Prognostic and predictive role of gene mutations in chronic lymphocytic leukemia: results from the pivotal phase III study COMPLEMENT1.

Next generation sequencing studies in Chronic lymphocytic leukemia (CLL) have revealed novel genetic variants that have been associated with disease characteristics and outcome. The aim of this study was to evaluate the prognostic value of recurrent molecular abnormalities in patients with CLL. Therefore, we assessed their incidences and associations with other clinical and genetic markers in the prospective multicenter COMPLEMENT1 trial (treatment naive patients not eligible for intensive treatment randomized to chlorambucil (CHL) vs. ofatumumab-CHL (O-CHL)). Baseline samples were available from 383 patients (85.6%) representative of the total trial cohort. Mutations were analyzed by amplicon-based targeted next generation sequencing (tNGS). In 52.2% of patients we found at least one mutation and the incidence was highest in NOTCH1 (17.0%), followed by SF3B1 (14.1%), ATM (11.7%), TP53 (10.2%), POT1 (7.0%), RPS15 (4.4%), FBXW7 (3.4%), MYD88 (2.6%) and BIRC3 (2.3%). While most mutations lacked prognostic significance, TP53 (HR2.02,p<0.01), SF3B1 (HR1.66,p=0.01) and NOTCH1 (HR1.39,p=0.03) were associated with inferior PFS in univariate analysis. Multivariate analysis confirmed the independent prognostic role of TP53 for PFS (HR1.71,p=0.04) and OS (HR2.78,p=0.02) and of SF3B1 for PFS only (HR1.52,p=0.02). Notably, NOTCH1 mutation status separates patients with a strong and a weak benefit from ofatumumab addition to CHL (NOTCH1wt:HR0.50,p<0.01, NOTCH1mut:HR0.81,p=0.45). In summary, TP53 and SF3B1 were confirmed as independent prognostic and NOTCH1 as a predictive factor for reduced ofatumumab efficacy in a randomized chemo (immune)therapy CLL trial. These results validate NGS-based mutation analysis in a multicenter trial and provide a basis for expanding molecular testing in the prognostic workup of patients with CLL. ClinicalTrials.gov registration number: NCT00748189.

From Biology to Therapy: The CLL Success Story.

Chemoimmunotherapy has been the standard of care for patients with chronic lymphocytic leukemia (CLL) over the last decade. Advances in monoclonal antibody technology have resulted in the development of newer generations of anti-CD20 antibodies with improved therapeutic effectiveness. In parallel, our knowledge about the distinctive biological characteristics of CLL has progressively deepened and has revealed the importance of B-cell receptor (BCR) signaling and upregulated antiapoptotic proteins for survival and expansion of malignant cell clones. This knowledge provided the basis for development of novel targeted agents that revolutionized treatment of CLL. Ibrutinib and idelalisib inhibit the Bruton tyrosine kinase (BTK) and phosphoinositide 3-kinase (PI3K) delta, respectively, thus interfering with supportive signals coming from the microenvironment via the BCR. These drugs induce egress of CLL cells from secondary lymphoid organs and remarkably improve clinical outcomes, especially for patients with unmutated immunoglobulin heavy-chain genes or with p53 abnormalities that do not benefit from classical treatment schemes. Latest clinical trial results have established ibrutinib with or without anti-CD20 antibodies as the preferred first-line treatment for most CLL patients, which will reduce the use of chemoimmunotherapy in the imminent future. Further advances are achieved with venetoclax, a BH3-mimetic that specifically inhibits the antiapoptotic B-cell lymphoma 2 protein and thus causes rapid apoptosis of CLL cells, which translates into deep and prolonged clinical responses including high rates of minimal residual disease negativity. This review summarizes recent advances in the development of targeted CLL therapies, including new combination schemes, novel BTK and PI3K inhibitors, spleen tyrosine kinase inhibitors, immunomodulatory drugs, and cellular immunotherapy.

Alkylphosphocholines and curcumin induce programmed cell death in cutaneous T-cell lymphoma cell lines.

While most patients with early-stage cutaneous T-cell lymphomas (CTCL) have a very good prognosis, the survival of patients with extensive tumour stage and visceral involvement remains extremely poor and necessitates the development of more effective treatment modalities. In this study, we evaluated the in vitro effects of two alkylphosphocholines (APCs, miltefosine and erufosine) and the polyphenolic compound curcumin on 5 human CTCL cell lines (Hut-78, HH, MJ, My-La CD4+ and My-La CD8+). All tested drugs showed considerable cytotoxic activity, as determined by the MTT dye reduction assay. The IC50 values of both APCs ranged from the low micromolar level (Hut-78 cells) to 60-80µM (HH cells). The IC50 values of curcumin ranged from 12 to 24µM. All tested drugs induced apoptosis, as ascertained by morphological changes, DNA fragmentation and activation of caspase cascades. Miltefosine and erufosine induced dephosphorylation of Akt in My-La CD8+ cells and phosphorylation of JNK in Hut-78 and My-La CD8+ cells. APCs increased the level of the autophagic marker LC3B in Hut-78 and MJ cells. Results from co-treatment with autophagy modulators suggested that the cytotoxicity of APCs in CTCL cells is mediated, at least in part, by induction of autophagy.

Interleukin-6, osteopontin and Raf/MEK/ERK signaling modulate the sensitivity of human myeloma cells to alkylphosphocholines.

Alkylphosphocholines are highly active against multiple myeloma (MM) cells in vitro and are devoid of myelotoxicity. Little is known about the determinants of MM cell responsiveness or resistance to these drugs. In this study we investigated the effects of disease-relevant cytokines, such as interleukin-6 (IL-6) and osteopontin (OPN), on the in vitro antimyeloma activity of erufosine and perifosine. The role of the Raf/MEK/ERK pathway was also studied. Exogenous IL-6 reduced the cytotoxicity of erufosine against OPM-2 cells and, to a smaller extent, against U-266 cells. This was accompanied by inhibition of apoptosis in OPM-2 cells. The efficacy of perifosine was similarly affected, but to a greater extent. IL-6 slightly enhanced the sensitivity of RPMI-8226 cells to erufosine, thus emphasizing the heterogeneity of MM. Induced overexpression of OPN isoforms made OPM-2 cells less sensitive to erufosine. In all cases of IL-6- or OPN-induced resistance, the effective concentrations of erufosine were still within the clinically achievable range. Like other alkylphosphocholines, erufosine enhanced Raf/MEK/ERK signaling in MM cells but in some cases this contributed to cytotoxicity.

A particular expression pattern of CD13 epitope 7H5 in chronic lymphocytic leukaemia--a possible new therapeutic target.

A total of 50 patients with chronic lymphocytic leukaemia (CLL), as well as the B-cell leukaemia cell lines MEC-1, JVM-3, and BV-173 were studied in order to assess the incidence of CD13/aminopeptidase N (APN) immunolabelling with a monoclonal antibody 7H5 compared to LeuM7 and to CD13 mRNA levels, and to correlate these data with the cytotoxic and apoptosis-induction activity of the natural phenolic APN inhibitor curcumin. CD13/APN was detected in a significant proportion of B-CLL patients (42/50, 84%), immunolabelled by 7H5 (42/50) ± LeuM7 (10/50). Molecular analysis for CD13 transcripts confirmed these data, resulting in a specific RT-PCR product in CD13 positive cases. Curcumin showed concentration-dependent cytoreductive efficacy and apoptosis-induction activity in all tested cell lines and primary cultures from CLL mononuclear cells. There was a clear tendency for a better response in CD13 positive cases. The incidence of CD13/APN in CLL suggests that the inhibition of APN/CD13 by curcumin may be an effective new molecular target for a more efficient therapy for these patients and warrants further investigations.

Erucylphospho-N,N,N-trimethylpropylammonium (erufosine) is a potential antimyeloma drug devoid of myelotoxicity.

PURPOSE: Erufosine is an i.v. injectable alkylphosphocholine which is active against various haematological malignancies in vitro. In the present study, its effects on multiple myeloma (MM) cell lines and on murine and human hematopoietic progenitor cells (HPCs) were investigated. METHODS: The following MM cell lines were used: RPMI-8226, U-266 and OPM-2. The cytotoxicity of erufosine against these cell lines was determined by the MTT-dye reduction assay. Bcl-2, Bcl-X(L) and pAkt expression levels, activation of caspases, as well as cleavage of PARP, were studied by Western blotting. Migration was evaluated by a modified Boyden-chamber assay. The haematologic toxicity of erufosine was assessed using clonogenicity assays with normal HPCs of murine or human origin. RESULTS: Significant cytotoxic activity of erufosine against the MM cell lines was found. Comparison of the characteristics of erufosine-induced cell death in the three cell lines revealed a complex mode of action with apoptotic mechanisms prevailing in OPM-2 cells and non-apoptotic mechanisms prevailing in U-266 cells. The sensitivity of the MM cell lines to erufosine-induced apoptosis correlated inversely with the Bcl-X(L) expression level. Erufosine participated in synergistic interactions with various drugs. Furthermore, it showed potent migration-inhibiting activity in RPMI-8226 cells. Erufosine was not toxic to normal HPCs of murine or human origin and even stimulated progenitors from human umbilical cord blood to form granulocyte/macrophage colonies. Moreover, erufosine ameliorated the toxicity of bendamustine to murine HPCs. CONCLUSIONS: Overall, the data presented reveal that erufosine could have potential as an antimyeloma drug and deserves further development.

Erucylphospho-N,N,N-trimethylpropylammonium shows substantial cytotoxicity in multiple myeloma cells.

Multiple myeloma (MM) is a frequent hematological malignancy that is incurable despite recent developments, such as proteasome and angiogenic inhibitors. Erucylphospho-N,N,N-trimethylpropylammonium (erufosine) is an i.v. injectable alkylphosphocholine with antineoplastic activity based on an unusual mode of action and is currently undergoing clinical trials in leukemia patients. The aim of this investigation was to evaluate the efficacy of erufosine in MM cells and to study the modulation of cell-death pathways. The cytotoxicity of erufosine against three MM cell lines (RPMI-8226, U-266, and OPM-2) was determined by the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide-dye reduction assay. All MM cell lines responded to erufosine, RPMI-8226 cells being most and U-266 being least sensitive. The respective IC(50) values were 3.2 and 16.2 micromol/L. Various cell-death characteristics were studied in response to erufosine, such as morphological changes, oligonucleosomal DNA fragmentation, caspase activation, and poly (ADP)-ribose polymerase cleavage. Erufosine was found to cause cell shrinkage, chromatin condensation, and caspase-8 and -3 activation. Taken together, our data indicate that erufosine is a potential antimyeloma drug eliciting specific features of apoptotic cell death in vitro.

The expression level of the tumor suppressor retinoblastoma protein (Rb) influences the antileukemic efficacy of erucylphospho-N,N,N-trimethylpropylammonium (ErPC3).

The alkylphosphocholine erucylphospho-N,N,N-trimethylpropylammonium (ErPC3) is a promising new drug for treating various types of cancer. Its mechanism of action is not yet fully understood but is related to the Rb tumor suppressor protein. In the present study, we investigated the role of decreased Rb expression levels for the antileukemic efficacy of ErPC3 in BV-173 and K-562 CML-derived cell lines. We used antisense technique to knock down Rb levels in the two cell lines in addition to ErPC3 treatment. Cells with reduced Rb expression showed a diminished sensitivity to ErPC3 exposure, as determined by MTT (BV-173 and K-562) and clonogenicity assays (K-562 only), if concentrations below the IC50 were used. The feasibility of Rb knockdown varied between BV-173 and K-562 cells, with the former being distinctly more sensitive than the latter. We conclude that sufficient Rb levels are important for the cytotoxic and anticlonogenic effects of ErPC3 at levels below the IC(50), but that higher concentrations of ErPC3 are less dependent on Rb status.