Detection of germ-cell tumor cells in the peripheral blood by nested reverse transcription-polymerase chain reaction for alpha-fetoprotein-messenger RNA and beta human chorionic gonadotropin-messenger RNA.

By establishing sensitive nested reverse transcription-PCRs for the detection of mRNA of alpha-fetoprotein (AFP) and beta human chorionic gonadotropin (betahCG), we investigated the presence of circulating tumor cells in the peripheral blood of 119 patients with germ-cell tumor. A total of 336 blood samples obtained before and during therapy were examined with regard to clinical applicability. The overall ratio of positive PCR results was 26.5% and was independent of the serum concentration of AFP and hCG/betahCG. No correlation of the positivity for AFP-mRNA to serum AFP level was found. In contrast, positive results in betahCG-PCR were twice as frequent in patients with elevated serum hCG/betahCG levels as in those with normal serum hCG/betahCG levels (P = 0.012). To develop a valid correlation to tumor stage, tumor histology, and serum level of tumor markers, a subgroup of 36 patients was evaluated before definite therapy. The subgroup revealed an overall ratio of 33.3% positive PCR results. The serum level of both of the markers did not correlate with the detection of corresponding mRNA in peripheral blood samples. However, positive betahCG-PCR results were found exclusively in patients with elevated serum hCG/betahCG (6 of 18 versus 0 of 18; P = 0.019). Patients with stage IIC/III germ-cell tumor demonstrated nearly twice the frequency of positive PCR results as patients with stage I tumor [7 (41.2%) of 17 versus 4 (23.5%) of 17] in this subgroup. With regard to histology, positive PCR results were found mostly in embryonal carcinoma.

Thymidylate synthase inhibitors in cancer therapy: direct and indirect inhibitors.

PURPOSE AND METHODS: Although fluoropyrimidines, in particular, fluorouracil (5-FU) and fluorodeoxyuridine (FdUrd), are active alone and in combination with other agents in a variety of human malignancies, therapeutic selectivity, resistance, and efficacy have been a major limitation in cancer therapy. Preclinical and clinical results in advanced and adjuvant colorectal cancers confirmed that the therapeutic efficacy of fluoropyrimidines, with thymidylate synthase (TS) as a primary target, can be improved significantly with leucovorin (LV) modulation. With the recognition that TS is an important therapeutic target, direct and specific inhibitors have been developed and are under intensive preclinical and clinical evaluation, primarily in patients with colorectal cancer, with demonstrable activity. The direct TS inhibitors have been shown to be potent, with a high level of specificity under therapeutic conditions for TS. This includes ZD1694, AG337, and LY231514. To date, although the therapeutic activity of both direct and indirect inhibitors of TS is similar, differences in the magnitude and profile of toxicity have been observed. A phase III comparative evaluation of a direct inhibitor of TS (ZD1694) with an indirect inhibitor (5-FU/LV) has been completed and showed similar activity but reduced toxicity in favor of ZD1694. RESULTS: Recognition that greater than 95% of the injected dose of 5-FU is rapidly inactivated by dihydropyrimidine dehydrogenase (DPD) to therapeutically inactive products, but with toxicity to normal tissues, led to the development of inhibitors of this enzyme with the aim to modify the therapeutic index of 5-FU. Several inhibitors in combination with 5-FU are under preclinical and clinical evaluation, including uracil and 5-chloro-2,4-dihydroxy pyridine, as modulators of 5-FU derived from its prodrug tegafur and 5-ethynyluracil as a modulator of 5-FU. CONCLUSION: In this review, an update of the present status of direct and indirect inhibitors of TS is discussed, as well as the future prospect for new drugs alone and in combination.

Phase I study of a weekly schedule of irinotecan, high-dose leucovorin, and infusional fluorouracil as first-line chemotherapy in patients with advanced colorectal cancer.

PURPOSE: To determine the maximum-tolerated dose (MTD) of a weekly schedule of irinotecan (CPT-11), leucovorin (LV), and a 24-hour infusion of fluorouracil (5-FU24h) as first-line chemotherapy in advanced colorectal cancer and to assess preliminary data on the antitumor activity. PATIENTS AND METHODS: Twenty-six patients with measurable metastatic colorectal cancer were entered onto this phase I study. In the first six dose levels, fixed doses of CPT-11 (80 mg/m2) and LV (500 mg/m2) in combination with escalated doses of 5-FU24h ranging from 1.8 to 2.6 g/m2 were administered on a weekly-times-four (dose levels 1 to 4) or weekly-times-six (dose levels 5 to 6) schedule. The dose of CPT-11 was then increased to 100 mg/m2 (dose level 7). RESULTS: Seventy-nine cycles of 5-FU24h/LV with CPT-11 were administered in an outpatient setting. No dose-limiting toxicities were observed during the first cycle at dose levels 1 to 6, but diarrhea of grade 4 (National Cancer Institute common toxicity criteria) was observed in three patients after multiple treatment cycles. Other nonhematologic and hematologic side effects, specifically alopecia and neutropenia, did not exceed grade 2. With the escalation of CPT-11 to 100 mg/m2 (dose level 7), diarrhea of grade 3 or higher was observed in four of six patients during the first cycle; thus, the MTD was achieved. Sixteen of 25 response-assessable patients (64%; 95% confidence interval, 45% to 83%) achieved an objective response. CONCLUSION: The recommended doses for further studies are CPT-11 80 mg/m2, LV 500 mg/m2, and 5-FU24h 2.6 g/m2 given on a weekly-times-six schedule followed by a 1-week rest period. The addition of CPT-11 to 5-FU24h/LV seems to improve the therapeutic efficacy in terms of tumor response with manageable toxicity.

Irinotecan in the treatment of colorectal cancer: clinical overview.

PURPOSE AND METHODS: For more than three decades, the therapeutic options for patients with advanced colorectal cancer have almost exclusively been based on fluoropyrimidines. With the recognition that topoisomerase-I (TOP-I) is an important therapeutic target in cancer therapy, irinotecan, a semisynthetic TOP-I-interactive camptothecin derivative, has been clinically established in the treatment of colorectal cancer. RESULTS: Irinotecan was investigated as second-line chemotherapy after prior treatment with fluorouracil (FU)-based regimens in two large randomized phase III trials comparing irinotecan with either best supportive care or an infusional FU/leucovorin (LV) regimen. The outcomes of these trials established irinotecan as the standard therapy in the second-line treatment of colorectal cancer. The therapeutic value of irinotecan in the first-line treatment of metastatic colorectal cancer was investigated in two large randomized phase III trials comparing the combination of irinotecan and FU/LV with FU/LV alone. Both trials demonstrated significant superior efficacy for the combination of irinotecan and FU/LV in terms of response rate, median time to disease progression, and median survival time. Consequently, the combination of irinotecan and FU/LV has been approved as first-line chemotherapy for patients with metastatic colorectal cancer and constitutes the reference therapy against which other treatment options must be tested in the future. CONCLUSION: In this review, the clinical rationale and update of the present clinical status of irinotecan in the treatment of colorectal cancer and future prospects of irinotecan-based combinations are discussed.

Gemcitabine in patients with relapsed or cisplatin-refractory testicular cancer.

PURPOSE: Despite generally high cure rates in patients with metastatic testicular germ cell tumors, patients with incomplete response to cisplatin-based first-line therapy or with relapsed disease after high-dose salvage chemotherapy have a very poor prognosis. This phase II study evaluates the use of gemcitabine in patients with intensively pretreated or cisplatin-refractory testicular germ cell cancers. PATIENTS AND METHODS: Thirty-five patients (median age, 33 years) were enrolled; 31 patients were fully assessable. All patients had metastatic nonseminomatous germ cell tumors; eight patients had extragonadal primary tumors. Twenty patients (63%) had lung metastases, and 12 patients (39%) had liver metastases. The median number of prior cisplatin-based chemotherapy cycles was seven; 22 patients (71%) had received high-dose chemotherapy with autologous stem-cell transplantation, and 19 patients (61%) had received treatment with paclitaxel. Seventeen patients (54%) were considered refractory or absolutely refractory to chemotherapy. RESULTS: Six of 31 assessable patients (19%) responded favorably to gemcitabine, 11 patients (35%) displayed no change, and 14 patients (45%) had disease progression. The median time to treatment failure was 4 months (range, 2 to 9+ months), and the median survival was 6 months (range, 2 to 23 months). Patients received a median of six gemcitabine applications. Ten patients (32%) required dose reductions, mainly owing to hematologic toxicity. Grade 3/4 granulocytopenia occurred in four patients (13%) and grade 3/4 thrombocytopenia in seven patients (22%). One case of severe sepsis was observed. CONCLUSION: Gemcitabine displays antitumor activity in intensively pretreated and refractory germ cell tumors. Responses were observed in approximately 20% of patients, including three of 22 patients after previous high-dose chemotherapy and one of four patients with mediastinal tumors. Gemcitabine may be a reasonable palliative option for intensively pretreated patients and should be further investigated to define its role in the risk-adapted treatment strategies for germ cell tumors.

Secondary leukemia following high cumulative doses of etoposide in patients treated for advanced germ cell tumors.

PURPOSE: High cumulative epipodophyllotoxin dosages are reported to be associated with an elevated risk for secondary acute myeloid leukemia (s-AML). This study examined the risk of s-AML following cumulative etoposide doses greater than 2 g/m2 in patients with metastatic germ cell tumors (GCT). PATIENTS AND METHODS: The incidence of s-AML was retrospectively assessed in patients treated within clinical trials between January 1986 and February 1996 at four university centers. All patients received high-dose chemotherapy (HDCT) plus autologous stem-cell support for metastatic GCT, including high cumulative etoposide doses (> 2 g/m2). Minimum patient follow-up was 12 months. Standardized morbidity ratio (SMR) was calculated to estimate the risk associated with high cumulative etoposide doses, as compared with the general population. RESULTS: A total of 302 patients with a median age of 29 years (range, 15 to 55) received a median cumulative etoposide dose of 5 g/m2 (range, 2.4 to 14 g/m2). Four cases of s-AML were observed, which resulted in a cumulative incidence of 1.3% (95% confidence interval [CI], 0.38% to 3.59%) at 52 months of median follow-up (range, 12 to 198). Two cases of secondary myelodysplasia (s-MDS) developed in patients with primary mediastinal GCT. Based on the observed four cases of AML, which are most likely etoposide-related, the risk for developing s-AML (SMR, 160 [95% CI, 43.7 to 411.2]) is significantly increased in comparison to the age-matched general population. CONCLUSION: Due to the low incidence of AML in the general population, the significantly elevated risk for developing s-AML affects only 1.3% of all patients who receive etoposide doses greater than 2 g/m2. HDCT, including etoposide doses greater than 2 g/m2, is associated with an acceptably low incidence of s-AML in patients with advanced GCT.

First-line high-dose chemotherapy compared with standard-dose PEB/VIP chemotherapy in patients with advanced germ cell tumors: A multivariate and matched-pair analysis.

PURPOSE: To compare first-line high-dose chemotherapy (HD-CT) with autologous blood stem-cell transplantation to standard-dose chemotherapy (SD-CT) in male patients with advanced germ cell tumors (GCTs), a matched-pair analysis was performed within a homogenous group of patients classified as having either Indiana advanced disease or a poor prognosis according to International Germ Cell Cancer Consensus Group (IGCCCG) criteria. PATIENTS AND METHODS: A multivariate analysis was performed that included 147 consecutive patients who had received sequential high-dose cisplatin, etoposide, and ifosfamide (VIP) therapy (HD-CT) in a German multicenter trial between 1993 and 1997 and 309 patients who had been treated with standard-dose cisplatin, etoposide, and bleomycin (PEB) or VIP chemotherapy (SD-CT) within two randomized trials at Indiana University between 1984 and 1992. RESULTS: Multivariate analysis demonstrated HD-CT to be significantly superior to SD-CT when adjustments were made for prognostic factors (P =.021). Primary tumor site (mediastinal v retroperitoneal/gonadal, P =.035) and presence of visceral metastases (P =.0004) were shown to be significant prognostic factors for overall survival. On the basis of these factors, as well as on tumor marker levels (good, intermediate, or poor, according to IGCCCG criteria), 146 of 147 HD-CT patients were fully matched to an SD-CT patient. Median follow-up was 21 months (range, 0 to 70 months) for the HD-CT patients and 22 months (range, 0 to 90 months) for the SD-CT patients. Two-year progression-free survival (75% v 59%) and overall survival (82% v 71%) were significantly prolonged in HD-CT patients (P =.0056 and P =.0184, respectively). CONCLUSION: The results indicate that first-line HD-CT in patients with poor-prognosis GCT may result in a significant improvement of progression-free and overall survival as compared with SD-CT. Salvage HD-CT seems not to compensate this survival advantage.

Cisplatin, etoposide, and ifosfamide salvage therapy for refractory or relapsing germ cell carcinoma.

Thirty patients with metastatic progressive germ cell carcinoma who failed to be cured with first-line cisplatin chemotherapy were treated with a salvage regimen consisting of cisplatin 20 mg/m2, etoposide 100 mg/m2, and ifosfamide 1.2 g/m2 (PEI) intravenously (IV), days 1 to 5 every 3 weeks. Ten patients (33%) were tumor-free at the end of therapy. Complete response (CR) was achieved with chemotherapy alone in four patients and with additional surgery in six patients (two necroses, two mature teratomas, two carcinomas). Six patients (20%) had normalization of tumor markers but unresectable residual disease (Rm-), and the remaining 14 patients (46%) failed to respond. Of 10 patients with CR, nine (90%) relapsed again (eight carcinomas, one mature teratoma). The median duration of CR was 3.5 months. The median survival of the whole group was 311 days (range, 110 to 996+). Currently, seven of 30 patients are alive, and five of them are without signs of progressive tumor. The response to prior cisplatin therapy predicted for response and survival after PEI salvage therapy. Of 14 patients with prior CR, eight (57%) achieved a second CR compared with one of 11 (9%) with prior unfavorable response (P = .039). The median survival for patients with prior favorable response was 400 days, compared with 251 days for patients with prior unfavorable response (P less than .001). Myelosuppression was dose-limiting, with leukopenia greater than grade 2 in 84% and thrombocytopenia greater than grade 2 in 51% of all cycles. This three-drug regimen can induce a second CR in one third of patients with relapsed or refractory germ cell carcinoma. Only those patients with prior favorable responses can expect to be cured by this salvage regimen, while patients with prior unfavorable response should be considered for alternative salvage approaches.

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

Different classes of EGFR inhibitors may have different potential to improve local tumour control after fractionated irradiation: a study on C225 in FaDu hSCC.

BACKGROUND AND PURPOSE: Previous experiments reported from this laboratory have shown that simultaneous application of the selective epidermal growth factor receptor tyrosine kinase (EGFR-TK) inhibitor BIBX1382BS during fractionated irradiation significantly prolonged growth delay of FaDu human squamous cell carcinoma but did not improve local tumour control. The present study investigates the effect of the EGFR monoclonal antibody (mAb) C225 on local tumour control of FaDu tumours after combined treatment with single dose and fractionated irradiation to address whether different classes of EGFR inhibitors have different potential to improve the outcome of radiotherapy in the same tumour model. MATERIAL AND METHODS: In unirradiated tumours, C225 was given either once or 4 times i.p. to the nude mice. Irradiation experiments were performed with graded single doses under clamp hypoxic conditions or with 30 fractions in 6 weeks with graded total doses under ambient blood flow. C225 was given 6h before or 6 h before and 2, 5 and 7 days after single dose irradiation. During fractionated irradiation C225 was given once per week. Experimental endpoints were tumour growth delay and local tumour control 120 after end of irradiation. RESULTS: C225 treatment resulted in prolongation of tumour growth delay after drug treatment alone as well as after single dose and fractionated irradiation. TCD50 values were reduced from 56.3 Gy [95% CI 50; 62 Gy] after single dose irradiation alone to 46.0 Gy [41;51] (enhancement ratio [ER]=1.22, P<0.01) after 1 C225 injection and 47.7 Gy [44; 51] after 4 injections of the drug (ER=1.18, P=0.06). After fractionated irradiation, tumour control dose 50% (TCD50) was 73.0 Gy [64; 82] in control tumours and 63.1 Gy [57; 69] after simultaneous C225 treatment, corresponding to an ER of 1.2 (P=0.01). CONCLUSION: Treatment of FaDu hSCC with the anti-EGFR mAb C225 resulted in a significant prolongation of tumour growth delay after single dose and fractionated irradiation. In contrast to previous results on the EGFR-TK inhibitor BIBX1382BS, this prolongation of growth delay translated into a slight but significant improvement of local tumour control. The data indicate that different classes of EGFR inhibitors may have different potential to improve the outcome of radiotherapy in the same tumour model.

Phase I clinical and pharmacokinetic study of titanocene dichloride in adults with advanced solid tumors.

This Phase I dose-escalation clinical trial of a lyophilized formulation of titanocene dichloride (MKT4) was conducted to determine the maximum tolerated dose, the dose-limiting toxicity (DLT), and pharmacokinetics of titanium (Ti) after a single i.v. infusion of MKT4. Forty patients with refractory solid malignancies were treated with a total of 78 courses. Using a modified Fibonacci scheme, 15 mg/m2 initial doses of titanocene dichloride were increased in cohorts of three patients up to level 11 (560 mg/m2) if DLT was not observed. The maximum tolerated dose was 315 mg/m2, and nephrotoxicity was DLT. Two minor responses (bladder carcinoma and non-small cell lung cancer) were observed. The pharmacokinetics of plasma Ti were assessed in 14 treatment courses by atomic absorption spectroscopy. The ratio for the area under the curve(0-infinity) in plasma and whole blood was 1.2. The following pharmacokinetic parameters were determined for plasma, as calculated in a two-compartment model: biological half-life t1/2beta in plasma was 22.8+/-11.2 h (xh +/- pseudo-SD), peak plasma concentration cmax approximately 30 microg/ml at a dose of 420 mg/m2, distribution volume Vss= 5.34+/-2.1 L (xa +/- SD), and a total clearance CItotal = 2.58+/-1.23 ml/min (xa +/- SD). There was a linear correlation between the area under the curve(0-infinity) of Ti in plasma and the titanocene dichloride dose administered with a correlation coefficient r2 of 0.8856. Plasma protein binding of Ti was in the 70-80% range. Between 3% and 16% of the total amount of Ti administered were renally excreted during the first 36 h. The recommended dose for Phase II evaluation is 240 mg/m2 given every 3 weeks with i.v. hydration to reduce renal toxicity.

Clinical phase I dose escalation and pharmacokinetic study of high-dose chemotherapy with treosulfan and autologous peripheral blood stem cell transplantation in patients with advanced malignancies.

A Phase I dose escalation and pharmacokinetic study of the alkylating cytotoxic agent treosulfan was conducted to evaluate the maximum tolerated dose and the dose-limiting toxicities in patients with advanced malignancies rescued by autologous peripheral blood stem cell transplantation. Twenty-two patients (15 ovarian and 7 other carcinomas/lymphomas) with a median age of 48 years were treated with 28 high-dose courses. Treosulfan was infused over 2 h at escalating doses from 20 to 56 g/m2, and pharmacokinetic parameters were analyzed. At 56 g/m2, three of six patients experienced dose-limiting toxicities: diarrhea grade III/IV in three patients; mucositis/stomatitis grade III in one patient; toxic epidermal necrolysis in one patient; and grade III acidosis in one patient. Other low-grade side effects, including erythema, pain, fatigue, and nausea/vomiting, were recorded. Two patients died within 4 weeks after treatment because of rapid tumor progression and fungal infection, respectively. Plasma half-life, distribution volume, and renal elimination of treosulfan were independent of dose, whereas the increase in area under the curve was linear up to 56 g/m2 treosulfan. The maximum tolerated dose of high-dose treosulfan is 47 g/m2. A split-dose or continuous infusion regimen is recommended for future high-dose trials. In consideration of antineoplastic activity and limited organ toxicity, inclusion of high-dose treosulfan in combination protocols with autologous peripheral blood stem cell transplantation seems worthwhile.

Raltitrexed (Tomudex) in combination with 5-fluorouracil for the treatment of patients with advanced colorectal cancer: preliminary results from phase I clinical trials.

The potential of raltitrexed (Tomudex) in combination with 5-fluorouracil (5-FU) as treatment for advanced colorectal cancer has been investigated in two phase I clinical trials. In the first study, raltitrexed was combined with bolus 5-FU; patients received raltitrexed as a 15-min infusion followed 24 h later by bolus 5-FU every 3 weeks. In the second study, 5-FU was administered as a weekly 24-h infusion for 5 weeks of a 6-week cycle and raltitrexed was given 15-min prior to 5-FU on days 8 and 29. The recommended dose for bolus 5-FU in combination with raltitrexed is likely to be 1200 mg/m2 as dose-limiting toxicity (DLT) of febrile neutropenia was observed at 1350 mg/m2, but escalation of raltitrexed above the dose used for single-agent use (3.0 mg/m2) continues. In the raltitrexed/infusional 5-FU study, dose escalation is also still continuing, but only in men as no DLT has been observed in men; 2 of 3 female patients had DLT of myelosuppression and diarrhoea at raltitrexed 3.0 mg/m2 and infusional 5-FU 2400 mg/m2. Raltitrexed had a significant effect on the pharmacokinetics of 5-FU irrespective of 5-FU regimen. Preliminary response data is encouraging with 53% of patients receiving raltitrexed/infusional 5-FU showing a partial response. In addition, significant disease stabilisation was observed in patients receiving raltitrexed combined with bolus 5-FU who had previously failed 5-FU therapy. Recruitment has recently commenced in two studies in which raltitrexed is combined with oral derivatives of 5-FU. In conclusion, preliminary data from these phase I studies indicate that the combination of raltitrexed and 5-FU is well tolerated and has encouraging clinical activity.

Titanocendichloride activity in cisplatin and doxorubicin-resistant human ovarian carcinoma cell lines.

The activity of a new organometallic compound, titanocendichloride, was evaluated in doxorubicin- and cisplatin-resistant human ovarian carcinoma cell lines in vitro. Titanocendichloride showed no cross resistance to doxorubicin in two multidrug resistant sublines of A2780. Furthermore, the cell line A2780 CP3, which is about 20-fold resistant to cisplatin was only 2.5-fold resistant to titanocendichloride, indicating a lack of cross resistance between the two metal compounds. These results were confirmed in vivo where titanocendichloride showed a much stronger inhibitory effect in cisplatin-resistant human ovarian carcinoma xenografts than cisplatin.

Activity of cytostatic drugs in two heterotransplanted human testicular cancer cell lines with different sensitivity to standard agents.

Two established human testicular cancer cell lines were used in a mouse xenograft model to assess the antitumour activity of 15 anticancer agents. Line H 12.1 was highly sensitive to cisplatin, bleomycin and vinblastine, resembling non-pretreated testicular tumours, whereas line H 23.1 showed resistance to cisplatin and vinblastine, comparable to tumours with acquired or intrinsic drug resistance. In line H 12.1 several drugs were highly active, including cyclophosphamide, ifosfamide, nimustine and vincristine; carmustine, vindesine, doxorubicin, epidoxorubicin, pirarubicin, mitoxantrone, carboplatin and iproplatin had only moderate activity. In line H 23.1 only cyclophosphamide, ifosfamide, nimustine, vincristine and bleomycin had antitumour activity. These two cell lines represent a useful model for preclinical evaluation of new agents with presumed activity in testis cancer.

Schedule-dependent antagonism of paclitaxel and cisplatin in human gastric and ovarian carcinoma cell lines in vitro.

Paclitaxel has demonstrated broad clinical activity in a variety of malignancies both alone and in combination with other chemotherapeutic agents. The in vitro cytotoxicity of paclitaxel and cisplatin alone, in combination and in sequence, were evaluated against established human gastric and ovarian carcinoma cell lines using 2-h drug exposure. The combination of cisplatin and paclitaxel was found to be additive or even synergistic when paclitaxel was given 24 h prior to cisplatin as demonstrated by isobologram analysis. However, when both drugs were given simultaneously or when cisplatin was given prior to paclitaxel, a strong antagonistic interaction was observed. This antagonism was evident for up to 72 h after a 2-h exposure to cisplatin. Pretreatment with cisplatin caused no alteration in [3H]paclitaxel uptake in HM2 gastric carcinoma cells, but resulted in decreased intracellular retention of paclitaxel. Since cisplatin treatment led to a reduction in cellular glutathione content in these cells and reduced levels of glutathione have been associated with protection against cytotoxicity of paclitaxel, cells were pretreated with L-buthionine sulfoximine (L-BSO). However, depletion of glutathione had no influence on the activity of paclitaxel. A significant accumulation of cells in S-phase was observed 24 h after cisplatin, which resolved after 48 h and resulted in a pronounced increase of G2M phase. These data demonstrate that the interactions of paclitaxel and cisplatin are highly schedule-dependent and applications of cisplatin simultaneously with or prior to paclitaxel may result in pronounced antagonism. These findings could have implications for the design of further clinical protocols.

Bilateral testicular tumours: prevalence and clinical implications.

In a series of 773 patients with the diagnosis of a testicular germ cell tumour, treated at Hannover University Medical School between 1972 and 1985 and with a median follow-up of 9 years (60-210 months), bilateral testicular tumours occurred in 27 (3.5%) patients. None of 157 patients receiving chemotherapy for metastatic disease of the first tumour developed a metachronous bilateral tumour. Of 24 patients with metachronous tumours 23 had stage I and 1 patient had stage II at the time of initial diagnosis. The second testicular tumour was stage I in 18 patients, stage II in 5 and stage IV in 1 patient. 3 patients (13%) relapsed after treatment for their second germ cell tumour (surveillance 13 patients, radiotherapy 7 patients, lymph node dissection 2 patients and chemotherapy 2 patients), 1 of which died after refusing further treatment. The cure rate was 96% in patients with bilateral disease. Routine biopsy of the contralateral testis to identify existing carcinoma in situ (CIS) is recommended. Patients with CIS must be informed about their increased risk of a second testicular tumour. Irradiation of CIS or close clinical follow-up might both constitute appropriate strategies for the management of these patients.

Phase I study of a weekly 1 h infusion of paclitaxel in patients with unresectable hepatocellular carcinoma.

The majority of patients with hepatocellular carcinoma will develop either unresectable or metastatic disease and, therefore, are candidates for systemic chemotherapy. Only a few chemotherapeutic agents have shown documented activity in the treatment of advanced hepatocellular carcinoma and there is clearly a need for the evaluation of new active drugs. Therefore, we performed a phase I trial with a weekly schedule of paclitaxel in patients with advanced hepatocellular carcinoma. 16 patients with documented progression of unresectable hepatocellular carcinoma were included. After premedication, paclitaxel was given as a 1 h infusion on days 1, 8, 15, 22, 29 and 36 representing one treatment cycle. The cycle was repeated every 50 days. The starting dose was 70 mg/m2 and the doses were escalated in steps of 10 mg/m2/week. A minimum of 3 patients were treated at each dose level. All treatment was given on an out-patient basis. Dose-limiting toxicity was reached at a dose of 100 mg/m2/week with 2 of 6 patients treated at that dose level having WHO grade 4 neutropenia. Other toxic side-effects were only mild. 1 partial response and 9 cases with disease stabilisation were observed in 16 patients with initially progressive disease. We, therefore, conclude that the recommended dose for a further phase II trial in patients with hepatocellular carcinoma is 90 mg/m2/week. These data indicate that paclitaxel given at this dose and schedule might have activity in hepatocellular carcinoma and further investigation in phase II trials is warranted.

A phase I/II study of a stepwise dose-escalated regimen of cisplatin, etoposide and ifosfamide plus granulocyte-macrophage colony-stimulating factor (GM-CSF) in patients with advanced germ cell tumours.

In order to improve the survival of patients with metastatic advanced disease germ cell tumours (according to Indiana University classification), 77 patients were treated by a stepwise dose-escalated combination regimen of platinum (P), etoposide (E) and ifosfamide (I) (PEI) followed by application of granulocyte-macrophage colony-stimulating factor (GM-CSF) (10 micrograms/kg subcutaneously per day at levels 2 and 3) starting the first day after chemotherapy for 10 consecutive days. The maximally tolerated dose was reached at the third dose level with P 30 mg/m2, E 200 mg/m2 and I 1.6 g/m2, all given for 5 days, once every 21 days, for a total of four cycles. Sixty-seven per cent of patients had three or more metastatic sites. Twenty-two per cent of patients had extragonadal primary tumours. 49 (65%) patients achieved complete remission, and 9 additional patients (12%) achieved marker normalisation with unresectable residual disease. After a median follow-up of 27 months, the overall survival is 80%, with 67% of patients remaining free from progression. The dose-limiting toxicities were WHO grades 3/4 mucositis/enteritis in 33% of patients and prolonged thrombocytopenia < 20.000/microliters (> 10 days). Adverse reactions to GM-CSF occurred in 13% of patients. The use of a single haematopoietic growth factor allowed only a moderate increase in dose intensity (factor 1.37). Peripheral blood stem cells will be additionally incorporated into the treatment protocol in order to deliver multiple cycles of an upfront dose-intensified PEI regimen in patients with "poor risk" germ cell tumours with less toxicity.

Deficient repair of cisplatin-DNA adducts identified in human testicular teratoma cell lines established from tumours from untreated patients.

Germ cell tumour lines appear generally more sensitive in vitro to cisplatin than other cultured cell lines, reflecting their clinical responsiveness. We proposed (Cancer Res 1988, 48, 3019-3024) that cisplatin hypersensitivity, expressed by a testicular teratoma line (SuSa), might be explained by an inability to repair platinated DNA. We have now quantitated cisplatin cytotoxicity by clonogenic assay, and platinum (Pt)-DNA adduct formation and removal immunochemically in four other testicular teratoma continuous cell lines (GCT46, GCT27 clone 4, H32 and H12.1), all established from tissue from non-drug-treated patients. For 1-h in vitro drug exposures, the cisplatin concentration required to reduce survival by 50% (IC50) ranged from 0.09 to 0.42 micrograms/ml (0.3-1.4 microM). Immediately following a 1-h exposure to 5 mu/ml cisplatin, total cellular platination levels ranged from 4.5 to 36.8 fmol Pt per microgram DNA, with lower platination occurring in the most sensitive lines. Following an 18-h post-treatment incubation period, the levels of the major cis-Pt-(NH3)2d(pGpG) (Pt-GG) adducts were not significantly reduced in any of the four lines, indicating a general deficiency in either the rate or extent of removal of these lesions. Deficient removal of the cis-Pt-(NH3)2d(pApG) adducts was also noted in two of the lines. DNA polymerase beta gene expression was comparable in all the tested testicular lines established from previously untreated patients, but markedly lower than that identified in the 833K testicular line, established from a drug-treated patient and identified earlier as proficient in Pt-GG adduct removal (Cancer Res 1988, 48, 3019-3024). Expression of the DNA excision repair genes ERCC-1 and XPBC/ERCC-3 was not significantly different in any of the five lines tested, including the 833K cell line. These data provide evidence of the apparent inability of testicular cell lines, derived from untreated tumours, to repair the major platinum-DNA intrastrand crosslinks, and so provide a biological basis for their hypersensitivity to cisplatin.