Topotecan plus cyclophosphamide in adults with relapsed or refractory pediatric-type sarcoma: a retrospective analysis from the German Sarcoma Medical Oncology Group (AIO).

BACKGROUND: To assess the efficacy and safety of topotecan and cyclophosphamide (TC) in adult patients with pediatric-type sarcoma subtypes who failed induction chemotherapy. PATIENTS AND METHODS: Patients with pediatric sarcoma subtypes, refractory to or relapsed after at least one prior induction chemotherapy, inoperable, ECOG PS 0-2, with measurable, progressive disease (PD), adequate organ functions, who have been treated with TC combination were retrospectively analysed within the AIO and SAREZ/BMBF network. RESULTS: Thirty-nine patients, median age 28 years (18-58), 14 females, 25 males, have been identified. All patients had received induction treatment according to (inter)national study protocols. Second-line TC was applied in 33 patients (≥3rd-line in 6 patients). Twenty-three patients had refractory disease (evidence of PD during induction chemotherapy); 8 patients experienced an early relapse within 6 months as well as 8 patients after more than 24 months (late relapse). A median of 3 cycles (range, 1-6) had been applied and antitumor activity was: CR 2.6 %, PR 7.9 %, and disease stabilisation (SD) 26.3 %. PR lasted 32.8 months and median duration in patients with SD was 5 months (range, 2.0-14.7). The 3/6-months progression-free rates were 43.2 and 18.9 %. CONCLUSIONS: Limited activity was seen in adult pts with refractory or relapsed pediatric-type sarcomas with the regimen which has proven activity in pediatric patients. Adults with refractory small cell sarcoma appear to have a similar dismal outcome as seen in pts with common adult-type histologies; however, a subset of patients has achieved long-lasting remissions on TC resulting in long-term survival.

Thermochemotherapy in patients with extremity high-risk soft tissue sarcomas (HR-STS).

PURPOSE: We report data from phase II trials examining the efficacy of multimodality treatment with neoadjuvant chemotherapy, hyperthermia, surgery, radiation and postoperative thermochemotherapy in adult patients with high-risk sarcomas of the extremities. PATIENTS AND METHODS: From 1991 to 2001 47 patients with high risk soft tissue sarcoma of the extremities were prospectively treated in two clinical trials with a treatment plan of four cycles of etoposide, ifosfamide and doxorubicin combined with regional hyperthermia followed by surgery, radiation and adjuvant chemotherapy. RESULTS: Objective response rate assessable in 39 patients was 21% (one complete and seven partial responses). A favourable histological response (>75% tumour necrosis) was observed in 34% of the 35 evaluable patients who had surgical resection. Median overall survival (OS) was 105 months. The five-year probability of local failure-free survival (LFFS), distant disease-free survival (DDFS), event-free survival (EFS) and OS were 48%, 55%, 35% and 57%, respectively. There were no significant differences between responders and non-responders of minimum temperatures (Tmin) and time-averaged temperatures achieved in 50% (T(50)) and 90% (T(90)) at all measured tumour sites. Response to this neoadjuvant regimen predicted for prolonged LFFS (p = 0.0123), but not for OS (p = 0.2). Limb preservation was achieved in 37 patients (79%) and did not result in inferior DDFS (52% versus 50%) or OS (61% versus 50%) at five years (p = 0.8) in comparison to patients who underwent amputation. CONCLUSION: Response to combined modality treatment with RHT and neoadjuvant chemotherapy was predictive for an improved LFFS and led to limb preservation in 79% of patients with extremity sarcomas.

Differential effects of ifosfamide on dendritic cell-mediated stimulation of T cell interleukin-2 production, natural killer cell cytotoxicity and interferon-gamma production.

Ifosfamide is a DNA-alkylating agent used frequently in chemotherapy of human malignancies. Ifosfamide and its major decomposition products deplete intracellular glutathione (GSH). Glutathione is the major intracellular thiol reductant that protects cells against oxidative injury. Ifosfamide depletion of intracellular GSH in human dendritic cells (DC), T cells and natural killer (NK) cells impairs their functional activity which can be restored by reconstituting GSH. Here we assessed the effect of ifosfamide on DC-mediated stimulation of NK cell proliferation via T cells and on direct DC stimulation of NK cell cytotoxicity and interferon (IFN)-gamma production. Indirect DC stimulation of NK cell proliferation via T cells and T cell-derived interleukin (IL)-2 were reduced by ifosfamide treatment of DC and reconstitution of GSH in DC restored both responses. When DC and NK cells were treated with ifosfamide, DC could overcome the negative effect of ifosfamide on NK cytotoxic function whereas NK cell IFN-gamma production was less efficiently restored. The ability of IL-2 activated NK cells to kill autologous immature DC or to induce DC maturation was reduced moderately by treatment of both cell types with ifosfamide. Overall, our results suggest that DC may stimulate anti-tumour effector cells in patients even if they had received treatment with chemotherapeutic agents such as ifosfamide.

Regional hyperthermia of the abdomen in conjunction with chemotherapy for peritoneal carcinomatosis: evaluation of two annular-phased-array applicators.

BACKGROUND: Peritoneal carcinomatosis is a stage of gynecological and gastrointestinal malignancies with poor prognosis. Options for enhancing the effect of standard chemotherapy, such as aggressive surgery and intraperitoneal chemotherapy, have limitations. In this phase I/II study, we evaluated regional hyperthermia of the pelvis and abdomen using the annular-phased-array technique as an adjunct to chemotherapy. METHODS: Forty-five patients with peritoneal carcinomatosis (with or without liver metastases) in colorectal cancer (CRC) (n = 16), ovarian cancer (OC) (n = 17), or gastric/pancreatic/biliary cancer (n = 12) underwent standard chemotherapy and regional hyperthermia. Most CRC patients received second-line chemotherapy. All OC patients were platinum resistant. Regional hyperthermia was applied using a SIGMA-60 applicator (OC), a SIGMA-Eye/MR applicator (CRC), or various ring applicators (gastric/pancreatic/biliary cancer). RESULTS: Abdominal regional hyperthermia was well tolerated, with acceptable acute discomfort and no long-term morbidity. The SIGMA-Eye/MR applicator achieved higher systemic temperatures (associated with higher systemic stress) and more effective heating of the upper abdomen; the SIGMA-60 applicator achieved higher temperatures (and power densities) in the pelvis. Three-year overall survival was encouraging for patients with CRC (22%) and OC (29%) but not gastric/pancreatic/biliary cancer. For the SIGMA-60 applicator (patients with OC), higher measured temperatures at the vaginal stump correlated with better outcome. CONCLUSIONS. The SIGMA-60 and SIGMA-Eye/MR applicators are feasible for abdominal heating and have low toxicity. The SIGMA-60 applicator is specifically suitable for malignancies with high pelvic burden; the SIGMA-Eye/MR applicator better heats the upper abdomen, including the liver. Further randomized investigations are warranted.

Promotion of cystine uptake, increase of glutathione biosynthesis, and modulation of glutathione status by S-2-(3-aminopropylamino)ethyl phosphorothioic acid (WR-2721) in Chinese hamster cells.

We recently found that exposure of cells to different aminothiols promotes cystine uptake and leads to an increase of cellular glutathione by new biosynthesis (Issels et al., Biochem. Pharmacol., 37: 881-888, 1988). Therefore, we further investigated whether the known radioprotective and chemoprotective aminothiol derivative S-2-(3-aminopropylamino)ethylphosphorothioic acid (WR-2721) or its dephosphorylated form (WR-1065) will lead to similar effects. In order to convert WR-2721 to the free thiol compound (WR-1065) in vitro, the medium also contained 20 U/ml alkaline phosphatase (AP). For uptake studies a modified McCoy's 5A medium supplemented with 0.1 mM [35S]cystine was used. In Chinese hamster ovary (CHO) and Chinese hamster ovarian carcinoma (OvCa) cells, WR-2721 exposure alone did not increase the cystine uptake relative to that of control (untreated) cells, while WR-2721 + AP enhanced the uptake of cystine more than twofold in both cell lines. The increase of cystine uptake was dependent on the time of exposure (0-60 min) and the concentrations of WR-2721 (0-8 mM) + AP. Half-maximal uptake of cystine was observed at concentrations of 0.69 and 0.57 mM WR-2721 in CHO and OvCa cells, respectively. Determination of both reduced (GSH) and oxidized (GSSG) cellular glutathione levels after the exposure (0-300 min) to WR-2721 + AP in CHO cells showed a depletion of GSH to less than 10% of the pretreatment value and a 4-fold reduction of the GSH/GSSG ratio. In contrast, in OvCa cells the amount of total glutathione rather increased with no significant change of the GSH/GSSG ratio by the exposure to WR-2721 + AP. Further analysis using high-performance liquid chromatography of cell extracts revealed that the relative amount of incorporated [35S]-cystine into glutathione was increased similarly in both cell lines. The data show that precursor availability and new biosynthesis of glutathione is enhanced by the exposure to WR-2721 + AP in vitro despite the differential modulation of the cellular glutathione status in the two cell lines. These findings may have important implications for the use of aminothiols like WR-2721 in various cells and tissues in regard of their response to chemotherapeutic agents, ionizing radiation and/or hyperthermia.

Enhancement of cysteamine cytotoxicity by hyperthermia and its modification by catalase and superoxide dismutase in Chinese hamster ovary cells.

Chinese hamster ovary cells were exposed to the sulfhydryl compound cysteamine at concentrations ranging from 0 to 8 mM for 120 min. No toxicity was found in cells maintained at 5 degrees during treatment; however, at 37 degrees and 44 degrees a paradoxical toxicity was observed, i.e., substantial toxicity was observed at cysteamine concentrations of 0.2 to 1 mM but decreased at higher drug concentrations. When drug-treated cells were exposed to a 30-min 44 degrees -heat treatment (surviving fraction, 0.15 in the absence of drug) toxicity was markedly enhanced. At 0.4 mM cysteamine, the surviving fraction was approximately 0.6 at 5 degrees, 0.01 at 37 degrees, and 0.00008 when the 44 degrees -heat treatment was also used. Cysteamine toxicity was not modified by the addition of superoxide dismutase (10 micrograms/ml) but was completely blocked by the addition of catalase (50 micrograms/ml) over the drug concentration range of 0.2 to 2.0 mM. Cysteamine autoxidation as measured by O2 uptake at 0.4 mM proceeds through hydrogen peroxide (H2O2) production as evidenced by the regeneration of O2 upon the addition of catalase. In contrast, at 4.0 mM cysteamine, O2 regeneration was not pronounced. The data suggest that the production of H2O2 is the first reaction step in the mechanism of cysteamine toxicity. The subsequent production of highly reactive oxygen species like hydroxyl radicals (.OH) from H2O2 in the presence of reduced metal (Fenton chemistry) probably leads to the observed cellular toxicity.

Influence of oxidative stress induced by cysteamine upon the induction and development of thermotolerance in Chinese hamster ovary cells.

Chinese hamster ovary cells exposed to the sulfhydryl compound cysteamine combined with heat treatment at 44 degrees C developed thermotolerance within 8 h. After initial treatment either with 15 min cysteamine (0.4 mM) at 37 degrees C immediately followed by 15 min heat at 44 degrees C or with 15 min cysteamine (0.4 mM) at 44 degrees C, the magnitude of thermotolerance developed was identical. The D0 of the subsequent 44 degrees C heat survival curves increased by factors of 8.9 and 7.9, respectively. The kinetics of thermotolerance induction and the time to reach the maximum of thermotolerance expression after combined cysteamine treatment at 44 degrees C for 15 min was found to be comparable to the effects of 44 degrees C treatment alone for 30 min. The synergistic effect of cysteamine with the conditioning heat treatment at 44 degrees C was blocked by catalase (50 micrograms/ml). Following initial treatment with cysteamine at 37 degrees C, cells became thermotolerant within 2 h. The D0 of the survival curves for 44 degrees C heat treatments increased with duration (t1 = min, 37 degrees C) of the cysteamine (0.4 mM) exposure; e.g., the D0 increased by factors of 1.5, 1.6, 2.2, and 2.6 for t1 = 30, 60, 90, and 120 min. The induction of thermotolerance by cysteamine at 37 degrees C was completely blocked by the addition of catalase (50 micrograms/ml), present during the initial period of drug treatment. Combined cysteamine and heat treatment at 44 degrees C, but also cysteamine exposure at 37 degrees C, enhanced synthesis of heat shock proteins. The data suggest that oxidative stress by cysteamine can be synergistic with the conditioning heat treatment at 44 degrees C which induces thermotolerance. At 37 degrees C, cysteamine itself induces thermotolerance and the enhanced synthesis of heat shock proteins under these conditions.

Temperature-dependent influence of thiols upon glutathione levels in Chinese hamster ovary cells at cytotoxic concentrations.

Chinese hamster ovary cells were exposed to the sulfhydryl compound cysteamine at different temperatures (5 degrees C, 37 degrees C, 44 degrees C) at concentrations known to generate activated oxygen species. At 37 degrees C, the cellular glutathione (GSH) content increased linearly over the time of drug exposure (2 h) as compared to untreated cells or to cells kept at 5 degrees C during drug treatment. The 2-4-fold increase in GSH induced by cysteamine was more rapid at 44 degrees C than at 37 degrees C and showed a saturation effect at the higher temperature. The elevation of GSH could be completely blocked by DL-buthionine-S,R-sulfoximine, an inhibitor of gamma-glutamylcysteine synthetase, or by incubation in a cystine-free medium during the period of drug treatment. The increased cellular GSH content induced by cysteamine alone at 37 degrees C or combined with heat at 44 degrees C decreased to the range of control values within 22 h after either treatment. Other thiols like cysteamine, namely cysteine, N-acetylcysteine, and dithiothreitol, were found to be similar in their potential to induce GSH elevation in Chinese hamster ovary cells. Cytotoxic effects of these sulfhydryl compounds were observed in the same concentration range as that for cysteamine (0-2 mM), but only if cells were plated at low densities (10(2)-10(4) cells/flask), and were completely blocked by the addition of catalase (50 micrograms/ml). In contrast, the elevation of GSH after thiol treatment (0.8 mM) was not modified by catalase. The data suggest that thiol treatment combined with hyperthermia leads to a rapid increase of GSH biosynthesis in Chinese hamster ovary cells which seems to be independent of the simultaneous generation of activated oxygen species by thiol autoxidation.

Ifosfamide plus etoposide combined with regional hyperthermia in patients with locally advanced sarcomas: a phase II study.

From July 1986 to July 1989, 40 patients (92% pretreated) with deep-seated, advanced soft tissue sarcomas (STS, 25 patients), Ewing's sarcomas (ES, eight patients), osteosarcomas (OS, three patients) and chondrosarcomas (ChS, four patients) were treated at the University of Munich in a protocol involving regional hyperthermia (RHT) combined with ifosfamide plus etoposide. A total of 265 RHT treatments (mean, 6.6 RHT per patient) were applied including 33 pelvic, four extremity, and three abdominal sites. The mean tumor volume was 537 cc (range, 50 to 2,980 cc). For systemic chemotherapy, all patients received ifosfamide (1.5 g/m2, days 1 to 5), etoposide (100 mg/m2, days 1, 3, and 5), and mesna (300 mg/m2 x 4, days 1 to 5) with RHT given only on days 1 and 5 in repeated cycles every 4 weeks. Acute toxicity consisted primarily of pain (57%) combined with local discomfort within the annular phased array applicator (AA) of the BSD hyperthermia system (BSD Medical Corp, Salt Lake City, UT). The average maximum systemic temperature was 37.4 +/- 0.5 degrees C, and there was no indication of enhanced bone marrow toxicity due to the addition of RHT to the systemic chemotherapy. Detailed thermal mapping by invasive thermometry was performed in all patients. In 38 assessable patients, the overall objective response rate was 37%: six complete responses (CRs), four partial responses (PRs), and four favorable histologic responses (FHRs) (95% confidence limits, 22% to 54%). Complete responders are alive and disease-free at 40, 35, 23, 19, 19, and 8 months. Of patients with PR and FHR, two died from metastatic disease after 4 and 17 months and one died from other disease after 27 months. The remaining five patients are stable at 37, 25, 21, 13, and 8 months. Eleven patients showed no change (NC), and 13 patients showed local tumor progression (PD). The mean observation time for all patients was 11.6 months. The time-averaged temperatures (Ts) of all RHT treatments calculated as 20% (T20), 50% (T50), or 90% (T90) of measured tumor sites differed significantly between responders and nonresponders (T20, P = .003; T50, P = .006; and T90, P = .004; respectively). These data support activity for ifosfamide-etoposide combined with RHT in pretreated patients with advanced sarcomas.

Ifosfamide induced stress response in human lymphocytes.

Ifosfamide, an isomer of cyclophosphamide, has been shown to be one of the most effective antineoplastic agents for the treatment of human malignancies. There is considerable evidence that the intracellular status of glutathione (GSH) plays a major role in modifying the cytotoxicity of ifosfamide in cells and tissues. We have studied the effects of 4-hydroperoxy-ifosfamide (4-OOH-IF) upon the proliferation of human peripheral blood lymphocytes (PBL) and the intracellular GSH content. The major finding was that occurrence of significant inhibition of [3H]-thymidine incorporation in interleukin-2 (IL-2) expanded PBL after exposure with 4-OOH-IF was accompanied by substantial depletion of intracellular GSH content in these cells. PBL seemed to be more sensitive to this drug induced effect comparing our results obtained in other cells (e.g. Ewing sarcoma, Chinese hamster ovary). In PBL 4-OOH-IF also induced rapid phosphorylation of the small heat shock protein (HSP27) signaling a similar type of stress response as reported for several other agents (e.g. arsenite, phorbol ester, tumor necrosis factor). Reconstitution of the depleted GSH content in PBL after treatment with 4-OOH-IF could be achieved by GSH-monoethylester and mesna within 24 hours of postincubation time. From these results we conclude that human lymphocytes are sensitive targets for ifosfamide induced metabolic stress during treatment. This might have further importance in regard to the immunological function of these cells.

Neoadjuvant chemotherapy combined with regional hyperthermia (RHT) for locally advanced primary or recurrent high-risk adult soft-tissue sarcomas (STS) of adults: long-term results of a phase II study.

In this phase II study, activity and safety of neoadjuvant regional hyperthermia (RHT) combined with chemotherapy was investigated in 59 patients with primary advanced or recurrent high-risk soft-tissue sarcoma (STS). Patients received four EIA cycles consisting of etoposide, ifosfamide and doxorubicin combined with RHT followed by surgical resection and adjuvant treatment. The overall objective response (OR) rate was 17%, with one complete (2%) and eight partial (15%) responses. In addition, 13 minor responses (25%) were seen. At time of surgery, complete necrosis (pCR) occurred in 6 patients and >75% necrosis (favourable histological response (FHR)) in 12 patients. At the completion of protocol treatment, 36 patients were rendered disease-free which was significantly associated with the initial radiographic and/or pathological tumour response (P=0.004). Treatment-related toxicity was acceptable overall. At a medium follow-up of 82 months, local treatment failure occurred in 33 patients, median overall survival (OS) was 52 months, and the 5-year survival rate was 49% (95% confidence interval (CI): 36-61%). OS which did not differ for extremity versus non-extremity STS (P=0.21) was better for patients responding to EIA combined with RHT (P<0.01).

Treatment of primary, recurrent or inadequately resected high-risk soft-tissue sarcomas (STS) of adults: results of a phase II pilot study (RHT-95) of neoadjuvant chemotherapy combined with regional hyperthermia.

The efficacy of thermochemotherapy in adult patients with primary, recurrent or inadequately resected non-metastatic high-risk soft-tissue sarcomas (STS) was assessed. 54 patients were prospectively treated with four cycles of etoposide, ifosfamide and doxorubicin (EIA) combined with regional hyperthermia (RHT) followed by surgery, another four cycles of EIA without RHT and external beam radiation. The objective response rate was 16% and at a median follow-up time of 57 months, the 4-year estimated rates of local failure-free survival (LFFS), distant metastasis-free survival (DMFS), event-free survival (EFS) and overall survival (OS) were 59% (95% confidence interval (CI) 45-73%), 59% (95% CI 44-73%), 26% (95% CI 14-38%) and 40% (95% CI 27-53%), respectively. OS was in favour of patients responding to neoadjuvant treatment (P=0.073). In comparison to a preceding phase II study including pre- and postsurgical thermochemotherapy (RHT-91), at a 4-year follow-up the RHT-95 study cohort showed an inferior LFFS rate (P=0.027), but this did not affect DMFS (P=0.558) or OS (P=0.126). Hence, postsurgical thermochemotherapy seems critical for local tumour control without affecting survival.

Gemcitabine: a modulator of intracellular nucleotide and deoxynucleotide metabolism.

Gemcitabine (2',2'-difluorodeoxycytidine, dFdC) is a deoxycytidine (dCyd) analog that extensively modulates intracellular CTP and dCTP metabolism. In Chinese hamster ovary (CHO) cells, a 4-hour exposure to gemcitabine (100 mumol/L) reduced cellular CTP and dCTP concentrations to 5.9% and 50%, respectively. Intracellular UTP concentrations increased, indicating a metabolic block at CTP synthetase. Pool-sizes of ATP and GTP remained unaffected. In contrast, a CHO mutant deficient in deoxycytidine kinase, and thus unable to accumulate dFdCTP, maintained its CTP pools under identical conditions, suggesting that the CTP pool depletion was dependent on dFdC phosphorylation. Neither 100 mumol/L arabinosylcytosine nor 5 mmol/L hydroxyurea affected CTP levels, indicating that inhibition of DNA synthesis by analog incorporation or by depletion of dNTP pools were not the causes of the CTP pool perturbation. Metabolic studies demonstrated that incorporation of [3H]uridine into the UTP pool was not impaired by dFdC treatment, whereas the specific activity of the CTP pools decreased as a function of increasing gemcitabine concentration and time of exposure. Comparable results were obtained using 3-deazauridine, a known inhibitor of CTP synthetase. We conclude that high cellular concentrations of dFdCTP deplete cellular CTP concentrations by inhibition of the dCTP pool and also may be a limiting factor for RNA synthesis.

Degradation of 2-(3-aminopropylamino)-ethanethiol (WR-1065) by Cu-dependent amine oxidases and influence on glutathione status of Chinese hamster ovary cells.

The radioprotective drug 2-(3-aminopropylamino)ethanethiol (WR-1065) can be degraded when incubated in cell culture medium in vitro. The degradation reaction consumes oxygen and results from the action of Cu-dependent amine oxidases present in the serum content of the medium. Analysis of the degradation products of WR-1065 demonstrates the formation of cysteamine, acrolein and H2O2. WR-2721, the inactive prodrug of WR-1065, is not a substrate for these enzymes. Extracellular degradation of WR-1065 by Cu-dependent amine oxidases leads to an intracellular depletion of glutathione (GSH) in Chinese hamster ovary (CHO) cells and to reduction of clonogenic cell survival. Addition of aminoguanidine, an inhibitor of Cu-dependent amine oxidases, protects CHO cells from the toxic effects of WR-1065 and under these conditions an increase of intracellular GSH levels occurs. These data demonstrate that WR-1065 can be degraded to toxic compounds by the presence of Cu-dependent amine oxidases which might have further implications for the clinical use of WR-2721.

Promotion of cystine uptake and its utilization for glutathione biosynthesis induced by cysteamine and N-acetylcysteine.

Chinese hamster ovary (CHO) cells obtain a high capacity to utilize cystine from the growth medium by exposure to cysteamine (2-mercaptoethylamine, MEA) or N-acetylcysteine (NAC). For uptake studies a modified McCoy's 5A medium supplemented with 0.1 mM [35S]cystine was used. The uptake of cystine was dependent on the time of exposure (0-60 min) and the concentrations of MEA or NAC (0-8 mM). At high concentrations of MEA or NAC, the uptake of cystine became saturated. Half-maximal uptake of cysteine was observed at concentrations of 0.12 mM MEA and 0.66 mM NAC, respectively. Increase in temperature (37-44 degrees) or pH (6.0-8.0) during MEA or NAC exposure further increased the cystine uptake. The increased uptake of cystine was not affected in the presence of glutamate or homocysteate which both inhibited the cystine uptake of control cells. Determination of both reduced (GSH) and oxidized (GSSG) cellular glutathione showed a twofold increase in MEA- or NAC-treated CHO cells. DL-buthionine-S,R-sulfoximine (BSO), an inhibitor of GSH biosynthesis completely blocked the promotion of cystine uptake by MEA and NAC. By further analysis using reversed-phase HPLC of cell extracts, more than 90% of the [35S] radioactive cystine taken up by the cells could be recovered within the pool of GSH. The results demonstrate that exposure of CHO cells with MEA and NAC leads to a promoted uptake of cystine from the culture medium and its rapid utilization for cellular GSH biosynthesis.

Definition of extracellular localized epitopes of Hsp70 involved in an NK immune response.

In order to define extracellular localized epitopes of Hsp70 on human tumor cells which are accessible to the immune system, six commercially available Hsp70-specific monoclonal antibodies (mAb) with different recognition sites were examined by immunological approaches. The recognition pattern of these antibodies was analyzed on purified recombinant Hsp70 proteins (rHsp70, Hsc70, DnaK), on lysates of Hsp70-expressing colon carcinoma cells (CX+) and on lysates of M21 rat-1 cells that overexpress human Hsp70 or Hsp70 fragments: ABgl (del 120-428) consisting of the C-terminal part and ASma (del 438-618) consisting of the N-terminal part of human Hsp70. All antibodies reacted equally well with rHsp70 and cytoplasmic Hsp70 derived from human tumor cells or M21 rat-1 cells. Only one antibody (MA3-007; Hsp70, Hsc70) detects a region localized within the ATPase domain of Hsp70 (amino acid 122-264) and reacts positively with the C-terminal deletion mutant ASma. All other antibodies, including RPN1197 are directed against the C-terminal peptide binding domain of Hsp70 and react positively with the N-terminal deletion mutant ABgl. Although all six antibodies detect full-length Hsp70 protein, derived from plasma membrane fractions of CX+ tumor cells, cell surface expressed Hsp70 on viable CX+ tumor cells, as determined by flowcytometry, is only recognized with the antibodies MA3-006 (Hsp70, Hsc70; 504-617), MA3-009 (Hsp70; 504-617) and RPN1197 (Hsp70). An estimation of the ratio of membrane-bound to cytoplasmic Hsp70 molecules revealed that 15-20% of total Hsp70 molecules are expressed on the plasma membrane. This tumor-selective cell surface expression of Hsp70 correlates with an increased sensitivity to lysis mediated by non-MHC restricted natural killer (NK) cells. We demonstrate that only antibodies directed against membrane-bound Hsp70 (MA3-006, MA3-009, RPN1197) inhibit NK-killing activity against Hsp70-expressing tumor cells. Taken together our data indicate that at least the C-terminal region 504-617, that contains at least one single alpha-helix (amino acid 512-536), has to be localized extracellularly and might be of importance for an NK-mediated anti-tumor immune response.

Improvement of local control by regional hyperthermia combined with systemic chemotherapy (ifosfamide plus etoposide) in advanced sarcomas: updated report on 65 patients.

From July 1986 to 1990, 65 patients with deep-seated, advanced sarcomas (43 soft-tissue sarcomas, 12 Ewing's sarcomas, 7 chondrosarcomas and 3 osteosarcomas) were entered in a protocol involving regional hyperthermia (RHT) combined with systemic ifosfamide and etoposide. RHT was produced by an electromagnetic deep regional heating device (BSD Medical Corporation, Salt Lake City, Utah). Of these patients, 62% (40 patients) had received ifosfamide-containing drug regimens before entering the RHT study, 26% (17 patients) were pretreated by surgery and/or radiation and 12% (8 patients) were treated primarily. A total of 426 RHT treatments (mean 6.6 RHT/patient) were applied predominantly within the pelvic region (82%) bearing relative large tumours (mean volume 500 cm3). For systemic chemotherapy, all patients received ifosfamide (1.5 g/m2, days 1-5), etoposide (100 mg/m2, days 1, 3, 5) and 2-mercaptoethanesulphonic acid (mesna; 300 mg/m2 x 4, days 1-5) with RHT only given on days 1 and 5 in repeated cycles every 4 weeks. Detailed thermal mapping by invasive thermometry was performed in all patients. In 61 patients evaluable for tumour control the overall objective response rate including 9 complete responders (CR), 4 partial responders (PR) and 8 patients with favourable histological response (FHR) was 34% (95% confidence limits, 23%-46%). Following CR, the patients are alive and remain disease-free (mean disease-free survival 15.6 months). Of the patients with PR and FHR, 3 died from metastatic and/or local disease after 4, 17, and 39 months, and 1 patient died from other disease (acute myelocytic leukemia) after 27 months. The other 8 patients remain stable at 29, 25, 17, 11, 10, 8, 7, and 6 months. Twenty-two patients revealed no change and 18 patients showed local tumour progression (PD). Side-effects of RHT were tolerable and there was no indication of enhanced bone marrow toxicity due to the addition of RHT to the systemic chemotherapy. By analysis of temperature parameters, the time-averaged temperatures of all RHT treatments calculated for 20% (T20), 50% (T50) or 90% (T90) of measured tumour sites differed significantly between responders (CR + PR + FHR) and non-responders (PD), respectively (T20, P = 0.001; T50, P = 0.0005; T90, P = 0.0001). the data further support a strong potential for ifosfamide plus etoposide combined with RHT in pretreated patients with advanced sarcomas.

Current trials and new aspects in soft tissue sarcoma of adults.

PURPOSE: For high-risk soft tissue sarcoma (HR-STS) of adults new treatment strategies are needed to improve outcome with regard to local control and overall survival. Therefore, systemic chemotherapy has been integrated either after (adjuvant) or before (neoadjuvant) optimal local treatment by surgery and radiotherapy in HR-STS. METHODS AND RESULTS: The Soft Tissue and Bone Sarcoma Group (STBSG) of the European Organization for Research and Treatment of Cancer (EORTC) is conducting an open randomized trial of adjuvant chemotherapy in high-grade primary or recurrent STS at any site (EORTC 62931). In all cases primary surgery should be curative in intent. All eligible patients are randomized after completion of definitive surgery to receive either radiotherapy alone with no further treatment (observation arm) or five cycles of doxorubicin (70 mg/m(2)) plus ifosfamide (5 g/m(2)) using G-CSF to support dose intensity followed by radiotherapy (chemotherapy arm). This more aggressive chemotherapy regimen within an adjuvant setting might retain sufficient antitumor activity to convert response rates into survival benefit. At present more than 220 patients have been recruited for this trial. To explain the rationale for the EORTC 62931 protocol, reported results of other clinical adjuvant protocols including a meta-analysis are given. In close collaboration with the European Society of Hyperthermic Oncology (ESHO) the STBSG has also initiated a randomized trial of neoadjuvant chemotherapy in primary or recurrent HR-STS as an EORTC Intergroup study. According to the inclusion criteria as defined (tumor size >or=5 cm + grade II or III + deep location + extracompartmental extension) for the EORTC 62961/ESHO RHT-95 Intergroup study, the majority of patients with HR-STS recruited for this pre- and postoperative multimodality treatment protocol cannot be cured by standard procedures. All eligible patients are randomized to receive either four cycles of EIA (etoposide 250 mg/m(2) + ifosfamide 6 g/m(2) + doxorubicin 50 mg/m(2)) within 12 weeks (chemotherapy arm) or the same EIA regimen combined with regional hyperthermia (RHT + chemotherapy arm). The patients then receive optimal local treatment using adequate surgery immediately followed by radiotherapy. Thereafter an additional four cycles of EIA chemotherapy are given with or without RHT according to the initial randomization. At present more than 150 patients have been recruited for this trial. The integration of RHT as a new potent treatment modality if combined with EIA chemotherapy as first-line treatment for well-defined risk groups is based upon encouraging long-term results of phase II studies both in pretreated patients with HR-STS and in those with locally advanced disease. CONCLUSIONS: In summary, significant prognostic variables recognized for patients with STS have been addressed in the design of two open phase III clinical trials on adjuvant and neoadjuvant chemotherapy. The best chance for offering such treatment strategies following evidence-based medicine criteria to eligible patients with HR-STS depends upon early contact with the coordinator of the individual protocol prior to any treatment.

Preoperative systemic etoposide/ifosfamide/doxorubicin chemotherapy combined with regional hyperthermia in high-risk sarcoma: a pilot study.

From November 1990 to September 1991, 23 adults with high-risk, nonmetastatic sarcomas (20 soft-tissue sarcomas and 3 chondrosarcomas) were entered in a pilot protocol (RHT-91) involving regional hyperthermia combined with systemic chemotherapy followed by surgery. Of these patients, 12 had undergone previous surgery and/or radiation, 5 had received previous multidrug chemotherapy, and 6 were previously untreated. A tumor size of > 8 cm and/or an extracompartmental tumor location (11 patients) or local recurrence (12 patients) were defined as high-risk factors in addition to tumor grading (21 patients had grade 2 or 3 sarcomas). Regional hyperthermia was produced by an electromagnetic deep-regional-heating device. For systemic chemotherapy, all patients received etoposide/ifosfamide/doxorubicin (EIA) and mesna, with regional hyperthermia being given only on days 1 and 4 in repeated EIA/regional hyperthermia cycles every 3 weeks. Tumor temperatures (range, 40 degrees-44 degrees C) were measured by invasive thermometry in all patients during each regional hyperthermia treatment. A total of 181 regional hyperthermia treatments were applied within the pelvic region (11 patients) or extremities (12 patients) bearing relatively large tumors (mean volume, 848 cm3). By the cutoff date for this analysis (October 15, 1991), 13 patients had undergone surgery after receiving 2-6 (mean, 3.8) cycles of EIA chemotherapy combined with regional hyperthermia; all tumors except one were resected without disfiguration. In 22 evaluable patients (minimum, 2 EIA plus regional hyperthermia cycles), the clinical response rate was 27%, with 6 patients showing partial responses (PRs). In addition, a pathologic response to preoperative thermochemotherapy was evaluable in 13 patients, with 4 responders (31%) having > 50% histologic necrosis. In all, 3 of the responders (1 PR and 2 patients with > 50% histologic necrosis) relapsed within 3 months of surgical resection. The other 7 responding patients (5 PRs and 2 patients with > 50% histologic necrosis) showed stable disease with local tumor control. The study (RHT-91) is continuing as a multicenter phase II trial (opened on November 19, 1991) in patients with high-risk soft-tissue sarcomas to test the potential of preoperative thermochemotherapy in regard to local control and survival.

[Operative therapy of abdominal and retroperitoneal sarcoma]. / Operative Therapie der abdominellen und retroperitonealen Sarkome.

Retroperitoneal soft tissue sarcomas are characterized by a high rate of local recurrence. Complete tumor resection is the only potentially curative therapeutic option. The concept of a systematic compartmental resection is to remove the tumor en bloc with a margin of uninvolved tissue and organs. This is frequently only achieved by multivisceral resection which often includes kidney, colon, pancreas and parts of the diaphragm or the psoas muscle. The adoption of such a policy of multivisceral organ resection improves the proportion of curative resections and, ultimately, results in lower local recurrence rates. The present article comprehensively describes the operative procedures, perioperative treatment and the oncological results of surgery for retroperitoneal sarcomas. The role of surgery in oncological treatment plans and the importance of specialized centres are outlined in detail.