Secondary malignancies in chronic myeloid leukemia patients after imatinib-based treatment: long-term observation in CML Study IV.

Treatment of chronic myeloid leukemia (CML) has been profoundly improved by the introduction of tyrosine kinase inhibitors (TKIs). Long-term survival with imatinib is excellent with a 8-year survival rate of ∼88%. Long-term toxicity of TKI treatment, especially carcinogenicity, has become a concern. We analyzed data of the CML study IV for the development of secondary malignancies. In total, 67 secondary malignancies were found in 64 of 1525 CML patients in chronic phase treated with TKI (n=61) and interferon-α only (n=3). The most common malignancies (n⩾4) were prostate, colorectal and lung cancer, non-Hodgkin's lymphoma (NHL), malignant melanoma, non-melanoma skin tumors and breast cancer. The standardized incidence ratio (SIR) for all malignancies excluding non-melanoma skin tumors was 0.88 (95% confidence interval (0.63-1.20)) for men and 1.06 (95% CI 0.69-1.55) for women. SIRs were between 0.49 (95% CI 0.13-1.34) for colorectal cancer in men and 4.29 (95% CI 1.09-11.66) for NHL in women. The SIR for NHL was significantly increased for men and women. An increase in the incidence of secondary malignancies could not be ascertained. The increased SIR for NHL has to be considered and long-term follow-up of CML patients is warranted, as the rate of secondary malignancies may increase over time.

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.

Evaluation of different protocols for gene transfer into non-obese diabetes/severe combined immunodeficiency disease mouse repopulating cells.

PURPOSE: Although gene transfer with retroviral vectors has shown distinct clinical success in defined settings, efficient genetic manipulation of hematopoietic progenitor cells remains a challenge. To address this issue we have evaluated different transduction protocols and retroviral constructs in the non-obese diabetes (NOD)/severe combined immunodeficiency disease (SCID) xenograft model. METHODS: An extended transduction protocol requiring 144 h of in vitro manipulation was compared to a more conventional protocol requiring 96 h only. RESULT: While pretransplantation analysis of cells transduced with a retroviral vector, expressing the enhanced green fluorescent protein (EGFP) marker gene, demonstrated significantly higher overall transduction rates for the extended protocol (33.6 +/- 2.3 vs. 22.1 +/- 3.8%), EGFP expression in CD34+ cells before transplantation (4.0 +/- 0.9 vs. 11.6 +/- 2.5%), engraftment of human cells in NOD/SCID bone marrow 4 weeks after transplantation (4.5 +/- 1.7 vs. 36.5 +/- 9.4%) and EGFP expression in these cells (0 +/- 0 vs. 11.3 +/- 2.8%) were significantly impaired. When the 96 h protocol was used in combination with the spleen focus forming virus (SFFV)/murine embryonic stem cell (MESV) hybrid vector SFbeta11-EGFP, high transduction rates for CD45+ (41.0 +/- 5.3%) and CD34+ (38.5 +/- 3.7%) cells prior to transplantation, as well as efficient human cell engraftment in NOD/SCID mice 4 weeks after transplantation (32.4 +/- 3.5%), was detected. Transgene expression was observed in B-lymphoid (15.9 +/- 2.0%), myeloid (36.5 +/- 3.5%) and CD34+ cells (10.1 +/- 1.5%). CONCLUSION: Our data show that CD34+ cells maintained in cytokines for multiple days may differentiate and loose their capacity to contribute to the haematological reconstitution of NOD/SCID mice. In addition, the SFFV/MESV hybrid vector SFbeta11-EGFP allows efficient transduction of and gene expression in haematopoietic progenitor cells.

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-beta-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.

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

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.

[Interdisciplinary cooperation in the treatment of complex patients with advanced testicular germ cell tumor]. / Interdisziplinäre Kooperation in der Therapie von komplexen Patienten mit fortgeschrittenem testikulärem Keimzelltumor.

Testicular germ cell tumors represent the classic example of a curable solid cancer even in the metastatic stage. Cure rates are as high as 95% and 80-85% in patients with good and intermediate prognosis; even in patients with poor prognosis cure rates of 50% have been achieved by interdisciplinary collaboration of all specialties involved in the management of testis cancer. Standardization of diagnosis and therapy should be further optimized due to the recently published interdisciplinary national and European guidelines. Besides realization of standardized guidelines, treatment of patients with extensive primary disease or recurrent germ cell tumors following standard therapy requires comprehensive knowledge in conservative and surgical management, which is basically only available at specialized cancer centers. Patients with complex findings, especially if associated with a poor prognosis according to IGCCCG, should be referred to specialized tertiary referral centers at a very early stage, since the cure rates depend not only on the consideration of guidelines but also on the expertise of the attending oncologist and surgeon. When treating these patients, one has to consider that inadequately administered chemotherapy (dosage, length of cycles, number of cycles) cannot be compensated for by surgery and that inadequately performed retroperitoneal lymphadenectomy or residual tumor resection cannot be compensated for by chemotherapy. In any case, suboptimal primary therapy will result in inferior cure rates and an unnecessarily increased mortality rate.

European consensus on diagnosis and treatment of germ cell cancer: a report of the European Germ Cell Cancer Consensus Group (EGCCCG).

Germ cell tumour is the most frequent malignant tumour type in young men with a 100% rise in the incidence every 20 years. Despite this, the high sensitivity of germ cell tumours to platinum-based chemotherapy, together with radiation and surgical measures, leads to the high cure rate of > or = 99% in early stages and 90%, 75-80% and 50% in advanced disease with 'good', 'intermediate' and 'poor' prognostic criteria (IGCCCG classification), respectively. The high cure rate in patients with limited metastatic disease allows the reduction of overall treatment load, and therefore less acute and long-term toxicity, e.g. organ sparing surgery for specific cases, reduced dose and treatment volume of irradiation or substitution of node dissection by surveillance or adjuvant chemotherapy according to the presence or absence of vascular invasion. Thus, different treatment options according to prognostic factors including histology, stage and patient factors and possibilities of the treating centre as well may be used to define the treatment strategy which is definitively chosen for an individual patient. However, this strategy of reduction of treatment load as well as the treatment itself require very high expertise of the treating physician with careful management and follow-up and thorough cooperation by the patient as well to maintain the high rate for cure. Treatment decisions must be based on the available evidence which has been the basis for this consensus guideline delivering a clear proposal for diagnostic and treatment measures in each stage of gonadal and extragonadal germ cell tumour and individual clinical situations. Since this guideline is based on the highest evidence level available today, a deviation from these proposals should be a rare and justified exception.

Impact of chemotherapy regimen and hematopoietic growth factor on mobilization and collection of peripheral blood stem cells in cancer patients.

Various chemotherapy regimens, combined with recombinant human granulocyte colony-stimulating factor(rhG-CSF) or recombinant granulocyte-macrophage CSF (rhGM-CSF) are used in cancer patients to mobilize and collect peripheral blood stem cells (PBSC). In this retrospective study, we evaluated and compared the efficacy of such regimens in 262 patients with different types of malignant diseases. The following chemotherapy regimens were applied: ifosfamide-etoposide-cisplatin or bleomycin (n = 96; mainly patients with testicular cancer); ifosfamide-etoposide plus or minus cytosine arabinoside (Ara-C) or vincristine (VCR)(n = 52; mainly patients with lymphoma); cyclophosphamide-anthracycline (n = 53; mainly patients with breast cancer); intermediate to high dose (ID-HD) cyclophosphamide (n = 37; mainly patients with breast or ovarian cancer. or multiple myeloma; and others (n = 24). rhG-CSF or rhGM-CSF, each at an average daily dose of 5 microg/kg body weight, were used in 166 and 96 patients, respectively. The study evaluated and compared the efficacy of these two cytokines. In patients receiving rhG-CSF, CD34+ cells could be collected earlier (median: day 14 versus day 16) and there was a significantly higher white blood cell count (WBC)(median 11,350 versus 5550/microl) and CD34+ cell count (median 88 versus 43/microl) at the start of apheresis, and a significantly higher CD34+ cell yield (median 7.4 x 10(6) versus 4.6 x 10(6)/kg) than in patients who receivedrhGM-CSF. Among the various chemotherapeutic regimens used, each combined with rhG-CSF, ifosfamide-etoposide plus or minus Ara-C or VCR mobilized a significantly higher number of CD34+ cells (median 119/microl) and produced a significantly higher harvest of these cells (median 13 x 10(6)/kg) than cyclophosphamide-anthracycline (median 87/microl and 7 x 10(6)/kg, respectively) or ID-HD cyclophosphamide (median 59/microl and 5 x I 0(6)/kg, respectively). Ifosfamide-etoposide plus or minus Ara-C or VCR was also superior to ifosfamide-etoposide-cisplatin or bleomycin (median 78/microl and 9 x 10(6)/kg, respectively), but at borderline significance. The outcome of PBSC mobilization and collection appeared to be negatively influenced by the number of relapses before the current salvage treatment. These data indicate that mobilization and collection of PBSCstrongly depend on the type of hematopoietic growth factor and chemotherapeutic regimen used. The data further show rhG-CSF is a more effective growth factor than rhGM-CSF and ifosfamide-etoposide-based regimens, particularly ifosfamide-etoposide plus or minus Ara-C or VCR, are highly effective regimens in mobilizing and collecting CD34+ cells.

Gene therapy for sarcoma.

Soft tissue sarcomas are mesenchymal tumors which respond poorly to systemic therapy. Recent studies suggest a higher response rate with an increased doxorubicin dosage. However, this was parallel with a profound hematotoxicity in 75% of patients. Transfer of the human multidrug resistance 1 (MDR1) gene to normal hematopoietic stem cells and transplantation may significantly reduce the hematotoxicity of anthracyclin-based chemotherapy. To test this concept of supportive gene therapy in advance of a clinical study, we transduced mobilized peripheral blood progenitor cells (PBPC) with the retroviral vector SF91m3 containing the human MDR1 gene, transplanted these cells to immune-deficient mice, allowed 6 weeks for engraftment to occur and treated the animals with MDR1-based chemotherapy. In the MDR1-transduced group the human leukocytes were significantly protected from the toxicity of chemotherapy (p < 0.05). While the gene transfer rate was in the range of 10% and thus comparable to recent clinical trials, the gene expression was 59% of transduced cells and thus significantly higher than previously reported for less-advanced vectors. On the other hand, ifosfamide, a drug which has been used successfully for stem cell mobilization, is active in soft tissue sarcoma. Due to these favorable characteristics sarcoma is an attractive target to test the efficacy of MDR1 gene therapy in a clinical setting. Gene therapeutic strategies may also be used to directly target sarcoma cells, e.g. by transfer of suicide genes. We found that adenoassociated virus 2 (AAV-2) vectors efficiently transduce human HS-1 and HT1080 sarcoma cells (>90%) while other tumor cell lines and primary human PBPC were less susceptible. The thymidine kinase (TK) suicide gene was cloned into an AAV-2 vector and a complete kill of TK-transduced HS-1 and HT1080 cells was observed following exposure to aciclovir or ganciclovir (GCV), while >90% of mock-transduced HS-1 cells survived at these dosages. Transplantation of those sarcoma cells to nonobese diabetic (NOD)/LtSz-severe-combined immunodeficient (scid)/scid (NOD/SCID) mice resulted in a survival of >5 months in the AAV-TK-transduced/GCV-treated group, while the mice in the mock-transduced/GCV-treated group had died after 3 weeks. These data show that soft tissue sarcomas are a particularly suitable model system for the development and clinical testing of new gene therapeutic concepts.

Protection of hematopoietic cells from O(6)-alkylation damage by O(6)-methylguanine DNA methyltransferase gene transfer: studies with different O(6)-alkylating agents and retroviral backbones.

Overexpression of O(6)-methylguanine DNA methyltransferase (MGMT) can protect hematopoietic cells from O(6)-alkylation damage. To identify possible clinical applications of this technology we compared the effect of MGMT gene transfer on the hematotoxicity induced by different O(6)-alkylating agents in clinical use: the chloroethylnitrosoureas ACNU, BCNU, CCNU and the tetrazine derivative temozolomide. In addition, various retroviral vectors expressing the MGMT-cDNA were investigated to identify optimal viral backbones for hematoprotection by MGMT expression. Protection from ACNU, BCNU, CCNU or temozolomide toxicity was evaluated utilizing a Moloney murine leukemia virus-based retroviral vector (N2/Zip-PGK-MGMT) to transduce primary murine bone marrow cells. Increased resistance in murine colony-forming units (CFU) was demonstrated for all four drugs. In comparison to mock-transduced controls, after transduction with N2/Zip-PGK-MGMT the IC50 for CFU increased on average 4.7-fold for ACNU, 2.5-fold for BCNU, 6.3-fold for CCNU and 1.5-fold for temozolomide. To study the effect of the retroviral backbone on hematoprotection various vectors expressing the human MGMT-cDNA from a murine embryonic sarcoma virus LTR (MSCV-MGMT) or a hybrid spleen focus-forming/murine embryonic sarcoma virus LTR (SF1-MGMT) were compared with the N2/Zip-PGK-MGMT vector. While all vectors increased resistance of transduced human CFU to ACNU, the SF1-MGMT construct was most efficient especially at high ACNU concentrations (8-12 microg/ml). Similar results were obtained for protection of murine high-proliferative-potential colony-forming cells. These data may help to optimize treatment design and retroviral constructs in future clinical studies aiming at hematoprotection by MGMT gene transfer.

Role of angiogenesis inhibitors in acute myeloid leukemia.

Neovascularization is increasingly recognized as an important factor in the pathogenesis of hematologic malignancies as well as solid tumors. The complex interactions between several cell types and numerous cytokine mediators suggest the involvement of autocrine and paracrine signaling mechanisms. Vascular endothelial growth factor (VEGF) in particular is critical to both stimulation of leukemic growth and proliferation of endothelial cells. Tyrosine kinase receptors specific for certain growth factors represent attractive target molecules for anticancer therapy. SU5416 is a competitive inhibitor of VEGF receptor subtypes VEGFR-1 and VEGFR-2 and stem cell factor receptor c-kit. Preclinical evidence shows that SU5416 effectively inhibits VEGF-induced endothelial cell proliferation and slows growth of subcutaneous solid tumor xenografts. This agent is in late-stage clinical trials in patients with solid tumors, and a Phase 2 study was recently initiated to evaluate its utility in the treatment of acute myeloid leukemia. In this Phase 2 study, investigators are seeking to determine the response rate to the antiangiogenic agent SU5416. Translational research in this study is intended to aid our understanding of the precise mechanisms by which SU5416 affects acute myeloid leukemia cells and the bone marrow microenvironment.

Long-term survival after induction therapy with idarubicin and cytosine arabinoside for de novo acute myeloid leukemia.

We treated 153 patients with de novo acute myeloid leukemia (AML) with two induction courses of conventional-dose cytosine arabinoside (ara-C) and idarubicin (AIDA) followed by either a third course of AIDA, high-dose ara-C or bone-marrow transplantation. The complete remission (CR) rate for all patients was 63.4%, with a higher CR rate for patients with a normal (versus unfavorable) karyotype (73.2% vs 52.5%; P=0.038). The probability of overall survival (OS) was 30.7% after 5 years (26.3% after 7 years). Improved OS at 5 years could be observed for patients up to 50 years old versus patients older than 50 years of age (37.6% vs 19.9%; P=0.001) and patients with a normal (versus unfavorable) karyotype (42.9% vs 14.1%; P=0.0016). Disease-free survival (DFS) after 5 years was 33.2% for all 97 CR patients and was significantly better for patients with a normal (versus unfavorable) karyotype (44.3% vs 12.3%; P= 0.003). Multivariate analysis revealed that the age for OS (P < 0.02) and the karyotype for both OS (P<0.03) and DFS (P< 0.05) were independent prognostic factors. In conclusion, AIDA is an effective and well-tolerated induction regimen (even in elderly patients) with a 5-year survival of more than 30% when combined with ara-C-containing postremission therapy. The karyotype is the most powerful prognostic factor for predicting the outcome of patients treated with this protocol.

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 SFbeta1-EGFP) or EGFP and a downstream (vector SFbeta1-EGFP-IRES) or upstream (vector SFbeta1-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 SFbeta1-EGFP vector (82.6 +/- 6.7 MFI) and the SFbeta1-EGFP-IRES vector (102.8 +/- 6.2 MFI) was higher than from SFbeta1-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 SFbeta1-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.

Optimized retroviral transduction protocol for human progenitor cells utilizing fibronectin fragments.

BACKGROUND: Retroviral transduction in the presence of fibronectin (FN) fragments has proven an efficient and clinically-applicable procedure for gene transfer into hematopoietic cells. So far, FN-based transduction protocols have been optimized primarily for transduction of stem cells, whereas for several therapeutic applications transduction of clonogenic progenitors (CFU) may be sufficient. METHODS: Transduction protocols for CFU were optimized by evaluating the effect of growth factors, timing of retroviral transduction, CD34-selection and heparin, using a neomycin-phosphotransferase (neo(R))-expressing retroviral vector. RESULTS: The presence of multiple growth factors during prestimulation and transduction, including the differentiating cytokines G-CSF or GM-CSF, substantially enhanced transduction of CFU. Best results were achieved when 24 h of prestimulation were followed by a 24-48 h transduction period in the presence of the CH-296 FN-fragment and IL-3, IL-11, SCF, erythropoietin (EPO), and GM-CSF. With this proto-col we observed highly efficient transduction of BM-derived CFU (90.7 +/- 8.8 % G 418-resistant colonies), even with retrovirus preparations of moderate infectious titer (5 x 10(4) - 2 x 10(5) CFU/mL). The number of CFU increased on average 2.6-fold (range 1.5-3.8) during the transduction procedure. Selection of CD34(+) cells prior to transduction did not improve transduction efficiency. Heparin, even in concentrations as low as 2.0 microg/mL, significantly inhibited transduction of CFU on FN-fragments. DISCUSSION: An optimized protocol for retroviral gene transfer into human clonogenic progenitor cells that allows highly efficient transduction, even with moderate titer retroviral vectors, is presented.

Prognostically orientated multimodality treatment including surgery for selected patients of small-cell lung cancer patients stages IB to IIIB: long-term results of a phase II trial.

Following mediastinoscopy, a prognostically orientated multimodality approach was chosen in selected small-cell lung cancer (SCLC) patients with hyperfractionated accelerated chemoradiotherapy (Hf-RTx) and definitive surgery (S). Stage IB/IIA patients had four cycles of cisplatin/etoposide (PE) and surgery. Stage IIB/IIIA patients had three cycles PE followed by one cycle concurrent chemoradiation including Hf-RTx and surgery. Most stage IIIB patients were not planned for surgery and had CTx followed by sequential RTx or one cycle concurrent CTx/RTx. Of 46 consecutive patients (stage IB six, IIA two, IIB/IIIA 22, IIIB 16) 43 (94%) showed an objective response. Twenty-three of patients (72%) planned for inclusion of S were completely resected (R0) (IB 6/6, IIA 2/2, IIB/IIIA 13/22, IIIB 2/2). Overall toxicity was acceptable--one patient died of septicaemia, no perioperative deaths occurred. Median follow-up of patients alive (n = 21) is 52 months (30+ - 75+). Median survival and 5-year survival rate of all patients are 36 months and 46%, in R0 patients 68 months and 63% (R0-IIB/IIIA/IIIB: not yet reached and 67%). This multimodality treatment including surgery proved highly effective with 100% local control and remarkable long-term survival after complete resection, even in locally advanced SCLC stages IIB/IIIA patients.

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.

Fanconi anemia and beta c deficiency-associated pulmonary alveolar proteinosis as two hereditary diseases of childhood which are potentially curable by stem cell gene therapy but require different therapeutic approaches.

Fanconi anemia (FANC) and pulmonary alveolar proteinosis associated with deficiency of the beta-chain common to the GM-CSF/IL3/IL5 receptors (beta c-PAP) are rare inherited disorders of childhood or adolescence. Hematopoietic stem cell gene therapy aiming at reintroducing the wildtype cDNA as a new concept for the treatment of hereditary diseases may be applicable to FANC and PAP, as both disorders can be successfully treated by allogeneic stem cell transplantation. However, there are important distinctions to be made between the two diseases: FANC seems to be a disorder with functional stem cell deficiency. Thus, introduction of the wildtype cDNA should provide an in vivo growth advantage to genetically corrected stem cells so that corrected cells and their progeny may expand in vivo and slowly repopulate the entire hematopoietic system. In beta c-PAP, the defect has no major impact on proliferation or differentiation of stem cells. Therefore, introduction of the wildtype gene will probably not provide any selective growth advantage and the percentage of corrected cells in the hematopoietic compartment depend on the percentage of stem cells initially transduced as the current technology only allows for transduction of stem cells with low efficiency. The introduction of a second selectable cDNA into the vector might be used to provide selective growth for modified cells and thus overcome a low gene transfer efficiency of stem cells. The correction of rare monogenetic diseases may serve as a model for gene therapy prior to attempts to treat more common and complex polygenetic diseases. The studies outlined here will be helpful envisioning new treatment strategies for other inherited monogenetic diseases such as mucopolysaccharidosis, Gauchers disease or adrenoleukodystrophy.

Thrombopoietin serum levels at the start of mobilization, collection, and transfusion of autologous peripheral blood stem cells.

Thrombopoietin (TPO) serum levels in 14 patients (9 male and 5 female, mean age 36 years, range 16 to 55 years) with breast cancer (n = 5), testicular cancer (n = 7), or lymphoma (n = 2), undergoing high dose chemotherapy with peripheral blood stem cell (PBSC) transplantation, were evaluated at the first day of the mobilization chemotherapy (1), at the day of the first apheresis (2), and at the day of stem cell transfusion (3). All patients have been pretreated (one to four regimens) and received chemotherapy and granulocyte colony stimulating factor (G-CSF) or granulocyte-macrophage colony stimulating factor (GM-CSF) both at 5 microg/kg body weight (bw). for stem cell mobilization. TPO was measured with a human TPO immunoassay. Mean TPO serum levels were: (1) 274+/-248.8 pg/ml (range 0 to 953 pg/ml), (2) 518+/-399.1 pg/ml (range 118 to 1,283 pg/ml), and (3) 556+/-506.4 pg/ml (range 147 to 1,570 pg/ml). The CD34+ cell concentration in the peripheral blood at the time of apheresis was 65+/-48.2/microl (7 to 148/microl), and the number of transfused CD34+ cells was 3.0+/-1.0x10(6)/kg bw (1.7 to 5.5x10(6)/kg bw). TPO levels showed some weak inverse correlation (r = -0.64) with the platelet counts at the day of the first apheresis that increased to -0.70 if a semilog correlation was done (plt[log] vs. TPO). The number of platelet transfusions after HDCT correlated to some degree (r = 0.61) with the TPO serum level at the day of PBSC transfusion. There was no correlation between any TPO serum level and the CD34+ cell concentration in the peripheral blood or neutrophil and platelet engraftment. We conclude from this study that TPO serum levels do not seem to correlate with the CD34+ cell concentration in the peripheral blood and the time to engraftment, although there was some weak correlation with the number of platelet transfusions.

Retroviral transduction of human CD34+ umbilical cord blood progenitor cells with a mutated dihydrofolate reductase cDNA.

Umbilical cord blood cells (UCB) have become a major target population for experimental and clinical studies using transfer of genes involved in inborn enzymatic diseases. Cord blood contains hematopoietic progenitor cells at a high frequency, and expanding these cells ex vivo generates sufficient numbers of hematopoietic precursors for transplantation into adults, e.g., as supportive treatment. As clinical reports about retroviral transduction into UCB cells have not been as encouraging as the first preclinical data, we have established a retroviral transduction system that allows expansion and selection of hematopoietic progenitor cells from UCB. CD34-enriched UCB cells were transduced with a retroviral vector encoding a mutated dihydrofolate reductase cDNA that confers MTX resistance. We observed increased resistance to MTX in transduced granulocyte macrophage-colony forming units (CFU-GM) after co-culture of CD34+ UCB cells with the virus-producing cell line, or after incubation with virus-containing supernatant. The supernatant-based transduction protocol included a prestimulation with recombinant interleukin-1 (rhIL-1), rhkit-ligand, and rhIL-3 to increase the percentage of cells in S phase to greater than 50%. Using this protocol we measured a 72-fold expansion of CFU-GM and a 2.5-fold selective advantage of transduced versus nontransduced progenitor cells after exposure to low-dose methotrexate in liquid culture. Polymerase chain reaction analysis revealed integration of proviral DNA into the majority of transduced colonies before and after ex vivo expansion. The retroviral vector and transduction protocol reported here provides an experimental system for selection and expansion of retrovirally transduced progenitor/stem cells from UCB that may help improve the efficiency of current clinical gene therapy strategies.

Preoperative chemotherapy followed by concurrent chemoradiation therapy based on hyperfractionated accelerated radiotherapy and definitive surgery in locally advanced non-small-cell lung cancer: mature results of a phase II trial.

PURPOSE: To evaluate the feasibility and efficacy of an intensive multimodality approach with combination chemotherapy, hyperfractionated accelerated chemoradiotherapy, and definitive surgery in prognostically unfavorable subgroups of locally advanced non-small-cell lung cancer stages IIIA and IIIB (LAD-NSCLC). PATIENTS AND METHODS: Following staging, including mediastinoscopy, 94 patients with inoperable LAD-NSCLC were treated preoperatively with chemotherapy (three courses of split-dose cisplatin and etoposide [PE]) followed by concurrent chemoradiotherapy (one course of PE combined with 45 Gy hyperfractionated accelerated radiotherapy). After repeat mediastinoscopy, patients underwent surgery 4 weeks postradiation. RESULTS: Of 94 consecutive patients (52 stage IIIA [> or = two lymph node levels involved] and 42 stage IIIB [no pleural effusion, no supraclavicular nodes]), 62 (66%) completed induction and underwent surgery. Complete resection (R0) was achieved in 50 (53% of all patients) and pathologic complete response (PCR) in 24 (26%). After a median follow-up of 43 months, the median survival time was 20 months for IIIA, 18 months for IIIB, and 42 months for R0 patients. Calculated survival rates at 4 years were 31%, 26%, and 46%. Two patients died of sepsis preoperatively and four died postoperatively of pleural empyema (n = 1), stump insufficiency (n = 2), and cardiac failure (n = 1). Other toxicities were acceptable-mainly hematologic during chemotherapy or chemoradiotherapy and esophagitis during chemoradiotherapy. CONCLUSION: This intensive multimodality treatment is feasible and demonstrates high efficacy in prognostically unfavorable LAD-NSCLC subgroups with high R0 rates and improved long-term survival compared with historical controls