Coil embolisation for massive haemoptysis in cystic fibrosis.

INTRODUCTION: Massive haemoptysis is a life-threatening event in advanced cystic fibrosis (CF) lung disease with bronchial artery embolisation (BAE) as standard of care treatment. The aim of our study was to scrutinise short-term and long-term outcomes of patients with CF and haemoptysis after BAE using coils. METHODS: We carried out a retrospective cohort study of 34 adult patients treated for massive haemoptysis with super selective bronchial artery coil embolisation (ssBACE) between January 2008 and February 2015. Embolisation protocol was restricted to the culprit vessel(s) and three lobes maximum. Demographic data, functional end-expiratory volume in 1 s in % predicted (FEV1% pred.) and body mass index before and after ssBACE, sputum colonisation, procedural data, time to transplant and time to death were documented. RESULTS: Patients treated with ssBACE showed significant improvement of FEV1% pred. after embolisation (p=0.004) with 72.8% alive 5 years post-ssBACE. Mean age of the patients was 29.9 years (±7.7). Mean FEV1% pred. was 45.7% (±20.1). Median survival to follow-up was 75 months (0-125). Severe complication rate was 0%, recanalisation rate 8.8% and 5-year-reintervention rate 58.8%. Chronic infection with Pseudomonas aeruginosa was found in 79.4%, Staphylococcus areus in 50% and Aspergillus fumigatus in 47.1%. DISCUSSION: ssBACE is a safe and effective treatment for massive haemoptysis in patients with CF with good results for controlling haemostasis and excellent short-term and long-term survival, especially in severely affected patients with FEV<40% pred. We think the data of our study support the use of coils and a protocol of careful and prudent embolisation.

Hepatic sinusoidal obstruction syndrome and short-term application of 6-thioguanine in pediatric acute lymphoblastic leukemia.

Long-term treatment with 6-thioguanine (6-TG) for pediatric acute lymphoblastic leukemia (ALL) is associated with high rates of hepatic sinusoidal obstruction syndrome (SOS). Nevertheless, current treatment continues to use short-term applications of 6-TG with only sparse information on toxicity. 6-TG is metabolized by thiopurine methyltransferase (TPMT) which underlies clinically relevant genetic polymorphism. We analyzed the association between hepatic SOS reported as a serious adverse event (SAE) and short-term 6-TG application in 3983 pediatric ALL patients treated on trial AIEOP-BFM ALL 2000 (derivation cohort) and defined the role of TPMT genotype in this relationship. We identified 17 patients (0.43%) with hepatic SOS, 13 of which with short-term exposure to 6-TG (P < 0.0001). Eight of the 13 patients were heterozygous for low-activity TPMT variants, resulting in a 22.4-fold (95% confidence interval 7.1-70.7; P ≤ 0.0001) increased risk of hepatic SOS for heterozygotes in comparison to TPMT wild-type patients. Results were supported by independent replication analysis. All patients with hepatic SOS after short-term 6-TG recovered and did not demonstrate residual symptoms. Thus, hepatic SOS is associated with short-term exposure to 6-TG during treatment of pediatric ALL and SOS risk is increased for patients with low-activity TPMT genotypes.

Social inequalities in the participation and activity of children and adolescents with leukemia, brain tumors, and sarcomas (SUPATEEN): a protocol for a multicenter longitudinal prospective observational study.

BACKGROUND: About 2000 children and adolescents under the age of 18 are diagnosed with cancer each year in Germany. Because of current medical treatment methods, a high survival rate can be reached for many types of the disease. Nevertheless, patients face a number of long-term effects related to the treatment. As a result, physical and psychological consequences have increasingly become the focus of research in recent years. Social dimensions of health have received little attention in health services research in oncology so far. Yet, there are no robust results that allow an estimation of whether and to what extent the disease and treatment impair the participation of children and adolescents and which factors mediate this effect. Social participation is of great importance especially because interactions with peers and experiences in different areas of life are essential for the development of children and adolescents. METHODS: Data are collected in a longitudinal, prospective, observational multicenter study. For this purpose, all patients and their parents who are being treated for cancer in one of the participating clinics throughout Germany will be interviewed within the first month after diagnosis (t1), after completion of intensive treatment (t2) and half a year after the end of intensive treatment (t3) using standardized questionnaires. Analysis will be done by descriptive and multivariate methods. DISCUSSION: The results can be used to identify children and adolescents in high-risk situations at an early stage in order to be able to initiate interventions tailored to the needs. Such tailored interventions will finally reduce the risk of impairments in the participation of children and adolescents and increase quality of life. TRIAL REGISTRATION: ClinicalTrials.gov: NCT04101123.

Genotyping circulating tumor DNA of pediatric Hodgkin lymphoma.

We used hybrid capture-targeted next-generation sequencing of circulating cell-free DNA (ccfDNA) of pediatric Hodgkin lymphoma (PHL) patients to determine pathogenic mechanisms and assess the clinical utility of this method. Hodgkin-Reed/Sternberg (HRS) cell-derived single nucleotide variants, insertions/deletions, translocations and VH-DH-JH rearrangements were detected in pretherapy ccfDNA of 72 of 96 patients. Number of variants per patient ranged from 1 to 21 with allele frequencies from 0.6 to 42%. Nine translocation breakpoints were detected. Genes involved in JAK/STAT, NFkB and PI3K signaling and antigen presentation were most frequently affected. SOCS1 variants, mainly deletions, were found in most circulating tumor (ct) DNAs, and seven of the nine translocation breakpoints involved SOCS1. Analysis of VH-DH-JH rearrangements revealed an origin of PHL HRS cells from partially selected germinal center B cells. Amounts of pretherapy ctDNA were correlated with metabolic tumor volumes. Furthermore, in all ccfDNA samples of 43 patients with early response assessment quantitative qPET < 3, indicative of a favorable clinical course, ctDNA was not detectable. In contrast, in five of six patients with qPET > 3, indicative of an unfavorable clinical course, ctDNA remained detectable. ccfDNA analysis of PHL is thus a suitable approach to determine pathogenic mechanisms and monitor therapy response.

Impact of rs12917 MGMT Polymorphism on [18F]FDG-PET Response in Pediatric Hodgkin Lymphoma (PHL).

PURPOSE: The enzyme O6-methylguanine-DNA methyltransferase (MGMT) is an important component of the DNA repair machinery. MGMT removes O6-methylguanine from the DNA by transferring the methyl group to a cysteine residue in its active site. Recently, we detected the single nucleotide polymorphism (SNP) rs12917 (C/T) in the MGMT sequence adjacent to the active site in Hodgkin lymphoma (HL) cell line KM-H2. We now investigated whether this SNP is also present in other HL cell lines and patient samples. Furthermore, we asked whether this SNP might have an impact on metabolic response in 2-deoxy-2-[18F]fluoro-D-glucose positron emission tomography ([18F]FDG-PET), and on overall treatment outcome based on follow-up intervals of at least 34 months. PROCEDURES: We determined the frequency of this MGMT polymorphism in 5 HL cell lines and in 29 pediatric HL (PHL) patients. The patient cohort included 17 female and 12 male patients aged between 4 and 18 years. After characterization of the sequence, we tested a possible association between rs12917 and age, gender, Ann Arbor stage, treatment group, metabolic response following two courses of OEPA (vincristine, etoposide, prednisone, and doxorubicin) chemotherapy, radiotherapy indication, and relapse status. RESULTS: We detected the minor T allele in four of five HL cell lines. 11/29 patients carried the minor T allele whereas 18/29 patients showed homozygosity for the major C allele. Interestingly, we observed significantly better metabolic response in PHL patients carrying the rs12917 C allele resulting in a lower frequency of radiotherapy indication. CONCLUSION: MGMT polymorphism rs12917 seems to affect chemotherapy response in PHL. The prognostic value of this polymorphism should be investigated in a larger patient cohort.

THROMBOTECT - a randomized study comparing low molecular weight heparin, antithrombin and unfractionated heparin for thromboprophylaxis during induction therapy of acute lymphoblastic leukemia in children and adolescents.

Thromboembolism is a serious complication of induction therapy for childhood acute lymphoblastic leukemia. We prospectively compared the efficacy and safety of antithrombotic interventions in the consecutive leukemia trials ALL-BFM 2000 and AIEOP-BFM ALL 2009. Patients with newly diagnosed acute lymphoblastic leukemia (n=949, age 1 to 18 years) were randomized to receive low-dose unfractionated heparin, prophylactic low molecular weight heparin (enoxaparin) or activity-adapted antithrombin throughout induction therapy. The primary objective of the study was to determine whether enoxaparin or antithrombin reduces the incidence of thromboembolism as compared to unfractionated heparin. The principal safety outcome was hemorrhage; leukemia outcome was a secondary endpoint. Thromboembolism occurred in 42 patients (4.4%). Patients assigned to unfractionated heparin had a higher risk of thromboembolism (8.0%) compared with those randomized to enoxaparin (3.5%; P=0.011) or antithrombin (1.9%; P<0.001). The proportion of patients who refused antithrombotic treatment as allocated was 3% in the unfractionated heparin or antithrombin arms, and 33% in the enoxaparin arm. Major hemorrhage occurred in eight patients (no differences between the groups). The 5-year event-free survival was 80.9±2.2% among patients assigned to antithrombin compared to 85.9±2.0% in the unfractionated heparin group (P=0.06), and 86.2±2.0% in the enoxaparin group (P=0.10). In conclusion, prophylactic use of antithrombin or enoxaparin significantly reduced thromboembolism. Despite the considerable number of patients rejecting the assigned treatment with subcutaneous injections, the result remains unambiguous. Thromboprophylaxis - for the present time primarily with enoxaparin - can be recommended for children and adolescents with acute lymphoblastic leukemia during induction therapy. Whether and how antithrombin may affect leukemia outcome remains to be determined.

Loss of interleukin-10 signaling and infantile inflammatory bowel disease: implications for diagnosis and therapy.

BACKGROUND & AIMS: Homozygous loss of function mutations in interleukin-10 (IL10) and interleukin-10 receptors (IL10R) cause severe infantile (very early onset) inflammatory bowel disease (IBD). Allogeneic hematopoietic stem cell transplantation (HSCT) was reported to induce sustained remission in 1 patient with IL-10R deficiency. We investigated heterogeneity among patients with very early onset IBD, its mechanisms, and the use of allogeneic HSCT to treat this disorder. METHODS: We analyzed 66 patients with early onset IBD (younger than 5 years of age) for mutations in the genes encoding IL-10, IL-10R1, and IL-10R2. IL-10R deficiency was confirmed by functional assays on patients' peripheral blood mononuclear cells (immunoblot and enzyme-linked immunosorbent assay analyses). We assessed the therapeutic effects of standardized allogeneic HSCT. RESULTS: Using a candidate gene sequencing approach, we identified 16 patients with IL-10 or IL-10R deficiency: 3 patients had mutations in IL-10, 5 had mutations in IL-10R1, and 8 had mutations in IL-10R2. Refractory colitis became manifest in all patients within the first 3 months of life and was associated with perianal disease (16 of 16 patients). Extraintestinal symptoms included folliculitis (11 of 16) and arthritis (4 of 16). Allogeneic HSCT was performed in 5 patients and induced sustained clinical remission with a median follow-up time of 2 years. In vitro experiments confirmed reconstitution of IL-10R-mediated signaling in all patients who received the transplant. CONCLUSIONS: We identified loss of function mutations in IL-10 and IL-10R in patients with very early onset IBD. These findings indicate that infantile IBD patients with perianal disease should be screened for IL-10 and IL-10R deficiency and that allogeneic HSCT can induce remission in those with IL-10R deficiency.

Nested stromal epithelial tumor of the liver--liver transplantation and follow-up.

BACKGROUND: The nested stromal epithelial tumor (NSET) of the liver is a rare tumor entity which is being reported in young girls. CASE REPORT: In our 16-year-old female patient, we have performed a liver transplantation (LTX) for a non-metastasizing non-resectable liver tumor. The patient was tumor free in the follow-up. At 28 months postoperatively, we detected lung metastases in the F18-FDG-PET/CT. The patient died 37 months after LTX from progressive pulmonary metastases. CONCLUSION: LTX should not have been generally recommended for NSET. Further statements about the value of LTX for NSET will be possible only after evaluation of the course of the disease in a larger number of transplanted patients.

Inflammatory bowel disease and mutations affecting the interleukin-10 receptor.

BACKGROUND: The molecular cause of inflammatory bowel disease is largely unknown. METHODS: We performed genetic-linkage analysis and candidate-gene sequencing on samples from two unrelated consanguineous families with children who were affected by early-onset inflammatory bowel disease. We screened six additional patients with early-onset colitis for mutations in two candidate genes and carried out functional assays in patients' peripheral-blood mononuclear cells. We performed an allogeneic hematopoietic stem-cell transplantation in one patient. RESULTS: In four of nine patients with early-onset colitis, we identified three distinct homozygous mutations in genes IL10RA and IL10RB, encoding the IL10R1 and IL10R2 proteins, respectively, which form a heterotetramer to make up the interleukin-10 receptor. The mutations abrogate interleukin-10-induced signaling, as shown by deficient STAT3 (signal transducer and activator of transcription 3) phosphorylation on stimulation with interleukin-10. Consistent with this observation was the increased secretion of tumor necrosis factor alpha and other proinflammatory cytokines from peripheral-blood mononuclear cells from patients who were deficient in IL10R subunit proteins, suggesting that interleukin-10-dependent "negative feedback" regulation is disrupted in these cells. The allogeneic stem-cell transplantation performed in one patient was successful. CONCLUSIONS: Mutations in genes encoding the IL10R subunit proteins were found in patients with early-onset enterocolitis, involving hyperinflammatory immune responses in the intestine. Allogeneic stem-cell transplantation resulted in disease remission in one patient.

Common acute lymphoblastic leukemia in a girl with genetically confirmed LEOPARD syndrome.

Germline mutations in PTPN11 gene cause Noonan syndrome and the clinically similar LEOPARD syndrome (LS). LS is a rare congenital developmental disorder characterized by multiple lentigines, cardiac abnormalities, facial dysmorphism, retardation of growth, and deafness. Mutations in exons 7 and 12 of the PTPN11 gene can be identified in nearly 90% of patients with LS. PTPN11 gene encodes for an ubiquitously expressed protein tyrosine phosphatase SHP-2 involved in a variety of intracellular signaling processes in development and hematopoiesis. Somatic PTPN11 mutations contribute to leukemogenesis in children with hematologic malignancies including juvenile myelomonocytic leukemia, acute lymphoblastic leukemia, acute myeloid leukemia, and myelodysplasia. Two cases of leukemia (acute myeloid leukemia) have been reported in children with LS. The authors describe for the first time a girl with genetically confirmed LEOPARD syndrome presenting with common acute lymphoblastic leukemia.

Prediction of broad spectrum resistance of tumors towards anticancer drugs.

PURPOSE: Drug resistance is a major obstacle in cancer chemotherapy. Although the statistical probability of therapeutic success is known for larger patient groups from clinical therapy trials, it is difficult to predict the individual response of tumors. The concept of individualized therapy aims to determine in vitro the drug response of tumors beforehand to choose effective treatment options for each individual patient. EXPERIMENTAL DESIGN: We analyzed the cross-resistance profiles of different tumor types (cancers of lung, breast, and colon, and leukemia) towards drugs from different classes (anthracyclines, antibiotics, Vinca alkaloids, epipodophyllotoxins, antimetabolites, and alkylating agents) by nucleotide incorporation and 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assays. Hierarchical cluster analysis and COMPARE analyses were applied. RESULTS: Tumors exert broad resistance profiles, e.g., tumors resistant to one drug tend to also be resistant to other drugs, whereas sensitive tumors reveal sensitivity towards many drugs. Interestingly, the broad spectrum resistance phenotype could reliably be predicted by doxorubicin alone. Expression of the ATP-binding cassette transporter P-glycoprotein (ABCB1, MDR1) and the proliferative activity of tumors were identified as underlying mechanisms of broad spectrum resistance. To find novel compounds with activity against drug-resistant tumors, a database with 2,420 natural products was screened for compounds acting independent of P-glycoprotein and the proliferative state of tumor cells. CONCLUSIONS: Tumors exert cross-resistance profiles much broader than the classical multidrug resistance phenotype. Broad spectrum resistance can be predicted by doxorubicin due to the multifactorial mode of action of this drug. Novel cytotoxic compounds from natural resources might be valuable tools for strategies to bypass broad spectrum resistance.

Pegylated-liposomal doxorubicin and oral topotecan in eight children with relapsed high-grade malignant brain tumors.

BACKGROUND: The combination of topoisomerase I and II chemotherapeutic agents has shown promising preclinical synergistic effects in the treatment of high-grade malignant brain tumors such as high-grade gliomas and choroid plexus carcinomas. To confirm the effectiveness of this treatment combination and determine its possible toxicity, we conducted a retrospective review of the charts of children who received the therapy. METHODS: Patients with relapsed malignant brain tumors who were given an individualized treatment of pegylated (PEG)-liposomal doxorubicin and topotecan were included in our study. PEG-liposomal doxorubicin was given intravenously at a dosage of 30-40 mg/m(2) over 4 h once every 4 weeks. Additionally, an intravenous formulation of topotecan was given orally twice daily and was increased on an individual basis from a starting dosage of 0.3 mg/m(2) per application to a total daily dosage of 0.6 mg/m(2). RESULTS: Eight patients were included. The main toxicity (NCI-CTC) after three cycles of the combination therapy was grade IV hematotoxicity (n = 3); grade III hematotoxicity (n = 2), grade III stomatitis (n = 1), grade III infection (n = 2), grade III diarrhea (n = 1); and grade II dermatitis (n = 1). In four patients, stable disease was achieved for 9, 23, more than 24, and more than 48 weeks, respectively. CONCLUSION: The schedule of PEG-liposomal doxorubicin with 30-40 mg/m(2) every 4 weeks in combination with oral topotecan resulted in tumor response, but the toxicity was high. An individualized increasing dose of PEG-liposomal doxorubicin 10-20 mg/m(2) every two weeks is now recommended.

Expression profiling of ATP-binding cassette transporters in childhood T-cell acute lymphoblastic leukemia.

A major issue in the treatment of T-cell acute lymphoblastic leukemia (T-ALL) is resistance to chemotherapeutic drugs. Multidrug resistance can be caused by ATP-binding cassette (ABC) transporters. The majority of these proteins have not yet been examined in T-ALL. Using a newly developed microarray for the simultaneous quantification of 38 ABC transporter genes, we observed a consistent overexpression of ABCA2/ABCA3 in clinical samples of ALL. Therefore, we analyzed the association of these two genes with drug resistance. Treatment of CCRF-CEM and Jurkat cells with methotrexate, vinblastine, or doxorubicin led to an induction of ABCA3 expression, whereas a significant increase of ABCA2 expression was only observed in Jurkat cells. To study the causal relationship of ABCA2/A3 overexpression with drug resistance, we applied RNA interference (RNAi) technology. RNAi specific for ABCA2 or ABCA3 led to a partial decrease of expression in these two ABC transporters. Upon cotreatment of RNAi for ABCA2 with methotrexate and vinblastine, a partial decrease of ABCA2 expression as well as a simultaneous increase of ABCA3 expression was observed. Vice versa, ABCA3 RNAi plus drugs decreased ABCA3 and increased ABCA2 expression. This indicates that down-regulation of one ABC transporter was compensated by the up-regulation of the other. Application of RNAi for both ABCA2 and ABCA3 resulted in a more efficient reduction of the expression of both transporters. As a consequence, a significant sensitization of cells to cytostatic drugs was achieved. In conclusion, ABCA2 and ABCA3 are expressed in many T-ALL and contribute to drug resistance.

ABCA3 as a possible cause of drug resistance in childhood acute myeloid leukemia.

BACKGROUND: A major issue in the treatment of acute myeloid leukemia (AML) is resistance to chemotherapeutic drugs. Multidrug resistance can be caused by ATP-binding cassette (ABC) transporters that function as drug efflux pumps. The majority of these proteins have not yet been examined in malignant diseases. EXPERIMENTAL DESIGN: A newly developed microarray for the simultaneous quantification of 38 ABC transporter genes and Taqman real-time PCR was used to analyze the expression of ABC transporters in pediatric AML and healthy bone marrow. Small interfering RNA was used to verify the role of ABCA3 in drug resistance. RESULTS: Using the microarray, we identified four new ABC transporters, which were overexpressed in many AML samples compared with healthy bone marrow: ABCA2, ABCA3, ABCB2, and ABCC10. The overexpression of these four genes was verified by real-time PCR in 42 samples from children with AML and 18 samples of healthy bone marrow. The median expression of ABCA3 was three times higher in 21 patients who had failed to achieve remission after the first course of chemotherapy than in a well-matched group of 21 patients who had achieved remission at this stage (P = 0.023). Incubation of cell lines with a number of different cytostatic drugs induced an up-regulation of ABCA3. Down-regulation of ABCA3 by small interfering RNA sensitized cells to doxorubicin. CONCLUSION: Our results show that ABCA2, ABCA3, ABCB2, and ABCC10 are overexpressed in childhood AML compared with healthy bone marrow. ABCA3 is the most likely transporter to cause drug resistance.

Are MTT assays the right tool to analyze drug resistance caused by ABC-transporters in patient samples?

Drug resistance can be caused by ATP-binding-cassette (ABC)-transporters which function as outward pumps for chemotherapeutic drugs. The aim of the present study was to analyze the association between eight ABC-transporters (BCRP, MDR1, SMRP, MRP1, MRP2, MRP3, MRP4, and MRP5) and in vitro drug resistance. Leukemic cells from 52 children with previously untreated acute leukemia (ALL: n=37; AML: n=15) were analysed. The expression of the ABC-transporters was measured by TaqMan real-time PCR. In vitro drug resistance to cytarabine, vincristine, tioguanine, daunorubicin, etoposide, dexamethasone, and prednisone was analysed with methyl-thiazol-tetrazolium (MTT) assays.MDR1 was weakly associated with resistance to vincristine (p<0.05) in AML samples. No other correlation between an ABC-transporter and a higher in vitro drug resistance was found. In vitro drug resistance was not associated with the simultaneous expression of a larger number of ABC-transporters.MTT assays are a widely used and validated method to analyse in vitro drug resistance but they may not be a useful tool to detect resistance which is caused by drug efflux in patient samples. If that is the case, MTT assays and the expression of ABC-transporters could provide complementary information on the drug resistance profile of patients with acute leukemia.

Complex chromosomal rearrangements in a secondary acute myeloblastic leukemia after chemotherapy in TRAPS.

We report on the cytogenetic findings from a patient with a de novo TNF-receptor-associated periodic syndrome (TRAPS), who showed first symptoms at the age of four months. Thus, he obtained a long-term therapy with cortisone, chlorambucile, methotrexate and cyclophosphamide. At the age of 14 he developed a secondary acute myeloblastic leukemia. Highly complex chromosomal rearrangements were detected after banding analysis. The exact definition of karyotype and the involved breakpoints could only be resolved after application of sophisticated multicolor-FISH techniques: 44,XY,-5,der(6)t(6;7)(6pter right curved arrow 6q12::7p22.2 right curved arrow 7pter or 7pter right curved arrow 7p22.2), dic(7;19)t(6;19;6;7;19;7;19)(19qter right curved arrow 19q12::7p13 right curved arrow 7p11.1::19q12 right curved arrow 19p12 or 19p12 right curved arrow 19q12::7p11.1 right curved arrow 7q21.3::6q12 right curved arrow 6q26::19p13.3 right curved arrow 19p12::6q26-6qter),dic(12;13)(13qter right curved arrow 13p11.2::12p13.1 right curved arrow 12qter),ace(12;13)(13pter right curved arrow 13p11.2::12p13.1 right curved arrow 12pter), -19. The simultaneous presence of two dicentric chromosomes has not been reported previously and is striking, as such chromosomes are suggested to be instable. However, such chromosomes are observed frequently after chemo- or radiotherapy and in secondary, i.e. therapy related AML (tAML). Thus, AML in this case may result from a long-term therapy of TRAPS with methotrexate, cyclophosphamide, chlorambucile and cortisone.

Identification of gene expression profiles predicting tumor cell response to L-alanosine.

The methylthioadenosine phosphorylase (MTAP) gene gained considerable interest as therapeutic target for tumors with the 9p21 deletion. This gene maps to 9p21 and loss of this chromosomal region in tumors offers an unique opportunity for chemoselective treatment, since MTAP is an important salvage enzyme for the formation of adenine that is needed for DNA synthesis. L-Alanosine, an antibiotic from Streptomyces alanosinicus, blocks the common de novo purine biosynthesis pathway and, thereby, inhibits tumor cells with MTAP deficiency. Normal cells escape the detrimental effects of L-alanosine due to their proficiency in the MTAP salvage pathway. The present analysis was undertaken to gain insights into the molecular architecture of tumor cells that determines the response to L-alanosine apart from the MTAP gene. Analysis of cell doubling times and IC(50) values for L-alanosine showed that slowly growing cell lines were more resistant to L-alanosine than rapidly growing ones. Mining the database of the National Cancer Institute (N.C.I.), for the mRNA expression of 9706 genes in 60 cell lines by means of Kendall's tau-test, false discovery rate calculation, and hierarchical cluster analysis pointed to 11 genes or expressed sequence tags whose mRNA expression correlated with the IC(50) values for L-alanosine. Furthermore, we tested L-alanosine for cross-resistance in multidrug-resistant cell lines which overexpress selectively either the P-glycoprotein/MDR1 (CEM/ADR5000), MRP1 (HL-60/AR), or BCRP (MDA-MB-231-BCRP) genes. None of the multidrug-resistant cell lines was cross-resistant to L-alanosine indicating that L-alanosine may be suitable to treat multidrug-resistant, refractory tumors in the clinic. Finally, the IC(50) values for L-alanosine of the 60 cell lines were correlated to the p53 mutational status and expression of p53 downstream genes. We found that p53 mutated cell lines were more resistant to L-alanosine than p53 wild type cell lines.

Analysis of single nucleotide polymorphism C3435T of the multidrug resistance gene MDR1 in acute lymphoblastic leukemia.

Multidrug resistance is an important mechanism responsible for refractoriness of leukemia and worse outcome of patients. Overexpression of the multidrug resistance gene, MDR1, is of prognostic relevance in acute myeloid leukemia, while its role in acute lymphoblastic leukemia (ALL) is still under debate. Single nucleotide polymorphisms (SNP) have been detected in the MDR1 gene. The C3435T polymorphism in this gene seems to have functional and clinical consequences. In the present investigation, we have analyzed the role of the C3435T SNP for drug resistance and prognosis of human ALL. The C3435T SNP was analyzed in 20 T-ALL cell lines and in blood samples from 53 ALL patients and 7 healthy donors. The cell line panel consisted of cell lines not prior exposed in vitro to cytostatic drugs as well as of drug-resistant lines which were selected in vitro by exposure to doxorubicin, vincristine, methotrexate, or hydroxyurea. We have developed a highly sensitive matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF-MS)-based genotyping approach to survey the C3435T SNP. Furthermore, mRNA expression was determined by real time reverse-transcribed polymerase chain reaction and doxorubicin sensitivity by a growth inhibition assay. Surprisingly, we did not find a significant correlation between C3435T homo- or heterozygote genotypes and MDR1 mRNA expression of cell lines or blood samples from patients and healthy donors. Furthermore, there was no relationship between the response of the cell lines to doxorubicin and the C3435T genotypes. Homo- or heterozygosity did not correlate to survival of patients in the Kaplan-Meier analysis. In conclusion, we do not have reason to assume that the C3435T SNP contributes to drug resistance of ALL and prognosis of ALL patients.

Molecular modes of action of artesunate in tumor cell lines.

A profound cytotoxic action of the antimalarial, artesunate (ART), was identified against 55 cancer cell lines of the U.S. National Cancer Institute (NCI). The 50% inhibition concentrations (IC50 values) for ART correlated significantly to the cell doubling times (P = 0.00132) and the portion of cells in the G0/G1 (P = 0.02244) or S cell cycle phases (P = 0.03567). We selected mRNA expression data of 465 genes obtained by microarray hybridization from the NCI data base. These genes belong to different biological categories (drug resistance genes, DNA damage response and repair genes, oncogenes and tumor suppressor genes, apoptosis-regulating genes, proliferation-associated genes, and cytokines and cytokine-associated genes). The constitutive expression of 54 of 465 (=12%) genes correlated significantly to the IC50 values for ART. Hierarchical cluster analysis of these 12 genes allowed the differentiation of clusters with ART-sensitive or ART-resistant cell lines (P = 0.00017). For exemplary validation, cell lines transduced with 3 of the 12 genes were used to prove a causative relationship. The cDNAs for a deletion-mutated epidermal growth factor receptor (EGFR) and for gamma-glutamylcysteine synthetase increased resistance to ART. The conditional expression of the CDC25A gene using a tetracycline repressor expression vector increased sensitivity toward ART. Multidrug-resistant cells differentially expressing the MDR1, MRP1, or BCRP genes were not cross-resistant to ART. ART acts via p53-dependent and- independent pathways in isogenic p53+/+ p21WAF1/CIP1+/+, p53-/- p21WAF1/CIP1+/+, and p53+/+ p21WAF1/CIP1-/- colon carcinoma cells.