Florence Cymbalista

Ibrutinib versus ofatumumab in previously treated chronic lymphoid leukemia

BACKGROUND In patients with chronic lymphoid leukemia (CLL) or small lymphocytic lymphoma (SLL), a short duration of response to therapy or adverse cytogenetic abnormalities are associated with a poor outcome. We evaluated the efficacy of ibrutinib, a covalent inhibitor of Brutons tyrosine kinase, in patients at risk for a poor outcome. METHODS In this multicenter, open-label, phase 3 study, we randomly assigned 391 patients with relapsed or refractory CLL or SLL to receive daily ibrutinib or the anti-CD20 antibody ofatumumab. The primary end point was the duration of progression-free survival, with the duration of overall survival and the overall response rate as secondary end points. RESULTS At a median follow-up of 9.4 months, ibrutinib significantly improved progression-free survival; the median duration was not reached in the ibrutinib group (with a rate of progression-free survival of 88% at 6 months), as compared with a median of 8.1 months in the ofatumumab group (hazard ratio for progression or death in the ibrutinib group, 0.22; P<0.001). Ibrutinib also significantly improved overall survival (hazard ratio for death, 0.43; P=0.005). At 12 months, the overall survival rate was 90% in the ibrutinib group and 81% in the ofatumumab group. The overall response rate was significantly higher in the ibrutinib group than in the ofatumumab group (42.6% vs. 4.1%, P<0.001). An additional 20% of ibrutinib-treated patients had a partial response with lymphocytosis. Similar effects were observed regardless of whether patients had a chromosome 17p13.1 deletion or resistance to purine analogues. The most frequent nonhematologic adverse events were diarrhea, fatigue, pyrexia, and nausea in the ibrutinib group and fatigue, infusion-related reactions, and cough in the ofatumumab group. CONCLUSIONS Ibrutinib, as compared with ofatumumab, significantly improved progression-free survival, overall survival, and response rate among patients with previously treated CLL or SLL. (Funded by Pharmacyclics and Janssen; RESONATE ClinicalTrials.gov number, NCT01578707.).

ERIC recommendations on TP53 mutation analysis in chronic lymphocytic leukemia

Recent evidence suggests that – in addition to 17p deletion – TP53 mutation is an independent prognostic factor in chronic lymphocytic leukemia (CLL). Data from retrospective analyses and prospective clinical trials show that ∼5% of untreated CLL patients with treatment indication have a TP53 mutation in the absence of 17p deletion. These patients have a poor response and reduced progression-free survival and overall survival with standard treatment approaches. These data suggest that TP53 mutation testing warrants integration into current diagnostic work up of patients with CLL. There are a number of assays to detect TP53 mutations, which have respective advantages and shortcomings. Direct Sanger sequencing of exons 4–9 can be recommended as a suitable test to identify TP53 mutations for centers with limited experience with alternative screening methods. Recommendations are provided on standard operating procedures, quality control, reporting and interpretation. Patients with treatment indications should be investigated for TP53 mutations in addition to the work-up recommended by the International workshop on CLL guidelines. Patients with TP53 mutation may be considered for allogeneic stem cell transplantation in first remission. Alemtuzumab-based regimens can yield a substantial proportion of complete responses, although of short duration. Ideally, patients should be treated within clinical trials exploring new therapeutic agents.

Improving efficiency and sensitivity: European Research Initiative in CLL (ERIC) update on the international harmonised approach for flow cytometric residual disease monitoring in CLL

Detection of minimal residual disease (MRD) in chronic lymphocytic leukaemia (CLL) is becoming increasingly important as treatments improve. An internationally harmonised four-colour (CLR) flow cytometry MRD assay is widely used but has limitations. The aim of this study was to improve MRD analysis by identifying situations where a less time-consuming CD19/CD5/κ/λ analysis would be sufficient for detecting residual CLL, and develop a six-CLR antibody panel that is more efficient for cases requiring full MRD analysis. In 784 samples from CLL patients after treatment, it was possible to determine CD19/CD5/κ/λ thresholds that identified cases with detectable MRD with 100% positive predictive value (PPV). However, CD19/CD5/κ/λ analysis was unsuitable for predicting iwCLL/NCI response status or identifying cases with no detectable MRD. For the latter cases requiring a full MRD assessment, a six-CLR assay was designed comprising CD19/CD5/CD20 with (1) CD3/CD38/CD79b and (2) CD81/CD22/CD43. There was good correlation between four-CLR and six-CLR panels in dilution studies and clinical samples, with 100% concordance for detection of residual disease at the 0.01% (10−4) level (n=59) and good linearity even at the 0.001–0.01% (10−5–10−4) level. A six-CLR panel therefore provides equivalent results to the four-CLR panel but it requires fewer reagents, fewer cells and a much simpler analysis approach.

Excess mortality after treatment with fludarabine and cyclophosphamide in combination with alemtuzumab in previously untreated patients with chronic lymphocytic leukemia in a randomized phase 3 trial

A French and Belgian multicenter phase 3 trial was conducted in medically fit patients with untreated chronic lymphocytic leukemia. Of 178 patients enrolled in the study, 165 were randomly assigned to receive 6 courses of oral fludarabine and cyclophosphamide (FC) in combination with rituximab (FCR; 375 mg/m(2) in cycle one, 500 mg/m(2) in all subsequent cycles) or alemtuzumab (FCCam; 30 mg subcutaneously injected on cycle days 1-3); each cycle was 28 days. Recruitment was halted prematurely because of excess toxicity; 8 patients died in the FCCam group, 3 from lymphoma and 5 from in-fection. Overall response rates were 91% with FCR and 90% with FCCam (P = .79). Complete remission rates were 33.75% with FCR and 19.2% with FCCam (P = .04). Three-year progression-free survival was 82.6% with FCR and 72.5% with FCCam (P = .21). Three-year overall survival was similar between the 2 arms at 90.1% in the FCR arm and 86.4% in the FCCam arm (P = .27). These results indicate that the FCCam regimen for the treatment of advanced chronic lymphocytic leukemia was not more effective than the FCR regimen and was associated with an unfavorable safety profile, representing a significant limitation of its use. This study is registered with www.clinicaltrials.gov as number NCT00564512.

Atrial fibrillation in CLL patients treated with ibrutinib. An international retrospective study.

Atrial fibrillation (AF) occurs in 5–9% of patients treated with ibrutinib for chronic lymphocytic leukaemia (CLL); the clinical consequences and optimal management are unclear. We retrospectively studied 56 CLL patients who received ibrutinib and developed AF. Median time to onset was 3·8 months. AF was persistent in 35/56 (62%) cases despite treatment. Clinical consequences included: three episodes of severe cardiac failure (one fatal) and one stroke; eight non‐thrombocytopenic patients (14%) experienced severe bleeding adverse events. Altogether, ibrutinib was permanently discontinued in 26/56 cases (46%). Data to guide optimal management are lacking and clinical practice guidelines are urgently needed.

Apigenin inhibits endothelial-cell proliferation in G2/M phase whereas it stimulates smooth-muscle cells by inhibiting P21 and P27 expression

Apigenin is a plant flavonoid that is thought to play a role in the prevention of carcinogenesis. However, its mechanism of action has not yet been elucidated. Because of the importance of angiogenesis in tumor growth, we investigated the effect of apigenin on endothelial and smooth‐muscle cells in an in vitro model. Apigenin markedly inhibited the proliferation, and, to a lesser degree, the migration of endothelial cells, and capillary formation in vitro, independently of its inhibition of hyaluronidase activity. In contrast, it strongly stimulated vascular smooth‐muscle‐cell proliferation. The molecular mechanisms of apigenin activity were analyzed in these 2 types of cells. Our results show that apigenin inhibits endothelial‐cell proliferation by blocking the cells in the G2/M phase as a result of the accumulation of the hyperphosphorylated form of the retinoblastoma protein. Apigenin stimulation of smooth‐muscle cells was attributed to the reduced expression of 2 cyclin‐dependent kinase inhibitors, p21 and p27, which negatively regulate the G1‐phase cyclin‐dependent kinase. Int. J. Cancer 85:691–696, 2000.

Expression level and differential JAK2-V617F–binding of the adaptor protein Lnk regulates JAK2-mediated signals in myeloproliferative neoplasms

Activating mutations in signaling molecules, such as JAK2-V617F, have been associated with myeloproliferative neoplasms (MPNs). Mice lacking the inhibitory adaptor protein Lnk display deregulation of thrombopoietin/thrombopoietin receptor signaling pathways and exhibit similar myeloproliferative characteristics to those found in MPN patients, suggesting a role for Lnk in the molecular pathogenesis of these diseases. Here, we showed that LNK levels are up-regulated and correlate with an increase in the JAK2-V617F mutant allele burden in MPN patients. Using megakaryocytic cells, we demonstrated that Lnk expression is regulated by the TPO-signaling pathway, thus indicating an important negative control loop in these cells. Analysis of platelets derived from MPN patients and megakaryocytic cell lines showed that Lnk can interact with JAK2-WT and V617F through its SH2 domain, but also through an unrevealed JAK2-binding site within its N-terminal region. In addition, the presence of the V617F mutation causes a tighter association with Lnk. Finally, we found that the expression level of the Lnk protein can modulate JAK2-V617F-dependent cell proliferation and that its different domains contribute to the inhibition of multilineage and megakaryocytic progenitor cell growth in vitro. Together, our results indicate that changes in Lnk expression and JAK2-V617F-binding regulate JAK2-mediated signals in MPNs.

A complementary role of multiparameter flow cytometry and high-throughput sequencing for minimal residual disease detection in chronic lymphocytic leukemia: an European Research Initiative on CLL study

In chronic lymphocytic leukemia (CLL) the level of minimal residual disease (MRD) after therapy is an independent predictor of outcome. Given the increasing number of new agents being explored for CLL therapy, using MRD as a surrogate could greatly reduce the time necessary to assess their efficacy. In this European Research Initiative on CLL (ERIC) project we have identified and validated a flow-cytometric approach to reliably quantitate CLL cells to the level of 0.0010% (10−5). The assay comprises a core panel of six markers (i.e. CD19, CD20, CD5, CD43, CD79b and CD81) with a component specification independent of instrument and reagents, which can be locally re-validated using normal peripheral blood. This method is directly comparable to previous ERIC-designed assays and also provides a backbone for investigation of new markers. A parallel analysis of high-throughput sequencing using the ClonoSEQ assay showed good concordance with flow cytometry results at the 0.010% (10−4) level, the MRD threshold defined in the 2008 International Workshop on CLL guidelines, but it also provides good linearity to a detection limit of 1 in a million (10−6). The combination of both technologies would permit a highly sensitive approach to MRD detection while providing a reproducible and broadly accessible method to quantify residual disease and optimize treatment in CLL.

Extended follow-up and impact of high-risk prognostic factors from the phase 3 RESONATE study in patients with previously treated CLL/SLL

In the phase 3 RESONATE study, ibrutinib demonstrated superior progression-free survival (PFS), overall survival (OS) and overall response rate (ORR) compared with ofatumumab in relapsed/refractory CLL patients with high-risk prognostic factors. We report updated results from RESONATE in these traditionally chemotherapy resistant high-risk genomic subgroups at a median follow-up of 19 months. Mutations were detected by Foundation One Heme Panel. Baseline mutations in the ibrutinib arm included TP53 (51%), SF3B1 (31%), NOTCH1 (28%), ATM (19%) and BIRC3 (14%). Median PFS was not reached, with 74% of patients randomized to ibrutinib alive and progression-free at 24 months. The improved efficacy of ibrutinib vs ofatumumab continues in all prognostic subgroups including del17p and del11q. No significant difference within the ibrutinib arm was observed for PFS across most genomic subtypes, although a subset carrying both TP53 mutation and del17p had reduced PFS compared with patients with neither abnormality. Reduced PFS or OS was not evident in patients with only del17p. PFS was significantly better for ibrutinib-treated patients in second-line vs later lines of therapy. The robust clinical activity of ibrutinib continues to show ongoing efficacy and acceptable safety consistent with prior reports, independent of various known high-risk mutations.

AMD3100 disrupts the cross-talk between chronic lymphocytic leukemia cells and a mesenchymal stromal or nurse-like cell-based microenvironment: pre-clinical evidence for its association with chronic lymphocytic leukemia treatments.

Background Interactions with the microenvironment, such as bone marrow mesenchymal stromal cells and nurse-like cells, protect chronic lymphocytic leukemia cells from spontaneous and drug-induced apoptosis. This protection is partially mediated by the chemokine SDF-1α (CXCL12) and its receptor CXCR4 (CD184) present on the chronic lymphocytic leukemia cell surface. Design and Methods Here, we investigated the ability of AMD3100, a CXCR4 antagonist, to sensitize chronic lymphocytic leukemia cells to chemotherapy in a chronic lymphocytic leukemia/mesenchymal stromal cell based or nurse-like cell based microenvironment co-culture model. Results AMD3100 decreased CXCR4 expression signal (n=15, P=0.0078) and inhibited actin polymerization/migration in response to SDF-1α (n=8, P<0.01) and pseudoemperipolesis (n=10, P=0.0010), suggesting that AMD3100 interferes with chronic lymphocytic leukemia cell trafficking. AMD3100 did not have a direct effect on apoptosis when chronic lymphocytic leukemia cells were cultured alone (n=10, P=0.8812). However, when they were cultured with SDF-1α, mesenchymal stromal cells or nurse-like cells (protecting them from apoptosis, P<0.001), chronic lymphocytic leukemia cell pre-treatment with AMD3100 significantly inhibited these protective effects (n=8, P<0.01) and decreased the expression of the anti-apoptotic proteins MCL-1 and FLIP. Furthermore, combining AMD3100 with various drugs (fludarabine, cladribine, valproïc acid, bortezomib, flavopiridol, methylprednisolone) in our mesenchymal stromal cell co-culture model enhanced drug-induced apoptosis (n=8, P<0.05) indicating that AMD3100 could mobilize chronic lymphocytic leukemia cells away from their protective microenvironment, making them more accessible to conventional therapies. Conclusions Taken together, these data demonstrate that interfering with the SDF-1α/CXCR4 axis by using AMD3100 inhibited chronic lymphocytic leukemia cell trafficking and microenvironment-mediated protective effects. Combining AMD3100 with other drugs may, therefore, represent a promising therapeutic approach to kill chronic lymphocytic leukemia cells.