Francesco Autore

Two types of BCR interactions are positively selected during leukemia development in the Eμ-TCL1 transgenic mouse model of CLL

Chronic lymphocytic leukemia (CLL) is a common B-cell malignancy characterized by a highly variable course and outcome. The disease is believed to be driven by B-cell receptor (BCR) signals generated by external antigens and/or cell-autonomous BCR interactions, but direct in vivo evidence for this is still lacking. To further define the role of the BCR pathway in the development and progression of CLL, we evaluated the capacity of different types of antigen/BCR interactions to induce leukemia in the Eμ-TCL1 transgenic mouse model. We show that cell autonomous signaling capacity is a uniform characteristic of the leukemia-derived BCRs and represents a prerequisite for CLL development. Low-affinity BCR interactions with autoantigens generated during apoptosis are also positively selected, suggesting that they contribute to the pathogenesis of the disease. In contrast, high-affinity BCR interactions are not selected, regardless of antigen form or presentation. We also show that the capacity of the leukemic cells to respond to cognate antigen correlates inversely with time to leukemia development, suggesting that signals induced by external antigen increase the aggressiveness of the disease. Collectively, these findings provide in vivo evidence that the BCR pathway drives the development and can influence the clinical course of CLL.

B-cell receptor configuration and adverse cytogenetics are associated with autoimmune hemolytic anemia in chronic lymphocytic leukemia

The development of autoimmune hemolytic anemia (AIHA) in patients with chronic lymphocytic leukemia (CLL) is associated with specific biological features. The occurrence of AIHA was hereby investigated in a retrospective series of 585 CLL patients with available immunoglobulin heavy chain variable (IGHV) gene status. AIHA occurred in 73 patients and was significantly associated with an IGHV unmutated (UM) status (P < 0.0001) and unfavorable [del(17)(p13) and del(11)(q23)] cytogenetic lesions (P < 0.0001). Stereotyped HCDR3 sequences were identified in 29.6% of cases and were similarly represented among patients developing or not AIHA; notably, subset #3 was associated with a significantly higher risk of AIHA than the other patients (P = 0.004). Multivariate analysis showed that UM IGHV, del(17)(p13) and del(11)(q23), but not stereotyped subset #3, were the strongest independent variables associated with AIHA. Based on these findings, we generated a biological risk score for AIHA development according to the presence of none (low risk), one (intermediated risk), or two (high risk) of the independent risk factors. Overall, our data indicate that UM IGHV status and/or unfavorable cytogenetic lesions are associated with the risk of developing secondary AIHA in CLL patients and suggest a possible role of specific stereotyped B‐cell receptor subsets in a proportion of cases. Am. J. Hematol. 2013.

Allele-specific loss and transcription of the miR-15a/16-1 cluster in chronic lymphocytic leukemia

Deregulation of the miR-15a/16-1 cluster has a key role in the pathogenesis of chronic lymphocytic leukemia (CLL), a clinically heterogeneous disease with indolent and aggressive forms. The miR-15a/16-1 locus is located at 13q14, the most frequently deleted region in CLL. Starting from functional investigations of a rare SNP upstream the miR cluster, we identified a novel allele-specific mechanism that exploits a cryptic activator region to recruit the RNA polymerase III for miR-15a/16-1 transcription. This regulation of the miR-15a/16- locus is independent of the DLEU2 host gene, which is often transcribed monoallellically by RPII. We found that normally one allele of miR-15a/16-1 is transcribed by RNAPII, the other one by RNAPIII. In our subset of CLL patients harboring 13q14 deletions, exclusive RNA polymerase III (RPIII)-driven transcription of the miR-15a/16-1 was the consequence of loss of the RPII-regulated allele and correlated with high expression of the poor prognostic marker ZAP70 (P=0.019). Thus, our findings point to a novel biological process, characterized by double allele-specific transcriptional regulation of the miR-15a/16-1 locus by alternative mechanisms. Differential usage of these mechanisms may distinguish at onset aggressive from indolent forms of CLL. This provides a basis for the clinical heterogeneity of the CLL patients carrying 13q14 deletions.

Bendamustine in combination with rituximab for elderly patients with previously untreated B-cell chronic lymphocytic leukemia: A retrospective analysis of real-life practice in Italian hematology departments

The front-line therapy for CLL young and fit patients is chemo-immunotherapy with fludarabine-cyclophosphamide-rituximab (FCR). FCR regimen results in a significant myelosuppression and high rates of early and late infections especially in elderly patients. German CLL study group compared FCR vs. bendamustine-rituximab (BR) in fit untreated patients. The response rates with BR or FCR were comparable, BR could be an alternative 1st-line treatment for elderly patients. Here we report retrospective data of 70 elderly (≥65 years) CLL patients from 12 Italian centers treated with BR as front-line therapy. The primary end points were overall response rate (complete remission/partial remission) and safety. Forty-seven males and 23 females, with a median age of 72 years, were included in the study. Eight patients were unfit for CIRS. The OR rate was 88.6% (31.4% CR and 57.2% PR). Progression free survival, treatment free survival and overall survival rates at 2-years were 79%, 90.3% and 89.6%, respectively. Only del17 was independent unfavorable parameter on the response rate and PFS. Our results indicate that BR front-line at standard dose provides a high response rate with a good safety profile, even if more than 50% of patients experienced a bendamustine dose reduction until 70 mg/m2.

Diagnostic and prognostic role of PET/CT in patients with chronic lymphocytic leukemia and progressive disease

In order to evaluate the predictive value of positron emission tomography–computed tomography (PET/CT) in discriminating the presence of a Richter’s syndrome (RS) or a second malignancy (SM), as well as to evaluate its prognostic value in patients with chronic lymphocytic leukemia (CLL), we retrospectively analyzed the data of 90 patients who, in the suspicion of a RS or a SM, underwent PET/CT followed by the biopsy of the involved tissue. The median maximum Standardized Uptake Value (SUVmax) in the presence of a CLL/small lymphocytic lymphoma, a diffuse large B-cell lymphoma (DLBCL), a Hodgkin lymphoma (HL), a SM were 3.5, 14.6, 7.0 and 6.3, respectively (P⩽0.0001). A SUVmax cutoff value ⩾5 showed a sensitivity, specificity, positive and negative predictive values of 88.2, 71.2, 51.3 and 94%, respectively, for the presence of a more aggressive disease (DLBCL, HL and SM). A SUVmax ⩾5 identified also a subset of treatment naive patients with an inferior progression-free survival (P=0.011) and overall survival (P=0.067). These findings suggest that PET/CT may helpfully integrate the biologically-based prognostic stratification of CLL. Prospective clinical trials including larger cohorts of patients are needed to conclusively define the role and prognostic impact of PET/CT in the routine management of CLL patients.

CHLORAMBUCIL PLUS RITUXIMAB AS FRONT-LINE THERAPY IN ELDERLY/UNFIT PATIENTS AFFECTED BY B-CELL CHRONIC LYMPHOCYTIC LEUKEMIA: RESULTS OF A SINGLE-CENTRE EXPERIENCE.

The current standard first line therapy for fit patients with B-CLL/SLL is based on combination of fludarabine-cyclophosphamide and rituximab. However, elderly patients or patients with comorbidities poorly tolerate purine analogue-based chemotherapy and they are often treated with Chlorambucil (Chl) only. However, complete response (CR) and overall response (OR) rates with Chl are relatively low. We now investigated whether the addition of Rituximab to Chl will improve the efficacy without impairing the tolerability in elderly and unfit patients. We included in our study 27 elderly or unfit patients that had not received prior therapy. All patients were treated with Chl (1mg/Kg per 28-day cycle for 8 cycles) plus Rituximab (375 mg/m2 for the first course and 500 mg/m2 for subsequent cycles until the 6th cycle). We obtained an OR rate of 74%. The most frequent adverse effect was grade 3–4 neutropenia, which occurred in 18.5% of the patients. Infections or grade 3–4 extra-hematological side effects were not recorded. None of the patients required reduction of dose, delay of therapy or hospitalization. Overall, these data suggest that Chl-R is an effective and well tolerated regimen in elderly/unfit patients with CLL.

Prevalence, characteristics and management of occult hepatitis B virus infection in patients with chronic lymphocytic leukemia: a single center experience.

Several reports have emphasized the risk of hepatitis B virus (HBV) reactivation in patients with lymphoproliferative disorders undergoing cytotoxic treatment. To determine the prevalence of occult B infection (OBI) in a population with chronic lymphocytic leukemia (CLL) and management with universal prophylaxis (UP) in all patients undergoing chemoimmunotherapy or targeted prophylaxis (TP) in patients experiencing seroreversion during therapy, we analyzed 397 patients with CLL from our database. The prevalence of OBI in our patients with CLL was 8.6% (34 patients). When comparing patients with OBI/CLL with those with CLL, we did not find any statistical difference among clinical–biological parameters and time dependent endpoints except for a lower peripheral blood lymphocyte count in the OBI/CLL group (p = 0.036). From 2000 to 2010 careful follow-up and TP were adopted; two out of 10 patients (20%) showed seroreversion. From June 2010 we adopted UP during and 12 months after immunosuppressive treatment in all patients with CLL with OBI; no evidence of seroreversion was detected.

New developments in the management of chronic lymphocytic leukemia: role of ofatumumab

Ofatumumab is one of the three anti-CD20 monoclonal antibodies currently available for the treatment of chronic lymphocytic leukemia (CLL). The US Food and Drug Administration (FDA) approved the use of ofatumumab in patients with CLL refractory to fludarabine and alemtuzumab in 2009, and the European Medicines Agency (EMA) granted approval for the same indication in 2010. Subsequent positive results of ofatumumab in combination with chlorambucil in treatment-naïve patients led the FDA in April 2014 to approve the use of this combination for first-line treatment of patients with CLL for whom fludarabine-based therapy is considered inappropriate. Later that year, the EMA approved the use of ofatumumab in combination with chlorambucil or bendamustine for the same indication. Ofatumumab has also shown potential as maintenance therapy for patients with relapsed CLL; an application to broaden the label for ofatumumab as maintenance therapy was submitted earlier this year to the EMA and FDA. Finally, ofatumumab has shown promising activity in combination with ibrutinib or idelalisib in relapsed/refractory CLL patients; combinations of ofatumumab with B-cell-receptor pathway inhibitors could represent another potential use of this antibody in the near future.

Autoimmune hemolytic anemia during bendamustine plus rituximab treatment in CLL patients: multicenter experience.

Luca Laurenti, Francesco Autore, Idanna Innocenti, Giovanni D’Arena, Marta Coscia, Patrizia Mondello, Patrizia Chiusolo, Silvia Bellesi, Dimitar G. Efremov, Simona Sica and Francesca Romana Mauro Catholic University of the Sacred Heart, Rome, Italy; IRCCS Centro Di Riferimento Oncologico Della Basilicata, Rionero in Vulture, Italy; Division of Hematology, Department of Molecular Biotechnology and Health Sciences, University of Torino, Torino, Italy; Department of Human Pathology, University of Messina, Messina, Italy; International Center for Genetic Engineering & Biotechnology, Monterotondo Scalo, Italy; Department of Cellular Biotechnologies and Hematology, Sapienza University, Rome, Italy

CD200 included in a 4-marker modified Matutes score provides optimal sensitivity and specificity for the diagnosis of chronic lymphocytic leukaemia

CD200, a transmembrane type Ia glycoprotein belonging to the immunoglobulin superfamily, has been shown to have a differential expression in B‐cell neoplasms. Here, we retrospectively assessed the diagnostic relevance of CD200 on 427 patients with B‐cell chronic neoplasms in leukemic phase (median age, 69 y; range, 35‐97 y). The final diagnosis based on the investigators assessment was chronic lymphocytic leukaemia (CLL) in 75% of cases and non‐CLL in 25% of cases. Sensitivity and specificity for the diagnosis of CLL (vs non‐CLL) were calculated for the following markers: CD200, CD5, CD22, CD23, CD79b, FMC7, and SmIg. CD23 was the only marker without a statistically significant difference between the investigator assessment and the flowcytometric analysis. The other markers were unable—when individually evaluated—to discriminate between CLL and non‐CLL, requiring the integration into a scoring system. The modified score no. 1 (addition of CD200) showed superimposable sensitivity and specificity compared with the Matutes score. The substitution of CD79b (modified score no. 2), surface membrane immunoglobulins (SmIg) (modified score no. 3), and CD79b and FMC7 (modified score no. 4) with CD200 showed that only the modified score no. 4 had both higher sensitivity and higher specificity compared with standard Matutes score. In conclusion, this work defines a simplified score, compared with the classical Matutes score, for the differential diagnosis of chronic B‐cell leukaemia—which only requires 4 markers instead of 5 (CD5, CD23, CD200, and SmIg).