Guidelines for time-to-event end point definitions in sarcomas and gastrointestinal stromal tumors (GIST) trials: results of the DATECAN initiative (Definition for the Assessment of Time-to-event Endpoints in CANcer trials)†.

BACKGROUND: The use of potential surrogate end points for overall survival, such as disease-free survival (DFS) or time-to-treatment failure (TTF) is increasingly common in randomized controlled trials (RCTs) in cancer. However, the definition of time-to-event (TTE) end points is rarely precise and lacks uniformity across trials. End point definition can impact trial results by affecting estimation of treatment effect and statistical power. The DATECAN initiative (Definition for the Assessment of Time-to-event End points in CANcer trials) aims to provide recommendations for definitions of TTE end points. We report guidelines for RCT in sarcomas and gastrointestinal stromal tumors (GIST). METHODS: We first carried out a literature review to identify TTE end points (primary or secondary) reported in publications of RCT. An international multidisciplinary panel of experts proposed recommendations for the definitions of these end points. Recommendations were developed through a validated consensus method formalizing the degree of agreement among experts. RESULTS: Recommended guidelines for the definition of TTE end points commonly used in RCT for sarcomas and GIST are provided for adjuvant and metastatic settings, including DFS, TTF, time to progression and others. CONCLUSION: Use of standardized definitions should facilitate comparison of trials' results, and improve the quality of trial design and reporting. These guidelines could be of particular interest to research scientists involved in the design, conduct, reporting or assessment of RCT such as investigators, statisticians, reviewers, editors or regulatory authorities.

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.

[Combined functional and morphological imaging of sarcomas: significance for diagnostics and therapy monitoring]. / Kombinierte funktionelle und morphologische Bildgebung bei Sarkomen : Stellenwert für Diagnostik und Therapiemonitoring.

(18)F-fluorodeoxyglucose positron-emission tomography (FDG-PET) and especially hybrid FDG-PET/CT is becoming more and more accepted for the clinical management of adult and pediatric patients with sarcomas. By integrating the CT component the specificity in particular but also the sensitivity of the modality are improved further. With PET/CT a complete staging including the detection of lung metastases is feasible in a single examination. For patients with primary bone and soft tissue sarcomas FDG-PET/CT is utilized for diagnosis, staging and restaging, metabolic tumor grading, guidance of biopsies, detection of tumor recurrence and therapy monitoring. Furthermore, it has been demonstrated that FDG uptake of the tumor prior to treatment and changes of FDG uptake after therapy significantly correlate with histopathologic response and survival of patients. Therefore, PET and PET/CT have a prognostic value. In the future new perspectives of hybrid PET/CT imaging will arise by introducing novel radiotracers and combined functional imaging of tumor metabolism and perfusion. High resolution MRI is essential for local evaluation of the primary tumor and preoperative planning with assessment of possible infiltration of vascular or neural structures. Contrast-enhanced MRI remains a key tool in the diagnosis of recurrent disease, especially in tumors which are not hypermetabolic. Dynamic contrast-enhanced MR sequences can significantly contribute to therapy monitoring. More research is necessary to prospectively compare dynamic contrast-enhanced MRI and FDG-PET/CT for evaluation of local and recurrent diseases.

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.

Imatinib does not induce cardiac left ventricular failure in gastrointestinal stromal tumours patients: analysis of EORTC-ISG-AGITG study 62005.

Recent publications have suggested that imatinib (Glivec) may be cardiotoxic. We have therefore assessed the largest study on the agent performed in patients with gastrointestinal stromal tumours, randomising a daily dose of 400mg versus 800 mg. 946 Patients were entered, 942 patients received at least one dose of imatinib. The median time on treatment was 24 months. A total of 24,574 exposure months could be analysed. We could not identify an excess of cardiac events in the study population. In 2 patients (0.2%) a possible cardiotoxic effect of imatinib could not fully be excluded. The current analysis of a large randomised prospective study could not confirm previous suggestions of imatinib induced cardiac toxicity.

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.

A comparison of the effects of ifosfamide vs. mafosfamide treatment on intracellular glutathione levels and immunological functions of immunocompetent lymphocyte subsets.

This study compares the effects of ifosfamide treatment with those of mafosfamide treatment with respect to important immunological functions and intracellular glutathione (GSH) levels of immunocompetent lymphocyte subsets such as cytotoxic T lymphocytes (CTLs) and natural killer (NK) cells. The proliferative and cytotoxic capacity of human peripheral blood lymphocyte (PBL) subsets was measured by a standard [3H] thymidine uptake assay and a [51Cr] release assay; the intracellular glutathione levels were determined by using an established HPLC method described by Reed. Following incubation of human PBLs with the activated forms of ifosfamide (4-OH-IF) and mafosfamide (4-OH-CP), the proliferative capacity of recombinant interleukin 2 (rIL-2)-stimulated PBLs was reduced by both drugs in a dose-dependent manner. However, a three-fold higher concentration of ifosfamide compared with mafosfamide is needed to achieve a comparable inhibition rate in the proliferative capacity of theses lymphocytes. Separation of PBLs into a CD3+ CTL and a CD3- NK subpopulation revealed that proliferative activity was reduced in both subpopulations in a dose-dependent manner by ifosfamide and mafosfamide. However, growth inhibition was much more pronounced in the CD3+ CTL compared with the CD3- NK cells. The intracellular GSH level in CTL, and to a lower extent in NK cells, was reduced more substantially following an ifosfamide treatment compared with a mafosfamide treatment. With respect to our previous finding that an ifosfamide-induced reduction of intracellular GSH levels correlates with decreased cytotoxic function, in this study we compared the effects of ifosfamide treatment with those of mafosfamide treatment on the cytolytic activity of lymphocyte subpopulations. The cytotoxic activity of CD3+ CTL against allogeneic target cells (B-lymphoblastoid cells) was significantly reduced after preincubation with either activated ifosfamide or mafosfamide. In contrast, the lysis of NK-sensitive tumor target cells (K562), mediated by CD3- NK cells is only affected if the effector cells are exposed to high concentrations (100 microM) of activated ifosfamide. The cytotoxic activity of NK cells pretreated with high concentrations of activated mafosfamide (33 microM) had no significant inhibitory effect on the cytotoxic function. Taken together, our findings were as follows: 1) A three-fold higher concentration of activated ifosfamide compared with mafosfamide results in a comparable inhibition of the proliferative activity, in vitro. 2) The intracellular GSH levels of unseparated rIL-2 activated lymphocytes were reduced by ifosfamide and mafosfamide at concentrations above 16 microM. 3) Separated NK cells compared with CTLs are more resistant to treatment with ifosfamide with respect to their intracellular GSH levels. This phenomenon is even more pronounced after treatment with mafosfamide. 4) The reduction in intracellular GSH levels after treatment with ifosfamide and mafosfamide could be correlated with a reduction in the cytotoxic activity.

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.