Jochen Casper

Phase I/II Study of Sequential Dose-Intensified Ifosfamide, Cisplatin, and Etoposide Plus Paclitaxel As Induction Chemotherapy for Poor Prognosis Germ Cell Tumors by the German Testicular Cancer Study Group

PURPOSE To evaluate the feasibility and the toxicity of sequential, dose-intensified chemotherapy combined with paclitaxel plus peripheral blood-derived hematopoietic stem-cell support (PBSC) for patients with untreated metastatic germ cell tumors (GCTs) who have poor International Germ Cell Consensus Cancer Group prognostic features. PATIENTS AND METHODS Paclitaxel was added to high-dose (HD) etoposide, ifosfamide, and cisplatin (VIP; etoposide 1,500 mg/m2, ifosfamide 10,000 mg/m2, and cisplatin 100 mg/m2; cumulative dose; days -6 through -2 per cycle) at three dose levels (135, 175, and 225 mg/m2) applied on day -6. Cycles were supported by PBSC and granulocyte colony-stimulating factor. One cycle of standard VIP was administered before start of HD-VIP plus paclitaxel cycles to collect autologous PBSC. RESULTS Fifty-two of 53 patients receiving 152 cycles were assessable. As expected, myelosuppression was the major adverse effect. Median durations of leukocytes less than 1,000/microL and thrombocytes less than 25,000/microL were 6 and 4 days, respectively, independently of the dose of paclitaxel applied. WHO grade 2 neurotoxicity and grade 3 encephalopathy were observed in 5% of patients each. Other main adverse effects observed were stomatitis, diarrhea, and obstipation. Seventy-nine percent of patients achieved a favorable response to chemotherapy plus secondary surgery. After a median follow-up time of 41 months in surviving patients, the calculated 2- and 5-year survival rates were 77.6% (95% CI, 65.4% to 89.9%) and 75.2% (95% CI, 62.5% to 87.8%), respectively. CONCLUSION Dose-intensive, sequential HD-VIP plus paclitaxel up to a dose of 225 mg/m2 in patients with poor prognosis GCT is a feasible approach. The regimen warrants investigation for its therapeutic potential in an expanded cohort of poor prognosis GCT patients.

Phase II study of bendamustine in patients with relapsed or cisplatin-refractory germ cell cancer.

Despite generally high cure rates in patients with metastatic germ cell cancer, patients with incomplete response to first-line cisplatin-based chemotherapy or with relapsed disease following high-dose salvage therapy exhibit a very poor prognosis. We investigated the efficacy and toxicity of bendamustine, a bifunctional alkylating benzimidol derivative with only partial cross-resistance to other alkylating agents such as ifosfamide or cyclophosphamide. Nineteen patients with cisplatin-refractory germ cell tumors (GCT) or relapse after high-dose chemotherapy plus autologous stem cell support were treated with bendamustine at a dose of 120 mg/m2 on 2 consecutive days at 3 week intervals. Patients had received a median of 9 (range 4-20) platinum-containing treatment cycles prior to bendamustine and 13 patients (68%) had previously received carboplatin/etoposide-based high-dose chemotherapy. One patient achieved a partial remission of only 6 weeks duration. No other responses were seen. Toxicity was low with one patient developing WHO grade 3 thromboyctopenia as the only WHO grade 3/4 toxicity observed. Hematologic toxicity was similar in patients pretreated with and without high-dose chemotherapy plus autologous stem cell support. We conclude that bendamustine has little or no clinically relevant activity in patients with cisplatin-refractory GCT or relapsed disease after high-dose chemotherapy.

Phase II study of carboplatin in untreated inoperable advanced stomach cancer

Fig. 1. Electron micrographs: A = large round endocrine granules with fine wavy limiting membrane, x 20 000; inset shows cross-cut profile of micro6laments (F) (up to 15 nm in diameter, x 22 000). B = neoplastic cell showing irregularly developed microvilli on the luminar surface, numerous endocrine granules and a pool of microfilamentous structures, x 7800. C = endocrine granules showing somatostatin (gold staining), x 13 350.

AMIFOSTINE DOES NOT ALTER THE ANTITUMOR ACTIVITY OF CISPLATIN IN A PRE-CLINICAL MODEL OF TESTICULAR CANCER

Testicular germ cell tumors are so exquisitely sensitive to cisplatin that the majority of patients with this cancer are now cured with modern platinum-based chemotherapy. In contrast to some other tumor types, testicular germ cell tumors are known to express alkaline phosphatases (ALP). Amifostine is an aminothiol pro-drug which is rapidly dephosphorylated by ALP at the cell surface of healthy tissues and which exerts a clinically proven protective effect against chemotherapy associated toxicity. The aim of this pre-clinical study was to assess the potential of amifostine to protect platinum-sensitive non-seminomatous germ cell tumor (NSGCT) nude mouse xenografts established from an ALP-positive embryonal carcinoma (EC) cell line, from the cytotoxicity of cisplatin when both were administered at their individual maximally tolerated doses (MTD). The %T/C values calculated at day 30 for nude mice carrying H12.1 NSGCT xenografts treated with amifostine alone, amifostine plus cisplatin or cisplatin alone were, respectively, 93, 3 or 3%. Mean tumor volumes were not significantly different between mice treated with the combination versus cisplatin alone at day 14 or 30. The results of this study revealed no evidence of tumor protection by amifostine, confirming previous results in other tumor types.

Characterization of rearranged Y chromosomes in human testicular tumor cell lines

Cytogenetic analysis of four cell lines established from two different human testicular tumors revealed rearranged or missing Y chromosomes. Southern blot analysis and in situ hybridization with different Y-derived human DNA sequences revealed the existence of Y chromosomal material even in a line without a cytogenetically visible Y chromosome and clarified the composition of Y marker chromosomes.

Platelet recovery and survival measured in patients by quantitative polymerase chain reaction of mitochondrial DNA.

Mitochondrial (mt) DNA markers have been identified as potential targets for the quantification of endogenous and allogeneic platelets (PLTs) in the blood of individuals who received transfusions. Our goal was to develop a routine polymerase chain reaction (PCR) assay for ex vivo monitoring of PLT survival in patients after transfusion.

Long-term remissions in metastatic malignant melanoma following chemotherapy and tamoxifen maintenance.

It is remarkable that patients 1 and 2 developed progressive disease under tamoxifen maintenance (after 17 and 7 months, respectively), but remained in complete remission following local therapy (exstirpation of a kidney metastasis and an adrenal body in patient 1; irradiation of the lumbar spine, the sacrum and the right humerus in patient 2) and continuation of tamoxifen therapy. Four out of these 6 patients had liver metastases, usually indicating a particularly poor prognosis. 2 of them (patients 1 and 2) have shown a continuous complete remission for 16 and 13 years, respectively. Up to now, to our knowledge, there has been only 1 case published with a continuous complete remission of liver metastases for 10 years (following vindesine and interferon) [1]. As potential action mechanisms of tamoxifen in malignant melanoma, induction of apoptosis and inhibition of angiogenesis [2], inactivation of the insulin-like growth factor-1 receptor [3], or inhibition of tumor cell invasion and metastasis through suppression of the PKC/MEK/ERK and PKC/PI3K/ Akt pathways [4] have been discussed. Tamoxifen monotherapy in metastatic melanoma has been reported to induce remissions in about 5% of the patients, in some cases after progression of the disease in the first months of therapy [5]. In combination with chemotherapy, tamoxifen failed to exert an additional therapeutic effect in most studies [6]. But, to our knowledge, no results are published about tamoxifen maintenance following chemotherapy. Given the good tolerability and low costs of the drug, it would be worthwhile to start a clinical trial with a larger number of patients to investigate the potential of tamoxifen maintenance in metastatic malignant melanoma, if possible.

Digital PCR to assess platelet recovery after stem cell transplantation

Engraftment of platelets in patients after human hematopoietic stem cell (HSC) transplantation serves as an early and relevant sign of the success of the transplantation. The time to platelet engraftment appears to be a valid predictor of the probability of complications in transplanted patients and especially for allogeneic HSC transplantation [1]. At least two different criteria of platelet engraftment, i.e. definitions according to the CIBMTR and EBMT, have been widely applied. Both criteria utilize daily platelet counts to assess the time to a threshold count of more than 20,000/μL platelets in peripheral blood, provided that platelet transfusions have been terminated for either at least three [2] (EBMT) or at least seven [3] (CIBMTR) consecutive days. Here, we describe the development of a digital PCR assay to specifically quantify graft-derived platelets versus transfused single-donor platelets. The performance of this assay was then tested with samples from allogeneic HSC transplant patients. Samples of 2 mL EDTA anticoagulated blood from patients were mixed with an equal volume of 0.9% NaCl and centrifuged at 100×g for 10 min without brake (Hettich Rotina 46S, Hettich, Tuttlingen, Germany). A final centrifugation step at 1569×g for 5 min followed. The pellet was resuspended in 500 μL phosphate buffered saline (PBS, Invitrogen Karlsruhe, Germany). The number of platelets in the PRP was adjusted with PBS to 5 × 10–2.5 × 10 to extract the mt DNA from these platelet suspensions (500 μL). The MagnaPure Large Volume protocol (MagnaPure compact, Roche Diagnostics GmbH, Mannheim, Germany) was used to extract the mt DNA. Elution volume was 50 μL. The PCR strategy was based on generic amplification primers and allele-specific probes. The probes were designed to specifically detect alleles carrying SNPs at positions 73, 195, 295, 310, 16069, 16399 and 16,516 of the non-coding regions HVR 1 and HVR 2 of the mt genome. Droplet generation, amplification and detection of PCR products were performed according to the manufacturer’s instructions (QX200 ddPCR System, Bio-Rad Laboratories GmbH, München, Germany) using 5 μL extracted DNA. Specificity of the assay was evaluated in mt DNA samples pre-typed by Sanger sequencing. Sensitivity was tested by dilution series, described in detail elsewhere [4]. The reproducibility of the background signal (“false positive droplets”) was determined by calculation of mean and standard deviation of n= 50 non-template controls and n= 20 positive controls each for FAMor VIClabelled mt DNA samples. Residual white blood cells and their mt DNA content could interfere with the platelet quantification based on the mt DNA PCR. Samples prepared for mt DNA analysis were, therefore, tested for the presence of genomic DNA as an indicator for residual white blood cells as described before [4]. The absolute count of platelets was calculated from the readout of the droplet digital PCR system (copies/μL) as: concentration (copies/μL)/genome mass (pg) × dilution factor (PRP preparation; PCR assay). The cut-off value for quantification of platelets for all mt SNPs was set to 1000 platelets/μL based on dilution series. The third of three consecutive days with increasing platelet counts was defined as day of engraftment. A digital PCR for SNP detection was developed and validated to monitor the platelet engraftment in patients after allogeneic stem cell transplantation. DNA samples from 30 patients examined in our previous study for quantification of endogenous platelets with real-time PCR were used to validate the digital PCR assay [4]. The quantification of platelets in 150 samples was congruent in * Andrea Doescher doescher@bsd-nstob.de