B-cell chronic lymphocytic leukemia (CLL) consists of 2 prognostic entities where cases with mutated immunoglobulin VH genes have better outcome than unmutated cases. VH-mutated CLLs display longer ...telomeres compared with unmutated cases and telomere length has been indicated to predict outcome, although the prognostic value of telomere length has not been fully established in CLL. We analyzed telomere length, VH gene mutation status, and clinical parameters in a large series of CLL. Telomere length was assessed by quantitative polymerase chain reaction (PCR), giving a very good correlation to telomere length estimated by Southern blotting (P < .001). The prognostic information given by mutation status (n = 282) and telomere length (n = 246) was significant (P < .001, respectively). Telomere length was a prognostic factor for stage A (P = .021) and stage B/C (P = .018) patients, whereas mutation status predicted outcome only in stage A patients (P < .001). Furthermore, mutated CLLs were subdivided by telomere length into 2 groups with different prognoses (P = .003), a subdivision not seen for unmutated cases (P = .232). Interestingly, the VH-mutated group with short telomeres had an overall survival close to that of the unmutated cases. Thus, by combining VH mutation status and telomere length, an improved subclassification of CLL was achieved identifying previously unrecognized patient groups with different outcomes. (Blood. 2005;105:4807-4812)
Purpose
The aim of this study was to evaluate a phenotypic cell panel with tumor cells from various patients and normal cells for preclinical profiles of antitumor efficacy and toxicity of anticancer ...drugs.
Methods
The antitumor activity of fourteen anticancer drugs was tested in over one hundred tumor samples from patients with solid or hematological malignancies. Drug activity against four normal cell types was used for the assessment of normal tissue toxicity. In vitro activity of the drugs was compared with indications approved by the Food and Drug Administration and established adverse event profiles.
Results
In general, in vitro drug activity in tumor cells from patients reflected known clinical activity of the drugs investigated. For example, the clinical activity of imatinib in chronic myeloid leukemia was clearly detected in the tumor panel. Further, and in accordance with clinical use, cisplatin and bortezomib showed high activity in ovarian cancer and myeloma samples, respectively. The normal cell models roughly reflected known clinical toxicity profiles and were able to detect differences in therapeutic index, e.g., between targeted drugs and classical cytotoxic agents. For example, the high tolerability of imatinib and the well-known renal toxicity of cisplatin were demonstrated.
Conclusions
In preclinical drug development, primary tumor cells from patients can be used for the prediction of cancer diagnosis–specific activity and may aid in the selection of diagnoses for clinical trials. By using tumor and toxicity panels together, information about therapeutic index may be derived, which may be useful when choosing among drug candidates with similar tumor effects.
Abstract 3457
Poster Board III-345
One of the hallmarks of the clinically heterogeneous disease chronic lymphocytic leukemia (CLL) is defective apoptosis, which is considered to contribute not only ...to cell accumulation but also to disease progression and resistance to therapy. In an effort to further investigate the ‘apoptotic profile' in CLL, we have utilized the multiplex ligation-dependent probe amplification (MLPA) method to analyze the expression profile of 35 apoptosis-related genes in 67 CLL cases including 49 peripheral blood, 9 lymph node, 7 bone marrow and 2 spleen samples. CLL cells derived from peripheral blood displayed a different overall expression profile when compared to peripheral blood mononuclear cells (PBMC) from five healthy controls, with several genes including the anti-apoptotic genes BCL2A1 and MCL1 and the pro-apoptotic genes BAX and BMF all being significantly differently expressed (>2 fold difference in median expression). Few differences were detected when comparing peripheral blood CLL samples (25/49 IGHV unmutated/IGHV3-21) to lymph node samples (6/9 unmutated/IGHV3-21) or to bone marrow samples (5/7 unmutated/IGHV3-21), with lower relative expression of BIK and BAX in peripheral blood samples. Furthermore, we correlated the expression profiles of all genes included in the assay with IGHV mutation status, IGHV3-21 gene usage and genomic aberrations, but overall few differences were evident between prognostically different subgroups. An exception to this was the BIK gene which showed significantly higher expression in unmutated/IGHV3-21 compared to mutated CLL. Additionally, MCL1 and BNIP3L displayed higher expression in 17p-deleted cases compared to 13q deletion/no aberration cases. High expression of HRK and BAX indicated a correlation with poor overall survival and shorter time to treatment. However, when performing real-time quantitative PCR on an extended patient cohort (n=112) only prediction of time to treatment could be confirmed for HRK and BAX (p=0.0035 and p=0.024, respectively). Finally, in order to validate our MLPA approach, we compared the gene expression levels of four genes in 19 CLL samples using both MLPA and real-time quantitative PCR, which revealed a significant correlation for all genes tested. In summary, MLPA is a quick and cost-effective method for pathway specific gene expression analysis and the present analysis of 67 CLL cases highlights the rather homogenous apoptosis gene expression profiles among different clinical subsets of the disease.
No relevant conflicts of interest to declare.
One of the most common dose limiting adverse effects in cancer treatment is myelotoxicity. The aim of this study was to develop an
in vitro method for measuring potential myelotoxic properties of a ...drug candidate in a high throughput setting. Human CD34
+ progenitor cells from umbilical cord blood were plated in 384-well microplates with drugs in liquid culture, supplemented with specific cytokines for the granulocytopoietic-macrophage lineage. After 7 or 14 days of proliferation and differentiation the cells were analyzed using the automated non-clonogenic fluorometric microculture cytotoxicity assay (FMCA). Two types of assays setups were evaluated, the FMCA-GM7 where cells were exposed to drugs directly after thawing and cytotoxicity measured on day 7 in contrast to the FMCA-GM14 where the cells were cultured 7 days prior to plating and drug exposure, with viability analysis on day 14 of differentiation. Drug sensitivity was similar in both assays and method validation was performed using 24 drugs with known myelotoxic profile (acyclovir, bortezomib, busulfan, carboplatin, chloramphenicol, chlorpromazine, cisplatin, cytarabine, clozapine, doxorubicin, erlotinib, etoposide, 5-fluorouracil, fludarabine, gefitinib, gemcitabine, hydroxyurea, imatinib, lomustine, melphalan, sorafenib, sunitinib, taxol and 6-thioguanine). The 50% inhibitory concentrations (IC
50) from the FMCA-GM7 and the FMCA-GM14 correlated highly (
r
=
0.83) and (
r
=
0.82), respectively, with IC
50 from the established clonogenic assay (CFU-GM), obtained from the literature. The current data suggests that the FMCA-GM could offer a simple and robust alternative to the CFU-GM assay in preclinical hematotoxicity studies.
Objectives: Gefitinib inhibits epidermal growth factor receptor (EGFR) signalling, but may also act by non‐EGFR dependent mechanisms. We have investigated the activity of gefitinib in haematological ...tumour cells, in particular acute myeloblastic leukaemia (AML).
Methods: Cytotoxic activity of gefitinib, alone or in combination with standard anti‐leukaemic drugs, was assessed by the short‐term fluorometric microculture cytotoxicity assay in tumour cells from 117 patients representing five haematological and five non‐haematological malignancies. In AML, the EGFR status was analysed by immunochemistry. Gefitinib‐induced apoptosis was investigated in a subset of AML samples, as well as in the leukaemia cell line MV‐4‐11, using a multiparametric high content screening assay. To confirm activation of caspase‐3 in cells treated with gefitinib, a blocking test was carried out in which MV4‐11 cells were pretreated with the specific caspase inhibitor DEVD‐FMK.
Results: Gefitinib showed highest cytotoxic activity in AML (n = 19) with many samples being sensitive at concentrations achievable in clinical practice (<10 μM), and no difference between previously untreated and relapsed patients. No correlation between the activity of gefitinib and standard antileukaemic drugs (cytarabine, doxorubicin, etoposide) was observed. Combining gefitinib with these drugs resulted in mainly additive or synergistic (etoposide) effects, with no evidence of sequence dependency. The AML cells did not express the EGFR. Gefitinib induced apoptosis, which was at least partly mediated by activation of the caspase‐3 pathway.
Conclusion: In vitro, gefitinib has significant cytotoxic activity in AML by inducing apoptosis through non‐EGFR dependent pathways.
For chronic lymphocytic leukemia (CLL) patients with poor-prognostic genomic aberrations the therapeutic options are limited. We used the Spectrum Collection library to identify compounds with ...anti-leukemia activity in high-risk CLL.
We identified substances with equal high cytotoxic activity in vitro in samples from poor-prognostic CLL (11q-/17p-, n=3) as compared to those from favourable-prognostic CLL (13q-, n=3). Cell survival was measured by fluorometric microculture cytotoxicity assay.
Out of 2,000 compounds, 65 had a similar effect in both prognostic groups. Fifteen compounds were selected for dose-response experiments in 16 additional CLL samples. Of these compounds, 12 continued to have similar cytotoxicity between prognostic subgroups. Additional experiments demonstrated that in CLL cells with 11q or 17p deletion, 5-azacytidine induced apoptosis in a dose-dependent manner and lipoprotein lipase expression was reduced following orlistat treatment.
Using primary cultures of cells from high-risk CLL patients for compound screening is a feasible approach and that 5-azacytidine and orlistat exemplify substances that exhibit cytotoxicity in poor-risk CLL.
Aberrant signal transduction by mutant or overexpressed protein kinases has emerged as a promising target for treatment of acute myeloid leukemia (AML). We here present a novel low molecular weight ...kinase inhibitor, AKN-032, targeting the FMS-like tyrosine kinase 3 (FLT3) and discovered in a new type of screening funnel combining the target therapy approach with sequential cellular screens. AKN-032 was identified among 150 selected hits from three different high throughput kinase screens. Further characterization showed inhibitory activity on FLT3 enzyme with an IC50 of 70nM. Western blot analysis revealed reduced autophosphorylation of the FLT3-receptor in AML cell line MV4-11 cells after exposure to AKN-032. Flow cytometry disclosed cytotoxic activity against MV4-11, but not against non-malignant 3T3-L1 fibroblast cells. Using a fluorometric microculture cytotoxicity assay, AKN-032 was tested against 15 cell lines and displayed a potent cytotoxic activity in AML cell lines MV4-11 (IC50=0.4μM) and Kasumi-1 (IC50=2.3μM). AKN-032 was also highly cytotoxic in tumor cells from AML patients in vitro. Furthermore, AKN-032 demonstrated significant antileukemic effect in a relatively resistant in vivo hollow fiber mouse model. No major toxicity was observed in the animals. In conclusion, AKN-032 is a promising new kinase inhibitor with significant in vivo and in vitro activity in AML. Results from the hollow fiber mouse assay suggest a favorable toxicity profile. Future studies will focus on pharmacokinetic properties, toxicity as well as further clarifying the mechanisms of action of AKN-032 in AML.
Bortezomib represents a new class of anti-cancer drugs, the proteasome inhibitors. We evaluated the in vitro activity of bortezomib with regard to tumour-type specificity and possible mechanisms of ...drug resistance in 115 samples of tumour cells from patients and in a cell-line panel, using the short-term fluorometric microculture cytotoxicity assay. Bortezomib generally showed dose–response curves with a steep slope. In patient cells, bortezomib was more active in haematological than in solid tumour samples. Myeloma and chronic myeloid leukaemia were the most sensitive tumour types although with great variability in drug response between the individual samples. Colorectal and kidney cancer samples were the least sensitive. In the cell-line panel, only small differences in response were seen between the different cell lines, and the proteasome inhibitors, lactacystin and MG 262, showed an activity pattern similar to that of bortezomib. The cell-line data suggest that resistance to bortezomib was not mediated by MRP-, PgP, GSH-; tubulin and topo II-associated MDR. Combination experiments indicated synergy between bortezomib and arsenic trioxide or irinotecan. The data support the current use of bortezomib but also points to its potential utility in other tumour types and in combination with cytotoxic drugs.
SDX-101 is the non-cyclooxygenase 2-inhibiting R-enantiomer of the non-steroid anti-inflammatory drug etodolac, and has anti-tumour activity in chronic lymphocytic leukaemia (CLL). SDX-308 and ...SDX-309 are more potent, structurally related indole-pyran analogues of SDX-101. The current study was performed to investigate and quantify the cytotoxic potentiating effects resulting from a combination of either SDX-101, SDX-308 or SDX-309 with standard cytotoxic agents used in the CLL treatment today.
The lymphoma cell line U937-gtb was used, together with primary tumour cells isolated from seven CLL patients. Combinations between chlorambucil and each one of the agents etodolac, SDX-101, SDX-308 and SDX-309 were studied. In addition, SDX-309 was combined with fludarabine, doxorubicin or vincristine. Both simultaneous and sequential exposures were explored using the median-effect method.
Most combinations were additive, which could be of clinical benefit since SDX-101 has been shown to be well tolerated. At the 70% effect level, synergy was observed between SDX-308 and chlorambucil in U937-gtb cells and in two-third of the CLL samples. Since chlorambucil is the most important drug in CLL therapy today and SDX-308 is presently targeted as the lead clinical candidate, this combination would be interesting for further studies. Vincristine and SDX-309 were synergistic in two-fourth of CLL samples.
To conclude, the non-COX-inhibiting etodolac-derivatives SDX-101, SDX-308 and SDX-309 are potential candidates for combination treatment of CLL. Especially, SDX-308 in combination with chlorambucil warrants further evaluation.
The mammalian target of rapamycin inhibitor rapamycin and its analogues show promising anticancer activity in various experimental tumor models and are presently evaluated in clinical trials. We, ...here, evaluated the in vitro activity of rapamycin with regard to tumor-type specificity and possible mechanisms of drug resistance in 97 tumor cell samples from patients and in a resistance-based cell line panel, using the fluorometric microculture cytotoxicity assay. Rapamycin was dose-dependently cytotoxic in patient tumor cells and in cell lines. In primary cells, rapamycin was more active in hematological than in solid tumor samples, with chronic lymphocytic leukemia (CLL) and acute lymphocytic leukemia being the most sensitive tumor types. Considerable inter-individual differences in sensitivity were apparent among CLL samples, but no difference was observed between IGHV mutated and unmutated CLL samples, whereas a tendency to lower rapamycin sensitivity was indicated for samples displaying poor-prognostic genomic markers. Combination experiments in CLL cells indicated that rapamycin acted synergistically with vincristine, cisplatin, chlorambucil and taxotere. These results and the clinically-experienced good tolerance to rapamycin analogues encourage clinical studies of rapamycin in CLL treatment as single agent but also in combination with, e.g., vincristine and chlorambucil.
Celotno besedilo
Dostopno za:
DOBA, IJS, IZUM, KILJ, NUK, PILJ, PNG, SAZU, UILJ, UKNU, UL, UM, UPUK
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