Genomic variation by whole-genome SNP mapping arrays predicts time-to-event outcome in patients with chronic lymphocytic leukemia: a comparison of CLL and HapMap genotypes.

Genomic abnormalities, such as deletions in 11q22 or 17p13, are associated with poorer prognosis in patients with chronic lymphocytic leukemia (CLL). We hypothesized that unknown regions of copy number variation (CNV) affect clinical outcome and can be detected by array-based single-nucleotide polymorphism (SNP) genotyping. We compared SNP genotypes from 168 untreated patients with CLL with genotypes from 73 white HapMap controls. We identified 322 regions of recurrent CNV, 82 of which occurred significantly more often in CLL than in HapMap (CLL-specific CNV), including regions typically aberrant in CLL: deletions in 6q21, 11q22, 13q14, and 17p13 and trisomy 12. In univariate analyses, 35 of total and 11 of CLL-specific CNVs were associated with unfavorable time-to-event outcomes, including gains or losses in chromosomes 2p, 4p, 4q, 6p, 6q, 7q, 11p, 11q, and 17p. In multivariate analyses, six CNVs (ie, CLL-specific variations in 11p15.1-15.4 or 6q27) predicted time-to-treatment or overall survival independently of established markers of prognosis. Moreover, genotypic complexity (ie, the number of independent CNVs per patient) significantly predicted prognosis, with a median time-to-treatment of 64 months versus 23 months in patients with zero to one versus two or more CNVs, respectively (P = 3.3 × 10(-8)). In summary, a comparison of SNP genotypes from patients with CLL with HapMap controls allowed us to identify known and unknown recurrent CNVs and to determine regions and rates of CNV that predict poorer prognosis in patients with CLL.

Rapid generation of human B-cell lymphomas via combined expression of Myc and Bcl2 and their use as a preclinical model for biological therapies.

Although numerous mouse models of B-cell malignancy have been developed via the enforced expression of defined oncogenic lesions, the feasibility of generating lineage-defined human B-cell malignancies using mice reconstituted with modified human hematopoietic stem cells (HSCs) remains unclear. In fact, whether human cells can be transformed as readily as murine cells by simple oncogene combinations is a subject of considerable debate. Here, we describe the development of humanized mouse model of MYC/BCL2-driven 'double-hit' lymphoma. By engrafting human HSCs transduced with the oncogene combination into immunodeficient mice, we generate a fatal B malignancy with complete penetrance. This humanized-MYC/BCL2-model (hMB) accurately recapitulates the histopathological and clinical aspects of steroid-, chemotherapy- and rituximab-resistant human 'double-hit' lymphomas that involve the MYC and BCL2 loci. Notably, this model can serve as a platform for the evaluation of antibody-based therapeutics. As a proof of principle, we used this model to show that the anti-CD52 antibody alemtuzumab effectively eliminates lymphoma cells from the spleen, liver and peripheral blood, but not from the brain. The hMB humanized mouse model underscores the synergy of MYC and BCL2 in 'double-hit' lymphomas in human patients. Additionally, our findings highlight the utility of humanized mouse models in interrogating therapeutic approaches, particularly human-specific monoclonal antibodies.

Clinical safety and pharmacological profile of the HLA-DR antibody 1D09C3 in patients with B cell chronic lymphocytic leukemia and lymphoma: results from a phase I study.

1D09C3 is a human monoclonal IgG4-type antibody against human leukocyte antigen-DR (HLA-DR) which has demonstrated pro-apoptotic activity against lymphoid tumors in vitro and in vivo. We report results from a phase I dose-escalation study which aimed to identify tolerated dosing, and the pharmacokinetic and pharmacodynamic profile of 1D09C3. Fourteen patients with relapsed/refractory B cell type leukemia/lymphoma were treated and followed after up to 4 weekly infusions of 1D09C3, administered in 6 dose levels at 0.25-8 mg/kg/day. Treatment was tolerated well with mostly mild side effects. The most common grade III-IV toxicities were hematological events observed in 4 patients. In one patient, treated at 8.0 mg/kg/day, a dose limiting toxicity occurred, identified as an invasive catheter-related infection. Adverse events resolved completely without long-term sequelae. 1D09C3 reduced peripheral blood B cells and monocytes by a median of 73-81 % in all patients, with a nadir reached 30-60 min after infusion and sustained for <96 h. Granulocytes and natural killer cells predominantly increased with variable time courses. Pharmacokinetic assessments showed detectable drug concentrations at doses 4-8 mg/kg/day and a terminal half-life of 0.7-7.9 h. Effective saturation of HLA-DR on peripheral blood B cells/monocytes was achieved, varying consistently with available serum concentrations and the cell-reducing activity of 1D09C3. In summary, 1D09C3 could be administered safely in patients with advanced B cell malignancies. Pharmacodynamic studies demonstrated a strong dose dependent but transient reduction of peripheral blood B cells and monocytes, consistent with a short drug serum availability.

A two-gene signature, SKI and SLAMF1, predicts time-to-treatment in previously untreated patients with chronic lymphocytic leukemia.

We developed and validated a two-gene signature that predicts prognosis in previously-untreated chronic lymphocytic leukemia (CLL) patients. Using a 65 sample training set, from a cohort of 131 patients, we identified the best clinical models to predict time-to-treatment (TTT) and overall survival (OS). To identify individual genes or combinations in the training set with expression related to prognosis, we cross-validated univariate and multivariate models to predict TTT. We identified four gene sets (5, 6, 12, or 13 genes) to construct multivariate prognostic models. By optimizing each gene set on the training set, we constructed 11 models to predict the time from diagnosis to treatment. Each model also predicted OS and added value to the best clinical models. To determine which contributed the most value when added to clinical variables, we applied the Akaike Information Criterion. Two genes were consistently retained in the models with clinical variables: SKI (v-SKI avian sarcoma viral oncogene homolog) and SLAMF1 (signaling lymphocytic activation molecule family member 1; CD150). We optimized a two-gene model and validated it on an independent test set of 66 samples. This two-gene model predicted prognosis better on the test set than any of the known predictors, including ZAP70 and serum ß2-microglobulin.

Bendamustine combined with rituximab in patients with relapsed and/or refractory chronic lymphocytic leukemia: a multicenter phase II trial of the German Chronic Lymphocytic Leukemia Study Group.

PURPOSE: The objective of this trial was to evaluate safety and efficacy of bendamustine combined with rituximab (BR) in patients with relapsed and/or refractory chronic lymphocytic leukemia (CLL). PATIENTS AND METHODS: Seventy-eight patients, including 22 patients with fludarabine-refractory disease (28.2%) and 14 patients (17.9%) with deletion of 17p, received BR chemoimmunotherapy. Bendamustine was administered at a dose of 70 mg/m(2) on days 1 and 2 combined with rituximab 375 mg/m(2) on day 0 of the first course and 500 mg/m(2) on day 1 during subsequent courses for up to six courses. RESULTS: On the basis of intent-to-treat analysis, the overall response rate was 59.0% (95% CI, 47.3% to 70.0%). Complete response, partial response, and nodular partial response were achieved in 9.0%, 47.4%, and 2.6% of patients, respectively. Overall response rate was 45.5% in fludarabine-refractory patients and 60.5% in fludarabine-sensitive patients. Among genetic subgroups, 92.3% of patients with del(11q), 100% with trisomy 12, 7.1% with del(17p), and 58.7% with unmutated IGHV status responded to treatment. After a median follow-up time of 24 months, the median event-free survival was 14.7 months. Severe infections occurred in 12.8% of patients. Grade 3 or 4 neutropenia, thrombocytopenia, and anemia were documented in 23.1%, 28.2%, and 16.6% of patients, respectively. CONCLUSION: Chemoimmunotherapy with BR is effective and safe in patients with relapsed CLL and has notable activity in fludarabine-refractory disease. Major but tolerable toxicities were myelosuppression and infections. These promising results encouraged us to initiate a further phase II trial evaluating the BR regimen in patients with previously untreated CLL.

Chronic lymphocytic leukemia with t(14;19)(q32;q13) is characterized by atypical morphologic and immunophenotypic features and distinctive genetic features.

The t(14;19)(q32;q13) involving the IGH@ and BCL3 loci is an infrequent cytogenetic abnormality detected in B-cell malignancies. We describe the clinicopathologic, cytogenetic, and molecular genetic characteristics of 14 cases of chronic lymphocytic leukemia/small lymphocytic lymphoma (CLL/SLL) with t(14;19)(q32;q13). All patients (10 men and 4 women) had lymphocytosis; 10 had lymphadenopathy. Blood and bone marrow lymphocytes were predominantly small, but cytologically and immunophenotypically atypical. In all cases, t(14;19) was found in the neoplastic stem line; it was the sole abnormality in 4. Ten cases showed additional cytogenetic abnormalities, including trisomy 12 in 9 and complex karyotypes in 7. Fluorescence in situ hybridization demonstrated IGH@/BCL3 fusion gene in all cases. In all cases, the IGHV genes were unmutated, but only 7 expressed ZAP70. Seven cases preferentially used IGHV4-39. Our results indicate that t(14;19)(q32;q13) identifies a subset of CLL/SLL with distinctive clinicopathologic and genetic features. Furthermore, t(14;19) may represent an early, possibly primary, genetic event.

LDOC1 mRNA is differentially expressed in chronic lymphocytic leukemia and predicts overall survival in untreated patients.

We previously identified LDOC1 as one of the most significantly differentially expressed genes in untreated chronic lymphocytic leukemia (CLL) patients with respect to the somatic mutation status of the immunoglobulin heavy-chain variable region genes. However, little is known about the normal function of LDOC1, its contribution to the pathophysiology of CLL, or its prognostic significance. In this study, we have investigated LDOC1 mRNA expression in a large cohort of untreated CLL patients, as well as in normal peripheral blood B-cell (NBC) subsets and primary B-cell lymphoma samples. We have confirmed that LDOC1 is dramatically down-regulated in mutated CLL cases compared with unmutated cases, and have identified a new splice variant, LDOC1S. We show that LDOC1 is expressed in NBC subsets (naive > memory), suggesting that it may play a role in normal B-cell development. It is also expressed in primary B-cell lymphoma samples, in which its expression is associated with somatic mutation status. In CLL, we show that high levels of LDOC1 correlate with biomarkers of poor prognosis, including cytogenetic markers, unmutated somatic mutation status, and ZAP70 expression. Finally, we demonstrate that LDOC1 mRNA expression is an excellent predictor of overall survival in untreated CLL patients.

The B cell antigen receptor in atypical chronic lymphocytic leukemia with t(14;19)(q32;q13) demonstrates remarkable stereotypy.

The t(14;19)(q32;q13) is a recurrent chromosomal translocation reported in a variety of B-cell leukemias and lymphomas, including chronic lymphocytic leukemia (CLL). CLL cases associated with t(14;19) often have atypical morphologic and immunophenotypic features and unmutated immunoglobulin heavy chain (IGH) variable region (V) genes, associated with an aggressive clinical course. We analyzed IGHV somatic mutation status and gene use in 11 patients with t(14;19)-positive CLL. All cases were unmutated, and the IGHV genes in 10 cases showed minimal deviation from germline sequences. In 7 of 11 patients, we found homologous heavy chain rearrangements using IGHV4-39; light chain analysis revealed identical IGKV1-39 use. Corresponding V-(D)-J sequences demonstrated remarkable stereotypy of the immunoglobulin heavy and kappa light chain complementarity determining region 3 (H/K CDR3) genes. These findings raise the possibility that specific antigen drive is involved in the clonal development and/or selection of t(14;19)(q32;q13)-positive CLL cells. Our findings support the hypothesis that stimulatory signals through specific antigen receptors may promote the expansion of either CLL precursor cells or CLL clones that harbor distinct chromosomal abnormalities.

First-line therapy with fludarabine compared with chlorambucil does not result in a major benefit for elderly patients with advanced chronic lymphocytic leukemia.

Although chronic lymphocytic leukemia (CLL) is a disease of elderly patients, subjects older than 65 years are heavily underrepresented in clinical trials. The German CLL study group (GCLLSG) initiated a multicenter phase III trial for CLL patients older than 65 years comparing first-line therapy with fludarabine with chlorambucil. A total of 193 patients with a median age of 70 years were randomized to receive fludarabine (25 mg/m(2) for 5 days intravenously, every 28 days, for 6 courses) or chlorambucil (0.4 mg/kg body weight [BW] with an increase to 0.8 mg/kg, every 15 days, for 12 months). Fludarabine resulted in a significantly higher overall and complete remission rate (72% vs 51%, P = .003; 7% vs 0%, P = .011). Time to treatment failure was significantly shorter in the chlorambucil arm (11 vs 18 months; P = .004), but no difference in progression-free survival time was observed (19 months with fludarabine, 18 months with chlorambucil; P = .7). Moreover, fludarabine did not increase the overall survival time (46 months in the fludarabine vs 64 months in the chlorambucil arm; P = .15). Taken together, the results suggest that in elderly CLL patients the first-line therapy with fludarabine alone does not result in a major clinical benefit compared with chlorambucil. This trial is registered with www.isrctn.org under identifier ISRCTN 36294212.

Consolidation with alemtuzumab improves progression-free survival in patients with chronic lymphocytic leukaemia (CLL) in first remission: long-term follow-up of a randomized phase III trial of the German CLL Study Group (GCLLSG).

Alemtuzumab has shown considerable activity in untreated and relapsed chronic lymphocytic leukaemia. We report our long-term experience in 21 patients within a randomized phase III trial investigating the role of alemtuzumab for consolidation therapy after first-line fludarabine +/- cyclophosphamide, which was stopped prematurely due to severe infections. However, after a median follow-up of 48 months, progression-free survival was significantly prolonged for patients receiving alemtuzumab consolidation compared to those with no further treatment (P = 0.004). Minimal residual disease (MRD) levels were persistently reduced after consolidation. Therefore, despite toxicity, MRD reduction by alemtuzumab consolidation translates into a significantly improved long-term clinical outcome.

Lyme disease in a patient with chronic lymphocytic leukemia mimics leukemic meningeosis.

BACKGROUND: Involvement of the central nervous system (CNS) is a rare complication of chronic lymphocytic leukemia (CLL) and seems to be more frequent in patients with Richter's syndrome or prolymphocytic transformation. Cases with leptomeningeal involvement reported in the literature mostly do not discuss the definition of CLL-associated meningeosis and the exclusion of neuroborreliosis. PATIENT AND METHODS: We present the case of a 75-year-old male patient who was admitted to a rural hospital with ataxia, disorientation, and signs of progressive CLL disease. He was diagnosed of suspicious meningeosis leukemica, and treatment was started with dexamethasone for leukemic CNS involvement. RESULTS: When referred to our center, careful immunophenotyping of the CNS lymphocytes as well as assessment for infectious causes of lymphocytic meningitis led to the diagnosis of Lyme disease/neuroborreliosis. An antibiotic regimen with ceftriaxone for 3 weeks resulted in complete remission of all symptoms. There was no need for CLL treatment. CONCLUSION: In conclusion, this case report should alert clinicians that lymphocytic meningeal involvement in CLL patients accounts for the rare leukemic meningeosis only if cerebrospinal fluid cells show a predominating immunophenotype of typical BCLL cells, i.e. by flow cytometry, and if any infectious cause including Lyme disease has been ruled out.