Jan Philippé

PHF6 mutations in T-cell acute lymphoblastic leukemia

Tumor suppressor genes on the X chromosome may skew the gender distribution of specific types of cancer. T-cell acute lymphoblastic leukemia (T-ALL) is an aggressive hematological malignancy with an increased incidence in males. In this study, we report the identification of inactivating mutations and deletions in the X-linked plant homeodomain finger 6 (PHF6) gene in 16% of pediatric and 38% of adult primary T-ALL samples. Notably, PHF6 mutations are almost exclusively found in T-ALL samples from male subjects. Mutational loss of PHF6 is importantly associated with leukemias driven by aberrant expression of the homeobox transcription factor oncogenes TLX1 and TLX3. Overall, these results identify PHF6 as a new X-linked tumor suppressor in T-ALL and point to a strong genetic interaction between PHF6 loss and aberrant expression of TLX transcription factors in the pathogenesis of this disease.

Small-molecule MDM2 antagonists as a new therapy concept for neuroblastoma.

Circumvention of the p53 tumor suppressor barrier in neuroblastoma is rarely caused by TP53 mutation but might arise from inappropriately increased activity of its principal negative regulator MDM2. We show here that targeted disruption of the p53-MDM2 interaction by the small-molecule MDM2 antagonist nutlin-3 stabilizes p53 and selectively activates the p53 pathway in neuroblastoma cells with wild-type p53, resulting in a pronounced antiproliferative and cytotoxic effect through induction of G(1) cell cycle arrest and apoptosis. A nutlin-3 response was observed regardless of MYCN amplification status. Remarkably, surviving SK-N-SH cells adopted a senescence-like phenotype, whereas CLB-GA and NGP cells underwent neuronal differentiation. p53 dependence of these alternative outcomes of nutlin-3 treatment was evidenced by abrogation of the effects when p53 was knocked down by lentiviral-mediated short hairpin RNA interference. The diversity of cellular responses reveals pleiotropic mechanisms of nutlins to disable neuroblastoma cells and exemplifies the feasibility of exploiting, by a single targeted intervention, the multiplicity of anticancer activities exerted by a key tumor suppressor as p53. The observed treatment effects without the need of imposing a genotoxic burden suggest that selective MDM2 antagonists might be beneficial for treatment of neuroblastoma patients with and without MYCN amplification.

P-glycoprotein is an independent prognostic factor predicting relapse in childhood acute lymphoblastic leukaemia : results of a 6-year prospective study

P‐glycoprotein (P‐gp), a cellular drug‐efflux pump, is thought to be one of the major causes of multidrug resistance (MDR) in malignancies. Since therapeutic strategies are being developed to circumvent drug resistance by inhibiting P‐gp function, large prospective studies evaluating the clinical relevance of P‐gp in childhood acute lymphoblastic leukaemia (ALL) are warranted. P‐gp expression was evaluated over a period of 6 years in 102 consecutive patients with de novo childhood ALL and in 35 children with relapse of ALL. Bone marrow and blood smears were studied immunocytochemically with two monoclonal antibodies at initial diagnosis and at relapse. P‐gp expression was found in 14 (14%) patients at initial diagnosis. After induction treatment, complete remission was achieved in 100/102 patients (98%), of whom 19 relapsed. Cumulative event‐free survival was significantly higher in the P‐gp‐negative group compared with the P‐gp‐positive population (Logrank P = 0.02). Multivariate analysis showed the results to be independent of age, WBC count and karyotype, and concomitantly underlined the importance of MDR1 phenotype detection in childhood ALL. P‐gp expression was more frequently found at relapse (34%) than at primary diagnosis (P = 0.01). In the relapsed patient group, P‐gp‐positive patients had a 2‐fold greater risk for adverse clinical outcome than the P‐gp‐negative relapsed patients. P‐gp expression was not induced by exposure to previous chemotherapy since the majority of P‐gp‐negative patients remained negative at relapse. P‐glycoprotein expression in newly diagnosed childhood ALL is an independent adverse prognostic parameter with a predictive value for relapse.

Quantification of apoptosis in lymphocyte subsets and effect of apoptosis on apparent expression of membrane antigens.

Annexin V binding to phosphatidylserine was evaluated by flow cytometry to examine apoptosis in different lymphocyte subsets of peripheral blood mononuclear cells after a 24 h in vitro culture period. We also applied a 2 Gy dose gamma-irradiation prior to incubation to evaluate the additional apoptogenic effect of radiation on the lymphocyte subsets. Overall, B lymphocytes showed the highest number of apoptotic cells, followed by T lymphocytes. Within the T lymphocytes, CD4-positive and CD45RA-negative cells were more prone to apoptosis than the CD8-positive and CD45RA-positive cells. Natural killer cells turned out to be most apoptosis-resistant. In the irradiated samples about twice as many apoptotic cells were found and the differences between lymphocyte subpopulations remained. Backgating of the annexin V-positive cells showed that these cells had a clearly decreased forward scatter signal. The antibody binding capacity (ABC) of lymphocyte membrane antigens was determined with CD3-fluorescein isothiocyanate (FITC), CD45RA-FITC, CD4-phycoerythrin (PE), CD8-PE, CD56-PE, and CD20-PE in viable and apoptotic cells. In the apoptotic cells a decrease of ABC was found for all antigens, except for CD20. There was no significant cell loss in the cultures. We conclude that the change in scatter and in ABC must be considered in immunophenotyping experiments on cells kept in culture for 24 h. If these changes are taken into account, percentages of subpopulations or the numbers of cells that stain positive for the studied markers do not significantly change.

Flow cytometry as a quantitative and sensitive method to evaluate low dose radiation induced apoptosis in vitro in human peripheral blood lymphocytes

Human peripheral blood lymphocytes, irradiated in vitro, die by an apoptotic process. The number of apoptotic cells after in vitro gamma-irradiation (0, 0.1, 0.2, 1, 2 and 5 Gy) was measured by flow cytometry using Annexin V and DiOC6 (a cationic dye) after 24 and 48 h incubation. The mean dose-response curves for apoptosis of six healthy volunteers obtained with both methods were steep below 1 Gy and flatter at higher doses. A slightly higher number of apoptotic cells was observed with DiOC6, compared to Annexin V. This can be assigned to a minor DiOC6-int/PI- population. Forty-eight hour cultures contained higher numbers of apoptotic cells compared with 24 h cultures. For both culture times, DiOC6 and Annexin V detected a statistically significant difference between a control sample and a 0.1 Gy irradiated one, illustrating the high sensitivity of the methods.

The αE-catenin gene ( CTNNA1 ) acts as an invasion-suppressor gene in human colon cancer cells

The acquisition of invasiveness is a crucial step in the malignant progression of cancer. In cancers of the colon and of other organs the E-cadherin/catenin complex, which is implicated in homotypic cell-cell adhesion as well as in signal transduction, serves as a powerful inhibitor of invasion. We show here that one allele of the αE-catenin (CTNNA1) gene is mutated in the human colon cancer cell family HCT-8, which is identical to HCT-15, DLD-1 and HRT-18. Genetic instability, due to mutations in the HMSH6 (also called GTBP) mismatch repair gene, results in the spontaneous occurrence of invasive variants, all carrying either a mutation or exon skipping in the second αE-catenin allele. The αE-catenin gene is therefore, an invasion-suppressor gene in accordance with the two-hit model of Knudsen for tumour-suppressor genes.

Array comparative genomic hybridization and flow cytometry analysis of spontaneous abortions and mors in utero samples

BackgroundIt is estimated that 10-15% of all clinically recognised pregnancies result in a spontaneous abortion or miscarriage. Previous studies have indicated that in up to 50% of first trimester miscarriages, chromosomal abnormalities can be identified. For several decades chromosome analysis has been the golden standard to detect these genomic imbalances. A major drawback of this method is the requirement of short term cultures of fetal cells. In this study we evaluated the combined use of array CGH and flow cytometry (FCM), for detection of chromosomal abnormalities, as an alternative for karyotyping.MethodsIn total 100 spontaneous abortions and mors in utero samples were investigated by karyotyping and array CGH in combination with FCM in order to compare the results for both methods.ResultsChromosome analysis revealed 17 abnormal karyotypes whereas array CGH in combination with FCM identified 26 aberrations due to the increased test success rate. Karyotyping was unsuccessful in 28% of cases as compared to only two out of hundred samples with inconclusive results for combined array CGH and FCM analysis.ConclusionThis study convincingly shows that array CGH analysis for detection of numerical and segmental imbalances in combination with flow cytometry for detection of ploidy status has a significant higher detection rate for chromosomal abnormalities as compared to karyotyping of miscarriages samples.

Changes in peripheral blood lymphocyte subsets in patients undergoing radiotherapy.

PURPOSE To investigate the changes in peripheral blood lymphocyte subpopulations in patients undergoing radiotherapy. MATERIALS AND METHODS In 8 patients undergoing external beam radiotherapy to the pelvis, the different lymphocyte subpopulations were followed during treatment. The lymphocyte populations were determined using two-colour flow cytometry. The study comprises the T-helper, T-suppressor/cytotoxic cells, the B-lymphocytes and natural killer (NK) cells. RESULTS The B-cells were characterized by a steep decrease at the beginning of the radiotherapy. They reached their lowest level at an equivalent total body dose of approximately 1.5 Gy and remained constant during the rest of the therapy (10% of the initial level). In T-cells (both T-helper and T-suppressor subsets) the steep decrease was less pronounced. T-lymphocytes reached a base level at 2.5 Gy equivalent total body dose (20% of the initial level). No significant differences between the T-helper and the T-suppressor/cytotoxic cells were observed. NK cells were characterized by a weak decline during the first weeks of therapy, being less pronounced than in the other populations. Near the end of therapy, the NK cells reached the level of the T-lymphocytes. CONCLUSION In vivo, NK cells were the most radioresistant and B-cells the most radiosensitive lymphocytes. No significant differences between T-helper and T-suppressor/cytotoxic cells were observed. These data are in agreement with the differences in apoptosis induction in peripheral blood lymphocyte subpopulations after in vitro gamma-irradiation of whole blood lymphocytes.

ABO-mismatch adult living donor liver transplantation using antigen-specific immunoadsorption and quadruple immunosuppression without splenectomy.

ABO‐incompatible (ABO‐I) liver transplantation is a controversial issue because of the generally less favorable outcome as compared to compatible transplants. Encouraging results have been shown by the introduction of new strategies to reduce posttransplant‐specific hemagglutinin (HA) titers with plasmapheresis, reinforced immunosuppression (IS), and the use of splenectomy. We describe a new protocol consisting of daclizumab (DAC) induction, mycophenolate mofetil (MMF)/tacrolimus (TAC)/steroids without splenectomy. Five recipients (mean age of 47 ± 14 yr) undergoing ABO‐I living donor liver transplantation (LDLT) were included in this protocol. Immunoadsorbent columns (Glycosorb ABO) were used for antigen‐specific immunoadsorption (ASI). The median follow‐up was 18.5 ± 10.5 months. ASI was very efficient in lowering HA titers (mean log2 immunoglobulin [Ig] M [IgM] and IgG values before and after ASI were 5.9 ± 2.8 and 1.2 ± 1.4 [P= 0.0038] and 6.5 ± 2.3 and 1.1± 1.9, respectively [P= 0.0001]). Persisting low HA titers were observed over time. No sepsis nor cytomegalovirus infection episodes were recorded. Acute cellular rejection (ACR) occurred in 1 recipient responding to steroid pulse therapy. Two grafts were lost in 2 patients due to technical failure during the first postoperative month. We conclude that ASI using Glycosorb ABO, quadruple immunosuppression including DAC and MMF provide high efficiency to lower HA titers over time, avoiding the need for splenectomy. ABO‐I LDLT can be performed with this adapted IS protocol. Liver Transpl 12:1412‐1417, 2006.

Anti-invasive activity of alkaloids and polyphenolics in vitro

Invasiveness, the ability of certain tumour cells to migrate beyond their natural tissue boundaries, often leads to metastasis, and usually determines the fatal outcome of cancer. The need for anti-invasive agents has led us to search for possibly active compounds among alkaloids and polyphenolics. One hundred compounds were screened in an assay based on the confrontation of invasive human MCF-7/6 mammary carcinoma cells with fragments of normal embryonic chick heart in vitro. Anti-invasive activity was frequently found among chalcones having a prenyl group. Six compounds were found to inhibit invasion when added to the culture medium at concentrations as low as 1 microM. For at least three of them the anti-invasive effect could be associated with a cytotoxic effect on the MCF-7/6 cells, but not on the heart tissue. This selective cytotoxicity was substantiated by different methods, such as histology and growth assays (volume measurements, cell counts, MTT and sulforhodamine B assays). The anti-invasive effects of the compounds could neither be ascribed to induction of apoptosis nor to the promotion of cell-cell adhesion. Our data indicate that among the alkaloids and polyphenolics a number of molecules can inhibit growth and invasion of human mammary cancer cells via selective cytotoxicity.