Lorenzo De Paoli

Mutations of NOTCH1 are an independent predictor of survival in chronic lymphocytic leukemia.

Analysis of the chronic lymphocytic leukemia (CLL) coding genome has recently disclosed that the NOTCH1 proto-oncogene is recurrently mutated at CLL presentation. Here, we assessed the prognostic role of NOTCH1 mutations in CLL. Two series of newly diagnosed CLL were used as training (n = 309) and validation (n = 230) cohorts. NOTCH1 mutations occurred in 11.0% and 11.3% CLL of the training and validation series, respectively. In the training series, NOTCH1 mutations led to a 3.77-fold increase in the hazard of death and to shorter overall survival (OS; P < .001). Multivariate analysis selected NOTCH1 mutations as an independent predictor of OS after controlling for confounding clinical and biologic variables. The independent prognostic value of NOTCH1 mutations was externally confirmed in the validation series. The poor prognosis conferred by NOTCH1 mutations was attributable, at least in part, to shorter treatment-free survival and higher risk of Richter transformation. Although NOTCH1 mutated patients were devoid of TP53 disruption in more than 90% cases in both training and validation series, the OS predicted by NOTCH1 mutations was similar to that of TP53 mutated/deleted CLL. NOTCH1 mutations are an independent predictor of CLL OS, tend to be mutually exclusive with TP53 abnormalities, and identify cases with a dismal prognosis.

The Prognostic Value of TP53 Mutations in Chronic Lymphocytic Leukemia Is Independent of Del17p13: Implications for Overall Survival and Chemorefractoriness

Purpose: Del17p13 predicts poor outcome and chemorefractoriness in chronic lymphocytic leukemia (CLL). Conversely, it is unknown whether TP53 mutations carry any prognostic value independent of del17p13. We tested the independent prognostic value of TP53 mutations in CLL. Experimental Design: The study was based on a consecutive series of 308 CLL. DNA sequencing of TP53 exons 2 to 10 and del17p13 interphase fluorescence in situ hybridization were done at CLL diagnosis. Study end points were survival and chemorefractoriness. Results: At diagnosis, TP53 mutations (n = 32) occurred in 31 of 308 (10.0%) patients. Of all CLL showing TP53 disruption by either mutation and/or deletion (n = 44), 10 cases (22.7%) showed TP53 mutations in the absence of del17p13. Multivariate analysis selected TP53 mutations (hazard ratio, 3.20; P = 0.002) as an independent predictor of overall survival after adjustment for del17p13. Also, multivariate analysis selected TP53 mutations (hazard ratio, 3.97; P < 0.001) as an independent predictor of chemorefractoriness after adjustment for del17p13. Compared with cases without TP53 alterations, CLL harboring any type of TP53 disruption (mutation only, del17p13 only, or both mutation and del17p13) uniformly displayed a high prevalence of unfavorable prognosticators and poor outcome. Analysis of sequential CLL samples showed the acquisition of new or additional TP53 alterations at the time of chemorefractoriness. Conclusions: These data show that (a) TP53 mutations are an independent predictor of short survival and chemorefractoriness, and (b) that CLL presenting with TP53 mutations without del17p13 fare as poorly as CLL carrying del17p13. Because CLL harboring TP53 mutations without del17p13 are currently not recognized by conventional diagnostic strategies, these results may be relevant for a comprehensive prognostic characterization of CLL.

Clinical impact of small TP53 mutated subclones in chronic lymphocytic leukemia

TP53 mutations are strong predictors of poor survival and refractoriness in chronic lymphocytic leukemia (CLL) and have direct implications for disease management. Clinical information on TP53 mutations is limited to lesions represented in >20% leukemic cells. Here, we tested the clinical impact and prediction of chemorefractoriness of very small TP53 mutated subclones. The TP53 gene underwent ultra-deep-next generation sequencing (NGS) in 309 newly diagnosed CLL. A robust bioinformatic algorithm was established for the highly sensitive detection of few TP53 mutated cells (down to 3 out of ∼1000 wild-type cells). Minor subclones were validated by independent approaches. Ultra-deep-NGS identified small TP53 mutated subclones in 28/309 (9%) untreated CLL that, due to their very low abundance (median allele frequency: 2.1%), were missed by Sanger sequencing. Patients harboring small TP53 mutated subclones showed the same clinical phenotype and poor survival (hazard ratio = 2.01; P = .0250) as those of patients carrying clonal TP53 lesions. By longitudinal analysis, small TP53 mutated subclones identified before treatment became the predominant population at the time of CLL relapse and anticipated the development of chemorefractoriness. This study provides a proof-of-principle that very minor leukemia subclones detected at diagnosis are an important driver of the subsequent disease course.

Biological and clinical risk factors of chronic lymphocytic leukaemia transformation to Richter syndrome

Predictors of chronic lymphocytic leukaemia (CLL) transformation to Richter syndrome (RS) are not established and were investigated in 185 consecutive CLL cases. Actuarial incidence of RS (n = 17; all diffuse large B‐cell lymphomas) at 10 years was 16·2% (95% confidence interval: 8·0–24·4%). At CLL diagnosis, prognosticators of RS by univariate analysis were IGHV homology ≥98% (P = 0·006), IGHV4‐39 usage (P < 0·001), del13q14 absence (P = 0·004), expression of CD38 (P < 0·001) and ZAP70 (P = 0·004), size (P < 0·001) and number (P < 0·001) of lymph nodes, advanced Binet stage (P = 0·002), and lactate dehydrogenase (P < 0·001). Multivariate analysis, performed separately for biological and clinical variables, identified CD38 expression [Hazard ratio (HR) = 4·26; P = 0·018], IGHV4‐39 usage (HR = 4·29; P = 0·018), and lymph node size ≥3 cm (HR = 9·07; P < 0·001) as independent RS prognosticators. A multivariate model simultaneously analysing biological and clinical variables identified lymph node size ≥3 cm (HR = 6·51; P = 0·001) and del13q14 absence (HR = 4·08; P = 0·031) as independent RS prognosticators. Risk factors of CLL transformation differed from risk factors of CLL progression. These results suggest that CD38 and del13q14 may identify biological subsets of CLL with different RS predisposition. Predominant nodal disease, CD38 expression, IGHV4‐39 usage, and absence of del13q14 may help in predicting RS at CLL diagnosis. Close monitoring and a careful biopsy policy are needed in patients carrying transformation risk factors.

The prognosis of clinical monoclonal B cell lymphocytosis differs from prognosis of Rai 0 chronic lymphocytic leukaemia and is recapitulated by biological risk factors

Monoclonal B‐cell lymphocytosis (MBL) is an asymptomatic monoclonal expansion of <5·0 × 109/l circulating CLL‐phenotype B‐cells. The relationship between MBL and Rai 0 CLL, as well as the impact of biological risk factors on MBL prognosis, are unknown. Out of 460 B‐cell expansions with CLL‐phenotype, 123 clinical MBL (cMBL) were compared to 154 Rai 0 CLL according to clinical and biological profile and outcome. cMBL had better humoral immune capacity and lower infection risk, lower prevalence of del11q22‐q23/del17p13 and TP53 mutations, slower lymphocyte doubling time, and longer treatment‐free survival. Also, cMBL diagnosis was a protective factor for treatment risk. Despite these favourable features, all cMBL were projected to progress, and lymphocytes <1·2 × 109/l and >3·7 × 109/l were the best thresholds predicting the lowest and highest risk of progression to CLL. Although IGHV status, CD38 and CD49d expression, and fluorescence in situ hybridization (FISH) karyotype individually predicted treatment‐free survival, multivariate analysis identified the presence of +12 or del17p13 as the sole independent predictor of treatment requirement in cMBL (Hazard ratio: 5·39, 95% confidence interval 1·98–14·44, P = 0·001). Overall, these data showed that cMBL has a more favourable clinical course than Rai 0 CLL. Given that the biological profile can predict treatment requirement, stratification based on biological prognosticators may be helpful for cMBL management.

Stereotyped B-cell receptor is an independent risk factor of chronic lymphocytic leukemia transformation to Richter syndrome.

Purpose: Few biological prognosticators are useful for prediction of Richter syndrome (RS), representing the transformation of chronic lymphocytic leukemia (CLL) to aggressive lymphoma. Stereotyped B-cell receptors (BCR) may have prognostic effect in CLL progression. We tested the prognostic effect of stereotyped BCR for predicting RS transformation. Experimental Design: The prevalence of stereotyped BCR was compared in RS (n = 69) versus nontransformed CLL (n = 714) by a case-control analysis. Subsequently, the effect of stereotyped BCR at CLL diagnosis on risk of RS transformation was actuarially assessed in a consecutive CLL series (n = 753). Results: RS (n = 69) displayed a higher prevalence of stereotyped BCR (P < 0.001) compared with nontransformed CLL. The actuarial risk of RS transformation was significantly higher in CLL carrying stereotyped BCR (P < 0.001). Among BCR subsets most represented in CLL, subset 8 using IGHV4-39/IGHD6-13/IGHJ5 carried the highest risk of RS transformation [hazard ratio (HR), 24.50; P < 0.001]. Multivariate analysis selected stereotyped BCR (HR, 3.33; P = 0.001) and IGHV4-39 usage (HR, 4.03; P = 0.004) as independent predictors of RS transformation. The combination of IGHV4-39 usage and stereotyped BCR in the same patient identified CLL with a very high risk of RS transformation (5-year risk, 68.7%). The risk carried by stereotyped BCR and IGHV4-39 usage was specific for RS transformation and had no effect on CLL progression without transformation. Conclusions: Analysis of BCR features may help identify CLL patients at risk of RS. A close monitoring and a careful biopsy policy may help early recognition of RS in CLL patients using stereotyped BCR, particularly if combined with IGHV4-39.

Molecular prediction of durable remission after first-line fludarabinecyclophosphamide-rituximab in chronic lymphocytic leukemia

Fludarabine, cyclophosphamide, and rituximab (FCR) has represented a significant treatment advancement in chronic lymphocytic leukemia (CLL). In the new scenario of targeted agents, there is an increasing interest in identifying patients who gain the maximum benefit from FCR. In this observational multicenter retrospective analysis of 404 CLL patients receiving frontline FCR, the combination of three biomarkers that are widely tested before treatment (IGHV mutation status, 11q deletion and 17p deletion; available in 80% of the study cohort) allowed to identify a very low-risk category of patients carrying mutated IGHV genes but neither 11q or 17p deletion that accounted for 28% of all cases. The majority of very low-risk patients (71%) remained free of progression after treatment and their hazard of relapse decreased after 4 years from FCR. The life expectancy of very low-risk patients (91% at 5 years) was superimposable to that observed in the matched normal general population, indicating that neither the disease nor complications of its treatment affected survival in this favorable CLL group. These findings need a prospective validation and may be helpful for the design of clinical trials aimed at comparing FCR to new targeted treatments of CLL, and, possibly, for optimized disease management.

CD49d expression is an independent risk factor of progressive disease in early stage chronic lymphocytic leukemia

Findings of this study suggest that CD49d expression may be useful for the initial prognostic assessment of patients with chronic lymphocytic leukemia in the initial stage. Identification of prognosticators for Binet A chronic lymphocytic leukemia is important for selecting patients with dismal prognosis. We analyzed CD49d expression in 140 consecutive Binet A chronic lymphocytic leukemia. At diagnosis, CD49d ≥30% (54/140, 38.6%) associated with proliferation markers, namely CD38 ≥30% (p=3.9×10−6), LDH (p=0.007) and β2-microglobulin (p=0.020). Univariate log-rank analysis identified CD49d ≥30% as a risk factor of treatment free survival (p=8.3x10−5), time to progression to a more advanced stage (p=4.7×10−4), and time to lymphocyte doubling (p=0.009). Multivariate analysis selected CD49d ≥30% as an independent treatment free survival predictor after adjustment for biological (HR 2.28; 95% CI 1.71–4.45, p=0.015) and both biological and clinical variables analyzed together (HR 3.33, 95% CI 1.61–6.90, p=0.001). Within Binet A subgroups harboring favorable biological variables (IGHV homology <98%, favorable karyotype, CD38 <30%, ZAP70 <20%) or clinical variables, CD49d ≥30% consistently identified a subset of patients with short treatment free survival. Our observations indicate CD49d ≥30% as a new marker for the initial prognostic assessment of Binet A chronic lymphocytic leukemia.

Triplet vs doublet lenalidomide-containing regimens for the treatment of elderly patients with newly diagnosed multiple myeloma

Lenalidomide-dexamethasone improved outcome in newly diagnosed elderly multiple myeloma patients. We randomly assigned 662 patients who were age ≥65 years or transplantation-ineligible to receive induction with melphalan-prednisone-lenalidomide (MPR) or cyclophosphamide-prednisone-lenalidomide (CPR) or lenalidomide plus low-dose dexamethasone (Rd). The primary end point was progression-free survival (PFS) in triplet (MPR and CPR) vs doublet (Rd) lenalidomide-containing regimens. After a median follow-up of 39 months, the median PFS was 22 months for the triplet combinations and 21 months for the doublet (P = .284). The median overall survival (OS) was not reached in either arms, and the 4-year OS was 67% for the triplet and 58% for the doublet arms (P = .709). By considering the 3 treatment arms separately, no difference in outcome was detected among MPR, CPR, and Rd. The most common grade ≥3 toxicity was neutropenia: 64% in MPR, 29% in CPR, and 25% in Rd patients (P < .0001). Grade ≥3 nonhematologic toxicities were similar among arms and were mainly infections (6.5% to 11%), constitutional (3.5% to 9.5%), and cardiac (4.5% to 6%), with no difference among the arms. In conclusion, in the overall population, the alkylator-containing triplets MPR and CPR were not superior to the alkylator-free doublet Rd, which was associated with lower toxicity. This study was registered at www.clinicaltrials.gov as #NCT01093196.

MGA, a suppressor of MYC, is recurrently inactivated in high risk chronic lymphocytic leukemia.

Although modern treatment strategies are highly effective in many cases of progressive chronic lymphocytic leukemia (CLL), a challenging subgroup of patients show poor response to standard regimens...