Axel Sauerbrey

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.

Expression of the BCRP gene (ABCG2/MXR/ABCP) in childhood acute lymphoblastic leukaemia

Summary.  The breast cancer resistance protein (BCRP), also known as mitoxantrone resistance protein (MXR) or placenta ABC protein (ABC‐P), is the second member of the ABCG subfamily of ABC transport proteins (gene symbol ABCG2). BCRP has been detected in acute myeloid leukaemia and in breast, colon and gastric cancer but there has been no reports regarding BCRP expression in acute lymphoblastic leukaemia (ALL). We report the first results of BCRP expression in childhood ALL. Sixty‐seven children (47 initial stage, 20 relapses) with ALL were analysed for BCRP gene expression by TaqMan real‐time polymerase chain reaction. The expression of BCRP in mononuclear cells obtained from the bone marrow (BM) and peripheral blood (PB) of healthy donors was also investigated. There was no relationship between BCRP expression and age, sex, initial blast cell count, prednisolone response or BM response on d 15 and 33. Patients with T‐lineage ALL showed a lower expression of BCRP (P = 0·044). Kaplan–Meier analysis of the relapse‐free interval showed no prognostic significance of BCRP expression when different levels of BCRP expression were used as cut‐off points. No significant difference in expression of BCRP mRNA was measured between initial‐stage and relapsed‐stage ALL or between normal MNC obtained from BM and ALL patients. The results indicate a low expression of BCRP in childhood ALL. Relationships between BCRP and clinical, molecular or in vivo resistance characteristics of the patients were not observed.

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.

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. [Mol Cancer Ther 2006;5(8):1986–94]

Therapy for non-Hodgkin lymphoma in children with primary immunodeficiency: analysis of 19 patients from the BFM trials.

BACKGROUND Non-Hodgkin lymphomas (NHL) represent an important complication of primary immunodeficiency (ID), posing new therapeutic challenges in this patient population. This study analyzes clinical data and therapy results of pediatric patients with primary ID and NHL in three consecutive NHL-BFM trials. PROCEDURE Retrospective analysis of children with primary ID and NHL, treated according to protocol NHL-BFM, was performed regarding clinical presentation, diagnostic features, therapy, and outcome. RESULTS From October, 1986, to April, 1997, 19 of 1,413 newly diagnosed patients with NHL were registered as suffering from primary ID. Age at diagnosis of NHL was lower in patients with ID. Six patients suffered from humoral ID, 13 patients from combined ID (ataxia teleangiectasia n = 3; Nijmegen breakage syndrome n = 4; PNP deficiency n = 1; IL2 receptor defect n = 1, other combined ID n = 4). Thirteen lymphomas were of B-cell and six of T-cell-lineage. Four of thirteen patients with combined ID were diagnosed with T-NHL, nine with B-NHL. Two of six patients with humoral ID presented with T-NHL and four with B-NHL. NHL entities differed significantly between ID and non-ID patients (P < or = 0.01): centroblastic and immunoblastic lymphomas (31.6% vs. 8.1%), anaplastic large cell lymphoma (26.3% vs. 10.7%), Burkitt lymphoma and B-ALL (21% vs. 47. 8%). Seventeen patients received polychemotherapy. Therapy-related toxicity was increased in ID- compared to non-ID-patients. Three patients died of sepsis; three died of tumor progression; one patient relapsed; one died of BMT-related toxicity; one died of second malignancy. Ten patients are in first continuous remission after a median follow-up of 4 years. CONCLUSIONS Curative treatment of NHL in the presence of primary ID is possible and should be attempted.

Prognostic implications of cyclins (D1, E, A), cyclin-dependent kinases (CDK2, CDK4) and tumor-suppressor genes (pRb, p16INK4A) in childhood acute lymphoblastic leukemia

Immunohistochemistry was used to analyze samples of 40 newly diagnosed childhood acute lymphoblastic leukemias (ALL) for their expression of cyclins (D1, E, A), cyclin‐dependent kinases (cdk2, cdk4) and tumor‐suppressor genes (pRb, p16INK4A), in order to discover whether or not the expression of these various proteins may be of prognostic relevance for the survival of children with ALL. Patients with ALL who were strongly positive for cyclin D1 had a lower probability of remaining in first continuous remission than ALL patients who were negative or weakly positive for this trait. There was also a significant correlation between expression of cyclin D1 and frequency of recurrence. For cyclin E and cyclin A, in contrast, there was no difference in the duration of relapse‐free‐intervals or the frequency of recurrence in patients. Children with cdk4‐positive ALL had a lower probability of remaining in first continuous remission than children with cdk4‐negative ALL. No prognostic relevance was found for cdk2. Patients with ALL who expressed pRb had a higher probability and patients who expressed p16 a lower probability of remaining in first continuous remission, but the results were not statistically significant. This investigation demonstrated that cyclin D1 and cdk4 were the most important prognostic factors for children with ALL, and that the combination of them showed the strongest prognostic relevance. Int. J. Cancer 74:508–512, 1997.

Messenger RNA Analysis of the Multidrug Resistance Related Protein (MRP1) and the Lung Resistance Protein (LRP) in de novo and Relapsed Childhood Acute Lymphoblastic Leukemia

In this study, 86 children (58 initial ALL and 28 children with relapsed disease) were investigated for lung resistance protein (LRP) and multidrug resistance related protein (MRP1)-mRNA expression by semiquantitative RT-PCR. The majority of investigated cases demonstrated variable LRP and MRP1 mRNA expression, when normalized for β -microglobulin expression. LRP and MRP1 mRNA expression may be coordinately regulated, as expression of both transcripts was found to be significantly correlated (p =0.0001). No differences of LRP and MRP expression were observed between initial and relapsed stage patients (LRP: p =0.89 and for MRP: p =0.09 ). The prognostic value of both resistance mechanisms was subjected to Kaplan-Meier analysis for event-free survival. For this analysis the patients were divided into groups with high or low LRP or MRP1 mRNA expression by utilizing the median value as the cut-off point. Overexpression of both resistance mechanisms had no prognostic significance in our retrospective study (log-rank test for LRP: p =0.12 and for MRP1: p =0.95 ), however, patients who showed high LRP expression exhibited a lower tendency of remaining in continuous first remission.

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 Kendalls 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.

US and MRI of gastrointestinal graft-versus-host disease

Abstract. Abdominal problems often complicate the clinical course after bone marrow transplantation. Graft-versus-host disease occurs as a complication of allogenic bone marrow transplantation. In this report, the findings of intestinal involvement are described and correlated with histopathological findings. Increased bowel-wall thickness and increased vascularity were shown by US. MRI demonstrated generalised increased bowel-wall thickness associated with bowel-wall enhancement after administration of IV gadolinium.

Expression of the Retinoblastoma Tumor Suppressor Gene (RB-1) in Acute Leukemia

In this report we review current studies concerning the RB-1 gene expression in acute leukemias. The RB-1 gene was analyzed in several studies by protein-, RNA and DNA-techniques in acute lymphoblastic leukemia (ALL) as well as in acute myelogenous leukemia (AML). The frequency of RB-1 inactivation in ALL-patients ranged between 30% and 64% in several studies. Structural abnormalities of the RB-1 gene were reported in 18% of ALL-patients and in 27% of Philadelphia chromosome-positive ALL, respectively. The proportion of AML-patients with absent RB-1 protein expression ranged between 19% and 55%. Structural RB-1-abnormalities in AML were predominantly reported in leukemias with monocytic differentiation. Furthermore, the prognostic value of an abnormal RB-1 gene expression was also estimated in some studies. In childhood ALL RB-1 inactivation was reported to have prognostic significance while in contrast, in another study on adults no prognostic value of RB-1 was found. In 4 out of 5 documented studies AML-patients with RB-1 inactivation generally had a poorer prognosis. In conclusion, RB-1 inactivation is frequently observed in acute leukemia. The prognostic value of low RB-1 expression is controversial but the majority of published studies found low RB-1 expression to be a negative prognostic predictor, in acute leukemia.