months (95% CI 185-303). Patient stratification based on 1st line-treatment regimens did not show any significant difference in clinical outcome. FISH analysis detected trisomy 12 in 16% (24/154) patients, 13q14 deletion in 45/154 (29%), 17p deletion in 6% (10/154) and 11q deletion in 3%(5/154). Abnormal karyotype was present in 37% (27/73) of samples including 6 cases which resulted negative at the FISH test. Presence of 17p deletion was associated with the worse overall survival in univariate analysis (81 months, 95%CI 56-117, p value=0.0001). NOTCH1 c.7544_7545delCT was present in 8% (7/99), in 21% of trisomy 12 cases and in 16,6% of patients with no other genetic prognostic markers. The presence of NOTCH1 mutation had no impact on outcome. Comparing baseline characteristics between patients with or without mutations, no significant differences were found for age, sex, stage, B symptoms, blood cell counts, LDH, ß2-microglobulin. TP53 mutations were found in 2 out of 33 patients (6%). TP53p.R181P and p.249_251del10ins3 were present in two cases with 17p deletion detected by FISH. Thirtynine patients (35%) displayed an unmutated IGHV status, 68 cases (61%) had a mutated IGHV gene while 4 cases had a borderline IGHV mutation status. Unmutated IGHV status was an indipendent prognostic factor for worse overall survival (median survival 110 months 95%CI 85-303, p value= 0,02). The IGHV gene family usage within the mutated group was VH4>VH3>VH2>VH1>VH7, whereas IGHV 3 was the most frequently used in the unmutated group, being expressed in 13 patients. IGHV 1-69 genes were present in 3 CLL patients overall (2 unmutated). Correlation between cytogenetic categories and mutation status showed that the poor prognostic marker 17p deletion was present in 4/36 (11,0%) of unmutated CLLs and in 2/55 (3,6%) of mutated cases while 13q14 deletion was statistically associated with the 45% of mutated cases (p=0.0089).

Conclusion
In our cohort of unselected patients NOTCH1 mutation didn’t affect outcome of CLL patients, while TP53 deletion/mutation and IGHV mutational status maintain a strong prognostic negative impact. Treatment heterogeneity is likely the reason of absence of difference in outcome in our study. In current clinical practice prognostic markers need yet to be validated in large clinical trials, not biased by stringent selection criteria.

Disclosures:
No relevant conflicts of interest to declare.