Impact of sex on the efficacy and safety of panitumumab plus fluorouracil and folinic acid versus fluorouracil and folinic acid alone as maintenance therapy in RAS WT metastatic colorectal cancer (mCRC). Subgroup analysis of the PanaMa-study (AIO-KRK-0212).

BACKGROUND: Clinical trials in metastatic colorectal cancer (mCRC) are usually conducted irrespective of sex. Sex-associated differences relating to safety and efficacy in the treatment of mCRC, however, are gaining interest. METHODS: PanaMa investigated the efficacy of panitumumab (Pmab) plus fluorouracil and folinic acid (FU/FA) versus FU/FA alone after induction therapy with six cycles of FU/FA and oxaliplatin plus Pmab in patients with RAS wild-type mCRC. In this post hoc analysis, the study population was stratified for sex. Evaluated efficacy endpoints during maintenance treatment were progression-free survival (PFS, primary endpoint of the trial), overall survival (OS) and objective response rate during maintenance therapy. Safety endpoints were rates of any grade and grade 3/4 adverse events during maintenance therapy. RESULTS: In total, 165 male and 83 female patients were randomized and treated. Male and female patients showed numerically better objective response rates with Pmab, without reaching statistical significance. Male patients derived a significant benefit from the addition of Pmab to maintenance treatment with regard to PFS [hazard ratio (HR) 0.63; 95% confidence interval (CI) 0.45-0.88; P = 0.006] that was not observed in female patients (HR 0.85; 95% CI 0.53-1.35; P = 0.491). The better PFS for male patients treated with Pmab did not translate into improved OS (HR 0.85; 95% CI 0.55-1.30; P = 0.452). Female patients showed numerically improved OS when treated with Pmab. There was no difference in the total of grade ≥3 adverse events during maintenance regarding sex (P = 0.791). Female patients, however, had a higher rate of any grade nausea, diarrhea and stomatitis. CONCLUSIONS: In the PanaMa trial, the addition of Pmab to maintenance treatment of RAS wild-type mCRC with FU/FA improved the outcome in terms of the primary endpoint (PFS) particularly in male patients. Female patients did not show the same benefit while experiencing higher rates of adverse events. Our results support the development of sex-specific protocols.

Study protocol of the FIRE-8 (AIO-KRK/YMO-0519) trial: a prospective, randomized, open-label, multicenter phase II trial investigating the efficacy of trifluridine/tipiracil plus panitumumab versus trifluridine/tipiracil plus bevacizumab as first-line treatment in patients with metastatic colorectal cancer.

BACKGROUND: Initial systemic therapy for patients with metastatic colorectal cancer (mCRC) is usually based on two- or three-drug chemotherapy regimens with fluoropyrimidine (5-fluorouracil (5-FU) or capecitabine), oxaliplatin and/or irinotecan, combined with either anti-VEGF (bevacizumab) or, for RAS wild-type (WT) tumors, anti-EGFR antibodies (panitumumab or cetuximab). Recommendations for patients who are not eligible for intensive combination therapies are limited and include fluoropyrimidine plus bevacizumab or single agent anti-EGFR antibody treatment. The use of a monochemotherapy concept of trifluridine/ tipiracil in combination with monoclonal antibodies is not approved for first-line therapy, yet. Results from the phase II TASCO trial evaluating trifluridine/ tipiracil plus bevacicumab in first-line treatment of mCRC patients and from the phase I/II APOLLON trial investigating trifluridine/ tipiracil plus panitumumab in pre-treated mCRC patients suggest favourable activity and tolerability of these new therapeutic approaches. METHODS: FIRE-8 ( NCT05007132 ) is a prospective, randomized, open-label, multicenter phase II study which aims to evaluate the efficacy of first-line treatment with trifluridine/tipiracil (35 mg/m2 body surface area (BSA), orally twice daily on days 1-5 and 8-12, q28 days) plus either the anti-EGFR antibody panitumumab (6 mg/kg body weight, intravenously on day 1 and 15, q28 days) [arm A] or (as control arm) the anti-VEGF antibody bevacizumab (5 mg/kg body weight, intravenously on day 1 and 15, q28 days) [arm B] in RAS WT mCRC patients. The primary objective is to demonstrate an improved objective response rate (ORR) according to RECIST 1.1 from 30% (control arm) to 55% with panitumumab. With a power of 80% and a two-sided significance level of 0.05, 138 evaluable patients are needed. Given an estimated drop-out rate of 10%, 153 patients will be enrolled. DISCUSSION: To the best of our knowledge, this is the first phase II trial to evaluate the efficacy of trifluridine/tipiracil plus panitumumab in first-line treatment of RAS WT mCRC patients. The administration of anti-EGFR antibodies rather than anti-VEGF antibodies in combination with trifluridine/tipiracil may result in an increased initial efficacy. TRIAL REGISTRATION: EU Clinical Trials Register (EudraCT) 2019-004223-20 . Registered October 22, 2019, ClinicalTrials.gov NCT05007132 . Registered on August 12, 2021.

F2A sequence linking MGMT(P140K) and MDR1 in a bicistronic lentiviral vector enables efficient chemoprotection of haematopoietic stem cells.

Chemoprotection of haematopoietic stem cells (HSCs) by gene therapeutic transfer of drug-resistance genes represents the encouraging approach to prevent myelosuppression, which is one of the most severe side effects in tumor therapy. Thus, we cloned and evaluated six different bicistronic lentiviral SIN vectors encoding two transgenes, MGMT(P140K) (an O(6)-benzylguanine-resistant mutant of methylguanine-DNA methyltransferase) and MDR1 (multidrug resistance 1), using various linker sequences (IRESEMCV, IRESFMDV and 2A-element of FMDV (F2A)). Expression of both transgenes in HL-60 and in K562 cells was assayed by quantitative real-time PCR. Combination therapy with ACNU plus paclitaxel in HL-60 cells and with carmustin (BCNU) plus doxorubicin in K562 cells resulted in the most significant survival advantage of cells transduced with the lentiviral vector HR'SIN-MGMT(P140K)-F2A-MDR1 compared with untransduced cells. In human HSCs, overexpression of both transgenes by this vector also caused significantly increased survival and enrichment of transduced cells after treatment with BCNU plus doxorubicin or temozolomide plus paclitaxel. In summary, we could show significant chemoprotection by overexpression of MDR1 and MGMT(P140K) with a lentiviral vector using the F2A linker element in two different haematopoietic cell lines and in human primary HSCs with various combination regimens. Consequently, we are convinced that these in vitro investigations will help to improve combination chemotherapy regimens by reducing myelotoxic side effects and increasing the therapeutic efficiency.

Importance of blood graft characteristics in auto-SCT: implications for optimizing mobilization regimens.

Although blood stem cells have been widely used to support high-dose therapy in the autologous setting, limited data are available on the effects of graft characteristics in patient outcomes other than haematopoietic engraftment. Retrospective studies suggest that patients who mobilize more CD34(+) cells have better outcomes than do patients who mobilize less well. Furthermore, immunological reconstitution may be important in terms of post-transplant outcome and is apparently affected by graft composition. There is accumulating evidence that the mobilization regimen used may be an important determinant of graft content. Plerixafor has been recently introduced combined with G-CSF in patients who mobilize poorly. In addition to enhancing mobilization of CD34(+) cells, there are indications that plerixafor may also affect other graft components. A combination of chemotherapy plus G-CSF with plerixafor has been shown to be very effective in stem-cell mobilization, but more data are needed in regard to other graft characteristics in this setting. Prospective studies are needed to evaluate whether higher CD34(+) doses or other modifications to graft composition translate into better long-term outcomes in the autologous setting. These studies are not only important in regard to defining the optimal stem-cell graft in the autologous setting, but also in identifying the optimal mobilization regimen.

Analysis of self-inactivating lentiviral vector integration sites and flanking gene expression in human peripheral blood progenitor cells after alkylator chemotherapy.

Abstract Hematotoxicity is a major and frequently dose-limiting side effect of chemotherapy. Retroviral methylguanine-DNA-methyltransferase (MGMT; EC 2.1.1.63) gene transfer to primitive hematopoietic progenitor cells (CD34(+) cells) might allow the application of high-dose alkylator chemotherapy with almost mild to absent myelosuppression. Because gammaretroviral vector integration was found in association with malignant or increased proliferation, novel lentiviral vectors with self-inactivating (SIN) capacity might display a safer option for future gene transfer studies. We assessed the influence of chemoselection on integration patterns in 1,3-bis(2-chloroethyl)-1-nitrosourea (BCNU)-treated and untreated human CD34(+) cells transduced with an SIN lentiviral vector carrying the MGMT(P140K) transgene, using ligation-mediated PCR (LM-PCR) and next-generation sequencing. In addition, for the first time, the local influence of the lentiviral provirus on the expression of hit and flanking genes in human CD34(+) cells was analyzed at a clonal level. For each colony, the integration site was detected (LM-PCR) and analyzed (QuickMap), and the expression of hit and flanking genes was measured (quantitative RT-PCR). Analyses of both treated and untreated CD34(+) cells revealed preferential integration into genes. Integration patterns in BCNU-treated cells showed mild, but not significant, differences compared with those found in untreated CD34(+) cells. Most importantly, when analyzing the local influence of the provirus, we saw no significant deregulation of the integration-flanking genes. These findings demonstrate that SIN vector-mediated gene transfer might display a feasible and possibly safe option for MGMT(P140K)-mediated chemoprotection of CD34(+) cells.

Chemoprotection of human hematopoietic stem cells by simultaneous lentiviral overexpression of multidrug resistance 1 and O(6)-methylguanine-DNA methyltransferase(P140K).

Myelotoxicity is a dose-limiting effect of many chemotherapeutic regimens. Thus, there is great interest in protecting human hematopoietic stem cells by the transfer of drug resistance genes. The main focus of this study was the simultaneous overexpression of multidrug resistance 1 (MDR1) and the O(6)-benzylguanine (O(6)-BG)-resistant mutant MGMT(P140K) (O(6)-methylguanine-DNA methyltransferase) with a bicistronic lentiviral vector (HR'SIN-MDR1-IRES-MGMT(P140K)), with regard to the capability to convey chemoprotection in the leukemia cell line, HL60, and human hematopoietic stem cells (CD34(+)). Combination therapy with O(6)-BG/1-(2-chloroethyl)-3-(4-amino-2-methylpyrimidine-5-yl)methyl-1-nitrosourea) (ACNU) plus paclitaxel showed a significant survival advantage of HL60 cells transduced with this combination vector. In CD34(+) cells, monotherapy with O(6)-BG/temozolomide (TMZ) resulted in an increased percentage of MGMT-positive cells (vs untreated cells) after transduction with HR'SIN-MDR1-IRES-MGMT(P140K) (28.3%). For combination therapy with O(6)-BG/temozolomide plus paclitaxel the increase was higher with the combination vector (52.8%) than with a vector expressing MGMT(P140K) solely (29.1%). With regard to MDR1-positive cells the protective effect of the combination vector (88.5%) was comparable to the single vector HR'SIN-MDR1 (90.0%) for monotherapy with paclitaxel and superior for combination therapy with O(6)-BG/temozolomide plus paclitaxel (84.6 vs 69.7%). In conclusion, the combination vector presents simultaneous protective effects of two drug-resistance genes, offering an opportunity to increase the cancer therapeutic index.

Mobilization of peripheral blood stem cells for autologous transplant in non-Hodgkin's lymphoma and multiple myeloma patients by plerixafor and G-CSF and detection of tumor cell mobilization by PCR in multiple myeloma patients.

This report describes the first investigational use of plerixafor in Europe and the determination of tumor cell mobilization by polymerase chain-reaction after plerixafor treatment in a subset of patients with multiple myeloma (MM). Thirty-five patients (31 MM and 4 NHL) received granulocyte colony-stimulating factor (G-CSF) (10 microg/kg) each morning for 4 days. Starting the evening of Day 4, patients recieved plerixafor 0.24 mg/kg. Apheresis was initiated 10-11 h later, in the morning of Day 5. This regimen of G-CSF treatment each morning before apheresis and plerixafor treatment in the evening was repeated for up to 5 consecutive days. Mobilization with plerixafor and G-CSF resulted in a median 2.6-fold increase in peripheral blood (PB) CD34+ cell count compared with before plerixafor treatment. All patients collected > or =2 x 10(6) CD34+ cells/kg and 32 of 35 patients collected > or =5 x 10(6) CD34+ cells/kg. After plerixafor treatment, 3 of 7 patients had a small increase and 4 of 7 patients had a small decrease in PB tumor cells. No G-CSF was given post transplant. The median number of days to polymorphonuclear leukocyte and platelet engraftment was 14.0 and 11.0, respectively. There were no reports of graft failure. Plerixafor was generally well tolerated. Mobilization of PB CD34+ cells was consistent with previous clinical trials. The addition of plerixafor did not significantly increase the relative number of PB MM tumor cells.

QuickMap: a public tool for large-scale gene therapy vector insertion site mapping and analysis.

Several events of insertional mutagenesis in pre-clinical and clinical gene therapy studies have created intense interest in assessing the genomic insertion profiles of gene therapy vectors. For the construction of such profiles, vector-flanking sequences detected by inverse PCR, linear amplification-mediated-PCR or ligation-mediated-PCR need to be mapped to the host cell's genome and compared to a reference set. Although remarkable progress has been achieved in mapping gene therapy vector insertion sites, public reference sets are lacking, as are the possibilities to quickly detect non-random patterns in experimental data. We developed a tool termed QuickMap, which uniformly maps and analyzes human and murine vector-flanking sequences within seconds (available at www.gtsg.org). Besides information about hits in chromosomes and fragile sites, QuickMap automatically determines insertion frequencies in +/- 250 kb adjacency to genes, cancer genes, pseudogenes, transcription factor and (post-transcriptional) miRNA binding sites, CpG islands and repetitive elements (short interspersed nuclear elements (SINE), long interspersed nuclear elements (LINE), Type II elements and LTR elements). Additionally, all experimental frequencies are compared with the data obtained from a reference set, containing 1 000 000 random integrations ('random set'). Thus, for the first time a tool allowing high-throughput profiling of gene therapy vector insertion sites is available. It provides a basis for large-scale insertion site analyses, which is now urgently needed to discover novel gene therapy vectors with 'safe' insertion profiles.

Enhanced complement resistance in drug-selected P-glycoprotein expressing multi-drug-resistant ovarian carcinoma cells.

Multi-drug resistance (MDR) is a major obstacle in cancer chemotherapy. There are contrasting data on a possible correlation between the level of expression of the drug transporter P-glycoprotein (P-gp) and susceptibility to complement-dependent cytotoxicity (CDC). We therefore investigated the sensitivity of human ovarian carcinoma cells and their P-gp expressing MDR variants to complement. Chemoselected P-gp expressing MDR cells showed increased resistance to CDC associated with overexpression of membrane-bound complement regulatory proteins (mCRP) and increased release of the soluble inhibitors C1 inhibitor and factor I. MDR1 gene transfection alone did not alter the susceptibility of P-gp expressing A2780-MDR and SKOV3-MDR cells to CDC. However, subsequent vincristine treatment conferred an even higher resistance to complement to these cells, again associated with increased expression of mCRP. Blocking the function of P-gp with verapamil, cyclosporine A or the anti-P-gp-antibody MRK16 had no impact on their complement resistance, whereas blocking of mCRP enhanced their susceptibility to complement. These results suggest that enhanced resistance of chemoselected MDR ovarian carcinoma cells to CDC is not conferred by P-gp, but is due at least partly to overexpression of mCRP, probably induced by treatment with the chemotherapeutic agents.

Multiple displacement amplification enables large-scale clonal analysis following retroviral gene therapy.

Analysis of the fate of retrovirally transduced cells after transplantation is often hampered by the scarcity of available DNA. We evaluated a promising method for whole-genome amplification, called multiple displacement amplification (MDA), with respect to even and accurate representation of retrovirally transduced genomic DNA. We proved that MDA is a suitable method to subsequently quantify engraftment efficiencies by quantitative real-time PCR by analyzing retrovirally transduced DNA in a background of untransduced DNA and retroviral integrations found in primary material from a retroviral transplantation model. The portion of these retroviral integrations in the amplified samples was 1.02-fold (range 0.2, to 2.1-fold) the portion determined in the original genomic DNA. Integration site analysis by ligation-mediated PCR (LM-PCR) is essential for the detection of retroviral integrations. The combination of MDA and LM-PCR showed an increase in the sensitivity of integration site analysis, as a specific integration site could be detected in a background of untransduced DNA, while the transduced DNA made up only 0.001%. These results show for the first time that MDA enables large-scale sensitive detection and reliable quantification of retrovirally transduced human genomic DNA and therefore facilitates follow-up analysis in gene therapy studies even from the smallest amounts of starting material.

New bioinformatic strategies to rapidly characterize retroviral integration sites of gene therapy vectors.

OBJECTIVE: Increasing use of retroviral vector-mediated gene transfer created intense interest to characterize vector integrations on the genomic level. Techniques to determine insertion sites, mainly based on time-consuming manual data processing, are commonly applied. Since a high variability in processing methods hampers further data comparison, there is an urgent need to systematically process the data arising from such analysis. METHODS: To allow large-scale and standardized comparison of insertion sites of viral vectors we developed two programs, IntegrationSeq and IntegrationMap. IntegrationSeq can trim sequences, and valid integration sequences get further processed with IntegrationMap for automatic genomic mapping. IntegrationMap retrieves detailed information about whether integrations are located in or close to genes, the name of the gene, the exact localization in the transcriptional units, and further parameters like the distance from the transcription start site to the integration. RESULTS: We validated the method using 259 files originating from integration site analysis (LM-PCR). Sequences processed by IntegrationSeq led to an increased yield of valid integration sequence detection, which were shown to be more sensitive than conventional analysis and 15 times faster, while the specificities are equal. Output files generated by IntegrationMap were found to be 99.8% identical with results retrieved by much slower conventional mapping with the ENSEMBL alignment tool. CONCLUSION: Using IntegrationSeq and IntegrationMap, a validated, fast and standardized high-throughput analysis of insertion sites can be achieved for the first time.

Efficient mobilization of peripheral blood stem cells following CAD chemotherapy and a single dose of pegylated G-CSF in patients with multiple myeloma.

High-dose chemotherapy followed by autologous blood stem cell transplantation is the standard treatment for myeloma patients. In this study, CAD (cyclophosphamide, adriamycin, dexamethasone) chemotherapy and a single dose of pegfilgrastim (12 mg) was highly effective in mobilizing peripheral blood stem cells (PBSCs) for subsequent transplantation, with 88% of patients (n = 26) achieving the CD34+ cell harvest target of > or = 7.50 x 10(6) CD34+ cells/kg body weight, following a median of two apheresis procedures (range 1-4) and with first apheresis performed at a median day 13 after CAD application (range 10-20). Patients treated with pegfilgrastim showed a reduced time to first apheresis procedure from mobilization compared with filgrastim-mobilized historical matched controls (n = 52, P = 0.015). The pegfilgrastim mobilization regimen allowed for transplantation of a median of 3.58 x 10(6) CD34+ cells/kg body weight while leaving sufficient stored cells for a second high-dose regimen and back-ups in most patients. Engraftment following transplantation was comparable to filgrastim, with a median time of 14 days to leucocyte > or =1.0 x 10(9)/l (range 10-21) and 11 days to platelets > or = 20 x 10(9)/l (range 0-15). The results of this study thus provide further support for the clinical utility of pegfilgrastim for the mobilization of PBSC following chemotherapy in cancer patients scheduled for transplantation.

Retroviral MDR1 gene transfer into marrow-engrafting human peripheral blood progenitor cells results in preferential transgene expression in the immature myeloid compartment rather than in mature myeloid progeny in vivo.

BACKGROUND: The objective of multidrug resistance-1 (MDR1) gene therapy is protection of the myeloid cell lineage. It is therefore important to examine the effect of retroviral transduction on myeloid maturation. Transfer of the human MDR1 gene can confer resistance to a variety of cytostatic drugs. For a safe application in humans it is paramount to follow-up the development of transduced cells. METHODS: We transduced human mobilized peripheral blood progenitor cells (PBPC) with a viral vector containing the human MDR1 cDNA and transplanted the transduced cells into non-obese diabetic severe combined immunodeficient (NOD/SCID) mice. The progeny of the transduced cells was analyzed in detail by flow cytometry. RESULTS: A detailed analysis by four-color flow cytometry showed that MDR1 transgene-expressing CD33+ myeloid cells were preferentially negative for the maturation-associated myeloid markers CD11b and CD10, while the untransduced CD33+ myeloid cells expressed significantly higher proportions of these Ag (P<0.01 each). There was no difference in the expression of B- or T-lymphoid Ag among the MDR1-transduced and untransduced lymphoid cells. DISCUSSION: These data indicate that retroviral MDR1 gene transfer results in preferential P-glycoprotein expression in myeloid progenitor cells, which is the target cell population for myelotoxicity of cytostatic drugs.

Retroviral vector insertions in T-lymphocytes used for suicide gene therapy occur in gene groups with specific molecular functions.

Graft-versus-host disease (GvHD) is a severe complication in the context of allogeneic stem cell transplantation and adoptive immunotherapy. The transfer of a suicide gene into donor T-lymphocytes (TLCs) allows selective elimination of GvHD-causing cells. As retroviral gene transfer into hematopoietic stem cells can induce leukaemia, there is an urgent need also to analyze retroviral integration sites in TLCs. We examined suicide gene-transduced TLCs in four grafts and from four transplanted patients. One-hundred and fifteen integration sites were detected in vitro. Of these 90 could be mapped to the human genome; 50% (45) were located in genes and 32% (29) were detected 10 kb upstream or downstream of transcription start sites. We found a significant overrepresentation of genes encoding for proteins with receptor activity, signal transducer activity, transcription regulator activity, nucleic acid binding activity and translation regulator activity. Similar data were obtained from patient samples. Our results point to preferred vector integration patterns, which are specific for the target cell population and probably independent of selection processes. Thus, future preclinical analysis of the integration repertoire with abundant amounts of transduced cells could allow a prediction also for the in vivo situation, where target cells are scarce.

Innovative strategies for PBPC mobilization.

Stem cell transplantation, whether autologous or allogeneic, improves the outcome of patients with a number of hematologic malignancies or solid tumors. A relevant proportion of these patients are excluded from this treatment because sufficient numbers of hematopoietic stem cells cannot be obtained by standard cytokine-assisted mobilization. In this article we review the physiology of peripheral blood progenitor cell (PBPC) mobilization and discuss the role of adhesion molecules, such as integrins and selectins, chemokines and their ligands, such as SDF-1alpha and CXCR4, and proteolytic enzymes. Based on this knowledge, several innovative pharmacologic approaches have been proposed to boost the stem cell harvest. Some of them (CTCE, C3a receptor agonist and GrobetaT) are still subject of pre-clinical development, others, such as chemokine receptor ligand AMD3100, have recently been introduced in clinical trials and already deliver promising results. It appears possible to harvest PBPC successfully in poor mobilizers and to cut down the number of collections required in the remaining PBPC donors.

New developments in stem cell biology and therapy. First meeting report from the working group of the German Society for Hematology and Oncology.

The call for the meeting which took place in Heidelberg 13 January 2005, resulted in a high number of contributions covering a diversity of topics: embryonal stem cell research; molecular signaling pathways; assay systems for primitive, mesenchymal and epithelial stem cells; markers for transdifferentiation; and theoretical considerations including biomathematical modeling of stem cell development. The program was rounded off by pre-clinical and clinical applications of stem cell therapies, including new mobilization agents, treatment of myocardial infarction and chemoprotective gene transfer to stem cells.

Synergistic activity of imatinib and 17-AAG in imatinib-resistant CML cells overexpressing BCR-ABL--Inhibition of P-glycoprotein function by 17-AAG.

Overexpression of BCR-ABL and P-glycoprotein (Pgp) are two of the known mechanisms of imatinib resistance. As combination therapy may allow to overcome drug resistance, we investigated the effect of combination treatment with imatinib and 17-allylamino-17-demethoxygeldanamycin (17-AAG), a heat-shock protein 90 (Hsp90) inhibitor, on different imatinib-sensitive and imatinib-resistant CML cell lines. In imatinib-sensitive cells, combination index (CI) values obtained using the method of Chou and Talalay indicated additive (CI=1) or marginally antagonistic (CI>1) effects following simultaneous treatment with imatinib and 17-AAG. In imatinib-resistant cells both drugs acted synergistically (CI<1). In primary chronic-phase CML cells additive or synergistic effects of the combination of imatinib plus 17-AAG were discernible. Annexin V/propidium iodide staining showed that the activity of imatinib plus 17-AAG is mediated by apoptosis. Combination treatment with imatinib plus 17-AAG was more effective in reducing the BCR-ABL protein level than 17-AAG alone. Monotherapy with 17-AAG decreased P-glycoprotein activity, which may increase intracellular imatinib levels and contribute to the sensitization of CML cells to imatinib. The results suggest that combination of imatinib and 17-AAG may be useful to overcome imatinib resistance in a clinical setting.

Prospects and RISC score of viral gene therapy for sarcoma.

Soft tissue sarcomas are a challenge for medical oncology and gene therapy. Protective and sensitising approaches that target normal and malignant tissue, respectively, both have their role for opening the therapeutic window. Recent data show that an intensive maintenance chemotherapy significantly reduces metastatic spread and improves disease-free survival in selected patient groups. However, delays of treatment due to cytopenia are frequent. Cytostatic drug resistance gene transfer to haematopoietic progenitor cells using retroviral vectors may allow further improvement of therapy results. In recent years, retroviral vector design, transduction techniques and engraftment capability of transduced cells have been optimised. Safety considerations of retroviral gene transfer have attracted public attention and can be addressed by analysis of genomic vector integration sites. A data bank project, 'retroviral insertion estimate of chromosomal integration' (RISC), containing > 200 integration sequences, has been set up by the authors' group to recognise critical genomic regions and genes involved with possible transforming capacity. Monitoring these parameters will allow the selection of the most suitable vectors for clinical application. Sarcoma cells seem to be highly susceptible to a variety of vectors, such as recombinant adeno-associated virus-2 (rAAV-2) vectors, adenoviral vectors or oncolytic herpes simplex viruses. Results from the first clinical trials with adenoviral vectors encoding for cytokines are promising. The other systems await further development towards clinical applications. Perspectives for further research are discussed in this review.

Dynamics of BCR-ABL mRNA expression in first-line therapy of chronic myelogenous leukemia patients with imatinib or interferon alpha/ara-C.

We sought to determine dynamics of BCR-ABL mRNA expression levels in 139 patients with chronic myelogenous leukemia (CML) in early chronic phase, randomized to receive imatinib (n=69) or interferon (IFN)/Ara-C (n=70). The response was sequentially monitored by cytogenetics from bone marrow metaphases (n=803) and qualitative and quantitative RT-PCR from peripheral blood samples (n=1117). Complete cytogenetic response (CCR) was achieved in 60 (imatinib, 87%) vs 10 patients (IFN/Ara-C, 14%) after a median observation time of 24 months. Within the first year after CCR, best median ratio BCR-ABL/ABL was 0.087%, (imatinib, n=48) vs 0.27% (IFN/Ara-C, n=9, P=0.025). BCR-ABL was undetectable in 25 cases by real-time PCR, but in only four patients by nested PCR. Median best response in patients with relapse after CCR was 0.24% (n=3) as compared to 0.029% in patients with continuous remission (n=52, P=0.029). We conclude that (i) treatment with imatinib in newly diagnosed CML patients is associated with a rapid decrease of BCR-ABL transcript levels; (ii) nested PCR may reveal residual BCR-ABL transcripts in samples that are negative by real-time PCR; (iii) BCR-ABL transcript levels parallel cytogenetic response, and (iv) imatinib is superior to IFN/Ara-C in terms of the speed and degree of molecular responses, but residual disease is rarely eliminated.