Michael Flasshove

Resection of residual disease in patients with metastatic gastrointestinal stromal tumors responding to treatment with imatinib

Gastrointestinal stromal tumors (GIST) are the most common mesenchymal tumors of the gastrointestinal tract. Long‐term survival of patients with metastatic disease has only been observed in patients with completely resected disease. Recently, the tyrosine kinase inhibitor imatinib has been found to yield responses in the majority of patients with metastatic GIST suggesting improved resectability in responding patients. Combined treatment approaches including resective surgery after imatinib treatment in patients with advanced metastatic disease have rarely been explored. We report a series of 90 patients with metastatic GIST in whom treatment with imatinib enabled 12 patients with mostly recurrent and extensive disease to be considered for resection of residual disease. In 11 of these patients, complete resection could be achieved. Viable tumor cells were found in all but one resected specimens suggesting that despite favorable radiological or clinical responses, imatinib is unlikely to induce pathological complete responses. Until more mature data from prospective trials are available, these data suggest that an early aggressive surgical approach should be considered for all patients with metastatic GIST. Further trials investigating a combined surgical and pre/postoperative treatment with imatinib in patients with advanced metastatic GIST are warranted.

Serum Free Light Chain Analysis and Urine Immunofixation Electrophoresis in Patients with Multiple Myeloma

Purpose: Retrospective studies have shown that immunoassays measuring free light chains (FLC) in serum are useful for diagnosis and monitoring of multiple myeloma. This study prospectively evaluates the use of FLC assays and, for the first time, investigates the relationship between serum FLC concentrations and the presence and detectability of Bence Jones (BJ) proteins in the urine. Patients and Methods: Three hundred seventy-eight paired samples of serum and urine were tested from 82 patients during the course of their disease. The sensitivities of serum FLC analysis and urine immunofixation electrophoresis (IFE) in detecting monoclonal FLC were compared. Serum FLC concentrations required for producing BJ proteins detected by IFE were determined. Results: Abnormal FLC were present in 54% of serum samples compared with 25% by urine tests. In abnormal serum samples for κ or λ, the sensitivity of IFE to detect the respective BJ proteins in urine were 51% and 35% and the median serum FLC concentrations required to produce detectable BJ proteins were 113 and 278 mg/L. Renal excretions of monoclonal FLC increased with serum concentrations, but excretions significantly decreased at high serum concentrations combined with renal dysfunction. Conclusion: Serum FLC assays are significantly more sensitive for detecting monoclonal FLC than urine IFE analysis. They also have the advantage of FLC quantification and are more reliable for monitoring disease course and response to treatment.

Hematoprotection and enrichment of transduced cells in vivo after gene transfer of MGMT(P140K) into hematopoietic stem cells.

The overexpression of mutant forms of O6-methylguanine-DNA-methyltransferase (MGMT), resistant to the MGMT inhibitor O6-benzylguanine (BG), protects hematopoietic cells from the toxicity of combined BG plus O6-alkylating agent chemotherapy. To evaluate the feasibility of this approach for clinically relevant O6-alkylating agents, combined therapy with BG and two chloroethylnitrosourea-type drugs, ACNU or BCNU, or the triazene derivative temozolomide (TMZ) was investigated in a murine bone marrow transplant model allowing transgenic expression of the highly BG-resistant MGMTP140K mutant. Whereas 20/20 control animals transplanted with nontransduced cells died of progressive myelosuppression during therapy, nearly all animals transplanted with MGMTP140K-transduced cells survived treatment with BG/ACNU (12/15), BG/TMZ (10/10), or BG/BCNU (5/5). In surviving animals, hematological parameters improved during chemotherapy and pretreatment levels were reestablished during or shortly after therapy. All animals showed enrichment of transgenic granulocytes (range: 15- to 101-fold) and lymphocytes (range: 16- to 55-fold) in peripheral blood, bone marrow, and spleen. No significant differences were observed between individual treatment groups. Serial transplants demonstrated protection in secondary recipients and confirmed the transduction of transplantable stem cells. Thus, these data demonstrate efficient protection from hematotoxicity and substantial enrichment of transgenic cells following MGMTP140K gene transfer and treatment with different O6-alkylating drugs.

Resistance to cytarabine and gemcitabine and in vitro selection of transduced cells after retroviral expression of cytidine deaminase in human hematopoietic progenitor cells

Overexpression of the detoxifying enzyme cytidine deaminase (CDD) renders normal and leukemic hematopoietic cells resistant to cytarabine (1-β-D-arabinofuranosylcytosine), and studies on murine cells have suggested transgenic CDD overexpression as a way to reduce the substantial myelotoxicity induced by the deoxycytidine analogs cytarabine and gemcitabine (2′,2′-difluorodeoxycytidine). We now have investigated CDD (over-)expression in the human hematopoietic system. Retroviral gene transfer significantly increased the resistance of CDD-transduced cord blood and peripheral blood-derived progenitor cells for doses ranging from 20–100 nM cytarabine and 8–10 nM gemcitabine. Protection was observed for progenitors of erythroid as well as myeloid differentiation, though the degree of protection varied for individual drugs. In addition, significant selection of CDD-transduced cells was obtained after a 4-day culture in 30–100 nM cytarabine. Thus, our data demonstrate that overexpression of CDD cDNA results in significant protection of human progenitors from cytarabine- as well as gemcitabine-induced toxicity, and allows in vitro selection of transduced cells. This strongly argues for a potential therapeutic role of CDD gene transfer in conjunction with dose-intensive cytarabine- or gemcitabine-containing chemotherapy regimen.

A Novel Nonobese Diabetic/Severe Combined Immunodeficient Xenograft Model for Chronic Lymphocytic Leukemia Reflects Important Clinical Characteristics of the Disease

We here describe a novel xenograft model of chronic lymphocytic leukemia (CLL) generated by infusion of human primary CLL cells into immunodeficient nonobese/severe combined immunodeficient (NOD/SCID) mice. Combined i.v. and i.p. injection of peripheral blood mononuclear cells (PBMC) from 39 patients with CLL resulted in highly reproducible splenic (37 of 39) and peritoneal (35 of 39) engraftment, which remained stable over a time span of 4 to 8 weeks. By comparison, recovery of leukemic cells from bone marrow (21 of 39) or peripheral blood (8 of 22) was substantially lower. The engraftment pattern of CLL PBMC 4 weeks posttransplant was correlated with clinical disease activity: infusion of PBMC from donors with Binet stage A, lymphocyte doubling time of >12 months, and normal lactate dehydrogenase (LDH) serum levels led to marked engraftment of T cells whereas comparably few tumor cells could be detected. In contrast, NOD/SCID mice receiving PBMC from donors with advanced stage Binet C, lymphocyte doubling time of <12 months, and elevated LDH serum levels exhibited predominant engraftment of tumor cells and comparably low numbers of T cells. These results suggest that this model reflects the heterogeneity and important clinical characteristics of the disease, and thus may serve as a tool for preclinical drug testing and investigation of the pathophysiology of CLL.

Thalidomide in combination with vincristine, epirubicin and dexamethasone (VED) for previously untreated patients with multiple myeloma

Abstract:  The present study aimed to evaluate the side‐effects and efficacy of thalidomide in combination with an anthracycline‐containing chemotherapy regimen in previously untreated myeloma patients. Thalidomide (400 mg/d) was combined with bolus injections of vincristine and epirubicin and oral dexamethasone (VED). Chemotherapy cycles were repeated every 3 wk until no further reduction in myeloma protein was observed, whereas the treatment with thalidomide was continued until disease progression. Thirty‐one patients were enrolled, 12 patients were exclusively treated with thalidomide in combination with VED and 19 patients additionally received high‐dose melphalan, for consolidation. Adverse events and response to therapy were assessed prior to treatment with high‐dose chemotherapy. Response to thalidomide combined with VED was complete remission in six patients (19%), partial remission in 19 patients (61%), stable disease in five patients (16%), and progressive disease in one patient (3.2%). Grade 3 and 4 adverse events consisted of leukocytopenia in 10 patients (32%), and thrombocytopenia and anemia in one patient each (3.2%). Neutropenic infections grade 3 and 4 occurred in seven (23%) and three patients (9.7%), respectively, including two patients (6.5%) who died from septic shock. Deep vein thrombosis occurred in eight patients (26%), constipation in 20 patients (65%), and polyneuropathy in 20 patients (65%). The probability of event‐free survival and overall survival in the whole group of patients at 36 months were 26 and 62%, respectively. In conclusion, the combination of thalidomide with VED appears to be highly effective in previously untreated patients with multiple myeloma, but it is associated with a high rate of thrombotic events, polyneuropathy, and neutropenic infections.

Type and position of promoter elements in retroviral vectors have substantial effects on the expression level of an enhanced green fluorescent protein reporter gene.

Purpose: Although gene transfer with retroviral vectors has already been applied to patients as part of clinical protocols, low expression of transgenes in target cells still remains a problem. Therefore, we compared various retroviral vectors using different promoters and backbones for expression of the enhanced green fluorescent protein (EGFP) reporter gene in fibroblasts and CD34+ cells. Methods: The N2A retroviral vector was used to test expression from the herpes simplex virus thymidine kinase promoter (vector N2A-TK-EGFP), a human phosphoglycerate kinase promoter (vector N2A-PGK-EGFP), and the SV40 promoter (vector N2A-SV-EGFP). Additional constructs used the spleen focus-forming virus (SFFV) long terminal repeat (LTR) as promoter and expressed EGFP alone (vector SFβ1-EGFP) or EGFP and a downstream (vector SFβ1-EGFP-IRES) or upstream (vector SFβ1-IRES-EGFP) internal ribosomal entry site. Results: For NIH 3T3 cells the fluorescence-activated cell sorting analysis revealed that the most active internal promoter was the SV40 promoter in the vector N2A-SV-EGFP (mean fluorescence intensity, MFI, 66.7 ± 0.4), followed by N2A-PGK-EGFP (26.3 ± 1.8 MFI), and N2A-TK-EGFP (4.8 ± 0.1 MFI). Expression from the SFβ1-EGFP vector (82.6 ± 6.7 MFI) and the SFβ1-EGFP-IRES vector (102.8 ± 6.2 MFI) was higher than from SFβ1-IRES-EGFP vector (15.5 ± 1.8 MFI). In human CD34-positive cells, the EGFP expression from all vectors was considerably lower than in fibroblasts with the SFβ1-EGFP vector still being four- to fivefold more active than the internal promoters tested. Conclusion: The SFFV LTR seems to allow a high expression of transgenes, as long as the transgene is not expressed downstream of an internal ribosomal entry site. Internal promoters may be useful for targeted gene expression in specific cell types, but the reduced level of expression from some internal promoters has to be taken into consideration.

Impact of the expression of P glycoprotein, the multidrug resistance-related protein, bcl-2, mutant p53, and heat shock protein 27 on response to induction therapy and long-term survival in patients with de novo acute myeloid leukemia

OBJECTIVE Resistance to chemotherapy-induced apoptosis and a multidrug-resistance phenotype is the major problem in the treatment of acute myeloid leukemia (AML). PATIENTS AND METHODS We recently demonstrated that the coexpression of at least two proteins, including P glycoprotein, multidrug resistance-related protein, bcl-2 (flow cytometry), p53 (luminometric immunoassay), and heat shock protein 27 (Western blotting), was predictive for response to induction therapy in de novo AML comparing leukemic blasts of 20 responders with 20 nonresponders. After long-term follow-up, we now present our evaluation on the prognostic significance of these proteins in leukemic blasts of 124 untreated AML patients with regard to the probability of remission (PoR) and overall survival (OS). RESULTS Analyzing leukemic blasts obtained from bone marrow samples, we found that no single protein significantly correlated with PoR or OS. In contrast, the coexpression of at least two of these proteins was predictive for reduced OS in univariate as well as multivariate analysis. Although we could not identify any particular protein combination predictive for reduced OS, those patients with no or only one protein expressed in their leukemic blasts had a survival probability of 48% in contrast to 24% in those patients with the coexpression of two or more proteins. Among the clinical markers, only response to chemotherapy had a significant effect on OS and age was of prognostic relevance for PoR. CONCLUSION We conclude that overexpression of only one protein possibly involved in resistance, is not sufficient to influence the prognosis for long-term survival in AML, whereas the expression of more than one protein is predictive for reduced OS. Protein combination seems to be individually different, and targeting only one protein in further clinical trials may not help to overcome multifactorial resistance.

Transfer of the cytidine deaminase cDNA into hematopoietic cells

In order to investigate whether transfer of the cytidine deaminase (CDD) cDNA would increase chemotherapy resistance to cytosine arabinoside (ara-C) we used a retroviral vector expressing both, neomycin phosphotransferase and the CDD cDNA, to transduce hematopoietic cells from cell lines and from murine bone marrow (BM). After coculture on producer clones with a viral titer of 1 x 10(5) CFU/ml and up to 3-fold increased CDD enzymatic activity, WEHI-3 cell line and primary hematopoietic cells were exposed to ara-C in clonogenic assays. A transduction efficiency of 34.8 +/- 6.2% could be determined for BM clonogenic progenitor cells by G418 resistance. We could observe significantly more colonies (77 +/- 3.1%) surviving from transduced primary BM cells than from mock cells (51.7 +/- 9.3%) at 10(-8) mol/l ara-C. At 10(-7) mol/l ara-C 8.7% of BM cells became absolutely resistant after retroviral transduction. Our data confirm that CDD represents another candidate gene for increasing resistance to cytotoxic drugs in hematopoietic cells.

Differential Expression of Drug Resistance-Related Genes between Sensitive and Resistant Blasts in Acute Myeloid Leukemia

Drug resistance constitutes a considerable problem in the therapy of acute myeloid leukemia (AML). In order to identify genes which might be related to drug resistance, we retrospectively studied gene expression patterns in blast populations of 14 patients with de novo AML, focusing on known or potential resistance mechanisms against cytosine arabinoside and anthracyclines. Following induction and postremission chemotherapy, 7 patients achieved a complete remission (CR) for more than 1 year, while 7 patients showed blast persistence (BP) after induction and salvage chemotherapy. Gene expression analysis was performed using RNA extracted from archived guanidine extracts and Affymetrix HGU133A gene chips. We utilized the Gene Ontology category Biological Process to select genes implicated in DNA metabolism, nucleoside and nucleotide metabolism and transport, reactive oxygen species metabolism, apoptosis and response to drugs and identified 32 differentially expressed genes. From this functional perspective, we found differences between the CR and BP groups with regard to nucleotide metabolism (PBEF1, G6PD; p = 0.048), apoptosis (TNFAIP3, TNFAIP8, MPO, BCL2A1, BAX, SON, BNIP3L; p = 0.039) and reactive oxygen species metabolism (SOD2, KIAA0179; p = 0.048). However, the attempt to construct a predictive model of chemoresistance failed. BP samples had a 2-fold higher expression of CD34 than CR samples. Thus, our findings are in line with reports describing differences in apoptosis resistance between CD34+ and CD34– blast populations. Taken together, our results suggest that drug resistance in AML is a heterogenous phenomenon that might be better defined by means of disturbed biological processes than by focusing on the alteration of the expression of distinct genes.