Pharmacological profiles of acute myeloid leukemia treatments in patient samples by automated flow cytometry: a bridge to individualized medicine.

BACKGROUND: We have evaluated the ex vivo pharmacology of single drugs and drug combinations in malignant cells of bone marrow samples from 125 patients with acute myeloid leukemia using a novel automated flow cytometry-based platform (ExviTech). We have improved previous ex vivo drug testing with 4 innovations: identifying individual leukemic cells, using intact whole blood during the incubation, using an automated platform that escalates reliably data, and performing analyses pharmacodynamic population models. PATIENTS AND METHODS: Samples were sent from 24 hospitals to a central laboratory and incubated for 48 hours in whole blood, after which drug activity was measured in terms of depletion of leukemic cells. RESULTS: The sensitivity of single drugs is assessed for standard efficacy (EMAX) and potency (EC50) variables, ranked as percentiles within the population. The sensitivity of drug-combination treatments is assessed for the synergism achieved in each patient sample. We found a large variability among patient samples in the dose-response curves to a single drug or combination treatment. CONCLUSION: We hypothesize that the use of the individual patient ex vivo pharmacological profiles may help to guide a personalized treatment selection.

Drug cross-resistance and therapy-induced resistance in chronic lymphocytic leukaemia by an enhanced method of individualised tumour response testing.

Previous results with individualised tumour response testing (ITRT) in vitro in chronic lymphocytic leukaemia (CLL) have consistently shown good correlation with patient response and survival. We describe here an improved test and report its use with samples from the Leukaemia Research Fund CLL4 randomised clinical trial and previously treated patients. ITRT was performed by the tumour response to anti-neoplastic compounds (TRAC) assay, a modification of the differential staining cytotoxicity (DiSC) assay. Improvements included drying drugs into wells before assay and using the Octospot system to cytocentrifuge eight spots of cells onto one microscope slide. We successfully tested 765/782 (98%) cellular blood samples received within 48 h of phlebotomy. Cross-resistance (Pearson's r > 0.7) in untreated CLL was found between similar drugs. Mitoxantrone (r = 0.31), cyclophosphamide (r = 0.35) and pentostatin (r = 0.29) had low cross-resistance with fludarabine. Treatment resulted in increased resistance to chlorambucil, cyclophosphamide, doxorubicin, mitoxantrone, corticosteroids, cladribine and fludarabine (P < 0.01) but not to pentostatin. These results provide further rationale for standard drug combinations such as fludarabine-mitoxantrone and fludarabine-mitoxantrone-cyclophosphamide and suggest possible pentostatin salvage in fludarabine-resistant patients. ITRT results could assist both in determining the best treatment for individual patients and in the design and rationale of future clinical trials.

Silencing of APAF-1 in B-CLL results in poor prognosis in the case of concomitant p53 mutation.

Apoptosis protease-activating factor 1 (APAF-1), a transcriptional target of p53, is a cytosolic adaptor protein that links the mitochondrial apoptosis pathway to the caspase cascade. Here, we aimed to study the impact of APAF-1 expression levels on cell death induced by anticancer drugs or ionizing irradiation (IR) and disease prognosis in B-type chronic lymphocytic leukemia (B-CLL) patients. Samples from 138 patients with B-CLL were investigated for APAF-1 expression and p53 mutations. The results were related to survival data, in vitro cytotoxicity of various cytotoxic drugs and IR and clinico-pathological data. Variable APAF-1 expression was observed in all investigated B-CLL samples. Reduction in APAF-1 expression was observed at both mRNA and protein level indicating transcriptional silencing whereas mutation of p53 or the immunoglobulin heavy chain variable genes (IgH(V)) had no impact on APAF-1 expression. Surprisingly, APAF-1 loss did not result in resistance to cytotoxic therapies. Likewise, APAF-1 downregulation on its own showed no impact on disease prognosis. Nevertheless, a poor prognosis was observed in patients with loss of APAF-1 expression and additional p53 mutation. Thus, loss of APAF-1 may become relevant when additional core apoptosis signaling components are disrupted.

In B-CLL, the codon 72 polymorphic variants of p53 are not related to drug resistance and disease prognosis.

BACKGROUND: A common sequence polymorphism at codon 72 of the p53 gene encoding either arginine or proline was recently shown to be functionally relevant for apoptosis induction in vitro. In B-type chronic lymphocytic leukemia (B-CLL), p53 gene mutations occur in a subset of patients and are associated with impaired survival and drug resistance. Here, we address the functional relevance of the codon 72 single nucleotide (SNP) polymorphism for cell death sensitivity following exposure to clinically employed cytotoxic drugs and gamma-irradiation. METHODS: 138 B-CLL samples were analysed by SSCP-PCR and sequencing for single nucleotide polymorphism at codon 72 of the p53 gene. The in vitro cytotoxicity assay (DiSC-assay) was performed with 7 drugs (chlorambucil, mafosfamide, fludarabine phosphate, methylprednisolone, doxorubicin, vincristine) or gamma-irradiation. RESULTS: Of the 138 B-CLL samples, 9 samples were homozygous for proline (Pro/Pro), 78 samples homozygous for arginine (Arg/Arg), and 49 samples heterozygous (Arg/Pro). No differences were found for patient survival and cell death triggered by 7 cytotoxic drugs or gamma-irradiation. CONCLUSION: These data indicate that polymorphic variants of p53 codon 72 are not clinically relevant for apoptosis induction or patient survival in B-CLL.

Ex vivo therapeutic index by drug sensitivity assay using fresh human normal and tumor cells.

Toxicity is a major deterrent to achieving substantial improvements in cancer management, since most anticancer drugs inadequately distinguish normal and neoplastic tissues. Improving the differential between beneficial and toxic effects of therapy--therapeutic index--is a major clinical objective, but therapeutic index for cytotoxic drugs is narrow. Fresh tumor and normal cells from 59 patients with acute myeloid leukemia, non-Hodgkin's lymphoma, ovarian cancer and cancers of unknown origin were tested for ex vivo drug sensitivity using apoptosis by morphology assays. Drugs tested included carboplatin, doxorubicin, vincristine, cytarabine, fludarabine, mafosfamide and etoposide. Therapeutic index was derived from the ratio of normal and tumor cell LC90s. Individual patient therapeutic index varied markedly for different drugs and drug therapeutic index varied from patient to patient ranging from extremely unfavourable (<0.001) through excellent (>1000) reflecting patient heterogeneity. Therapeutic index for each drug was consistent with clinical expectations. Significantly, there was no relationship between normal and tumor cell LC90s. We conclude that further laboratory and clinical evaluation is required but the derived ex vivo therapeutic index could enhance choice of chemotherapy by reducing toxicity and/or improving efficacy.

High dose methylprednisolone can induce remissions in CLL patients with p53 abnormalities.

Abnormalities of the p53 gene are known to confer detrimental effects in chronic lymphocytic leukaemia (CLL) and are associated with short survival. We have used high dose methylprednisolone (HDMP) to treat 25 patients with advanced refractory CLL of whom 45% had p53 abnormalities shown by one or more methods: flow cytometry, fluorescent in situ hybridisation and direct DNA sequencing. Fifteen were resistant to fludarabine and 16 were non-responders to their most recent therapy. Methylprednisolone had a cytotoxic effect on lymphocytes from 95% of cases assessed by an ex vivo apoptotic drug sensitivity index (DSI). HDMP was given alone or in combination with other drugs: vincristine, CCNU, Ara-C, doxorubicin, mitoxantrone and chlorambucil, according to the results of DSI. Three patients were treated twice and each treatment was analysed separately. The overall response rate was 77% with a median duration of 12 months (range 7 -23+). Responders included 5/10 with abnormal p53, of which two achieved nodular PR. Patients with p53 abnormalities fared worse than those with normal p53. There were no differences in response according to whether HDMP was used alone or in combination. Nine of the 22 evaluable patients (3 NR and 6 PR) have died from progressive disease or transformation. Main toxicity was infection in 7/25 patients. Event free and overall survival were significantly better in responders vs non-responders ( P>0.0001 and P=0.04 respectively). Patients with a DSI of 100% to steroids had a better overall and event free survival, but this was not statistically significant. This study demonstrates that HDMP alone or in combination with other agents is a useful treatment strategy in refractory CLL including patients with p53 abnormalities.

Parameters affecting the ex vivo cytotoxic drug sensitivity of human hematopoietic cells.

The drug sensitivity of normal cells provides a baseline for determining the therapeutic index, and therefore the effectiveness, of cytotoxic drugs, yet little is known about the factors that affect normal cell chemosensitivity. Some parameters are known to have a profound effect on tumor cell sensitivity. The purpose of this study was to determine how cytotoxic drug sensitivity of hematopoietic cells isolated from cancer patients was affected by various parameters. These included previous chemotherapy (yes or no), sex, age, tumor type (leukemias or solid tumors), sample source (blood, bone marrow, serous effusions, or tumor biopsies) and predominant cell lineage (lymphoid, myeloid, macrophage, or mixed). Mononuclear cells isolated from blood, bone marrow, serous effusions, and tumor biopsies were incubated for four days with a median of 16 drugs. The differential staining cytotoxicity assay, an ex vivo apoptotic drug sensitivity test in which cell survival is determined morphologically, was used to assess normal hematopoietic and tumor cell response to cytotoxic drugs. One hundred forty-six specimens yielded hematopoietic cell chemosensitivity results with 3-36 drugs. Compared with tumor cells, there was far less interpatient variation in chemosensitivity of hematopoietic cells. Mean hematopoietic cell drug sensitivity showed little variation due to previous chemotherapy, sex, age, tumor type, and sample source or cell lineage. We therefore concluded that cytotoxic drug sensitivity of hematopoietic cells from a variety of sources could be used for assessment of therapeutic index. Drug therapeutic index results are a valuable tool in identifying novel cytotoxic agents and individually tailored chemotherapy regimens.