Clinical validity of new genetic biomarkers of irinotecan neutropenia: an independent replication study.

The overall goal of this study was to provide evidence for the clinical validity of nine genetic variants in five genes previously associated with irinotecan neutropenia and pharmacokinetics. Variants associated with absolute neutrophil count (ANC) nadir and/or irinotecan pharmacokinetics in a discovery cohort of cancer patients were genotyped in an independent replication cohort of 108 cancer patients. Patients received single-agent irinotecan every 3 weeks. For ANC nadir, we replicated UGT1A1*28, UGT1A1*93 and SLCO1B1*1b in univariate analyses. For irinotecan area under the concentration-time curve (AUC0-24), we replicated ABCC2 -24C>T; however, ABCC2 -24C>T only predicted a small fraction of the variance. For SN-38 AUC0-24 and the glucuronidation ratio, we replicated UGT1A1*28 and UGT1A1*93. In addition to UGT1A1*28, this study independently validated UGT1A1*93 and SLCO1B1*1b as new predictors of irinotecan neutropenia. Further demonstration of their clinical utility will optimize irinotecan therapy in cancer patients.

Translocation and rearrangement of myeloperoxidase gene in acute promyelocytic leukemia.

Acute promyelocytic leukemia (subtype M3) is characterized by malignant promyelocytes exhibiting an abundance of abnormally large or aberrant primary granules. Myeloperoxidase (MPO) activity of these azurophilic granules, as assessed by cytochemical staining, is unusually intense. In addition, M3 is universally associated with a chromosomal translocation, t(15;17)(q22;q11.2). In this report, the MPO gene was localized to human chromosome 17 (q12-q21), the region of the breakpoint on chromosome 17 in the t(15;17), by somatic cell hybrid analysis and in situ chromosomal hybridization. By means of MPO complementary DNA clones for in situ hybridization and Southern blot analysis, the effect of this specific translocation on the MPO gene was examined. In all cases of M3 examined, MPO is translocated to chromosome 15. Genomic blot analyses indicate rearrangement of MPO in leukemia cells of two of four cases examined. These findings suggest that MPO may be pivotal in the pathogenesis of acute promyelocytic leukemia.

4-Hydroperoxycyclophosphamide purging of breast cancer from the mononuclear cell fraction of bone marrow in patients receiving high-dose chemotherapy and autologous marrow support: a phase I trial.

We designed an ex vivo bone marrow treatment for breast cancer patients receiving high-dose chemotherapy and autologous bone marrow support (ABMS), using 4-hydroperoxycyclophosphamide (4-HC), an active derivative of cyclophosphamide with known activity against breast cancer. This phase I bone marrow purging trial used ficoll-separated mononuclear cells (MNC) (devoid of granulocytes and RBCs), as opposed to the buffy coat. Twenty-five patients with metastatic breast cancer were studied. Patients received three cycles of the Adriamycin (doxorubicin; Adria Laboratories, Columbus, OH), fluorouracil, and methotrexate (Duke AFM) regimen, followed by marrow harvest. An MNC fraction of marrow was prepared and treated with 4-HC in concentrations of 20 micrograms/mL (four patients), 40 micrograms/mL (four patients), 60 micrograms/mL (nine patients), or 80 micrograms/mL (eight patients) and cryopreserved. Patients then received high-dose systemic cyclophosphamide, cisplatin, and carmustine, followed by infusion of the purged marrow. The study end point was marrow engraftment, defined as WBC count greater than 1,000 cells per microliter. At the first three dose levels (20, 40, and 60 micrograms/mL 4-HC), there was no significant delay in time to engraftment (19, 20, and 23 days, respectively) compared with the unpurged historical controls (17 days). At 80 micrograms/mL, engraftment was significantly delayed compared with the lower concentrations (P = .027), and further escalation of 4-HC was not attempted. A significant correlation was observed between the time of leukocyte engraftment and the 4-HC concentration (P = .017). With a methylcellulose-based tissue culture assay, we demonstrated a statistically significant correlation between the colony-forming unit-granulocyte-macrophage (CFU-GM) content in the purged marrow and the days to engraftment. Ninety-five percent of patients responded clinically to the entire program, 55% of them completely. Longer follow-up is required to assess the ultimate benefit of intensive therapy on long-term survival.

Phase I study of topotecan and cisplatin in patients with advanced solid tumors: a cancer and leukemia group B study.

PURPOSE: The objectives of this phase I trial were to determine the dose-limiting toxicities (DLTs) of the novel topoisomerase I inhibitor topotecan combined with cisplatin, to define the maximum-tolerated doses (MTDs) of the combination without and with the use of filgrastim, and to define recommended doses for phase II trials. PATIENTS AND METHODS: Patients with advanced solid tumors were eligible if they had normal bone marrow, renal, and hepatic function and had not previously been treated with platinum compounds. Topotecan was administered intravenously on days 1 through 5 and cisplatin was administered intravenously on day 1 of a 21-day cycle. The topotecan dose was fixed at 1.0 mg/m2/d on the first four dose levels, and cisplatin was escalated in 25-mg/m2 increments from 25 to 100 mg/m2 without filgrastim. After encountering DLT, the dose of cisplatin was decreased by one level and topotecan dose escalation was attempted. After defining the MTD without growth factor, the study proceeded with escalating cisplatin doses to define the MTD with filgrastim 5 micrograms/kg subcutaneously (SC) daily starting on day 6 of treatment. Priming with filgrastim 5 micrograms/kg SC on days -6 to -2 before the first course was explored last. RESULTS: Of 38 patients entered, 37 were eligible, 35 assessable for toxicity in the first course, and 28 assessable for response. The principal toxicity was grade 4 neutropenia, which had to last more than 7 days to be considered dose-limiting. No DLT was observed at the starting cisplatin dose of 25 mg/m2 (dose level 1). On level 2 (cisplatin 50 mg/m2, one patient had dose-limiting neutropenia and one patient had grade 3 renal toxicity. On level 3 (cisplatin 75 mg/m2), two patients had dose-limiting neutropenia. Therefore, cisplatin dose escalation was stopped. On dose level 5 (cisplatin 50 mg/m2 and topotecan 1.25 mg/m2/d), one patient had grade 4 neutropenia that lasted more than 7 days and one patient died of neutropenic sepsis. The remaining dose levels used topotecan 1.0 mg/m2/d plus cisplatin 75 mg/m2 (level 6) and 100 mg/m2 (levels 7 and 8) with filgrastim. No DLT was observed on level 6. On level 7, two patients had dose-limiting neutropenia and one patient had grade 3 hyperbilirubinemia. Priming with filgrastim on level 8 demonstrated no obvious advantage over level 7, and one patient had grade 4 thrombocytopenia that lasted more than 7 days. Three patients with non-small-cell lung cancer achieved a partial response and one patient with breast cancer had a complete response. CONCLUSION: Topotecan and cisplatin in combination cause more neutropenia than expected from either drug given alone at the same dosage. The recommended phase II doses are topotecan 1.0 mg/m2/d for 5 days in combination with cisplatin 50 mg/m2 on day 1 without filgrastim or cisplatin 75 mg/m2 on day 1 with filgrastim support.

Relationship between toxicity and obesity in women receiving adjuvant chemotherapy for breast cancer: results from cancer and leukemia group B study 8541.

PURPOSE: We examined data from a large clinical trial to determine if chemotherapy dosing according to actual body weight places obese patients at greater risk of toxicity. PATIENTS AND METHODS: Cancer and Leukemia Group B (CALGB) study 8541, a randomized study of schedule and dose of adjuvant cyclophosphamide, doxorubicin, and fluorouracil (CAF) for stage II breast cancer patients with positive regional lymph nodes, provided data on 1,435 women for analysis. Using body-mass index (BMI), we classified a woman as obese if her BMI was > or = 27.3 kg/m2; doses were considered weight-based if within 5% of values calculated using actual weight. Our primary analysis concerned toxicity risks during cycle 1. RESULTS: Among women who received weight-based doses of the most dose-intensive CAF regimen, 47% of obese and 51% of nonobese women experienced severe hematologic toxicity (grade > or = 3) during cycle 1 (P = .51, two-tailed). The overall risk ratio (obese v nonobese) of treatment failure among women who received weight-based doses during cycle 1 was 1.02 (95% confidence interval, 0.83 to 1.26), stratified by treatment and adjusted for number of positive nodes, menopausal status, hormone receptor status, and tumor size. The overall adjusted failure risk ratio (weight-based v reduced initial doses) was 0.73 (95% confidence interval, 0.53 to 1.00) among obese women. CONCLUSION: Obese patients initially dosed (within 5%) by actual weight did not experience excess cycle 1 toxicity or worse outcome compared with nonobese women dosed similarly. The data suggest that obese women who received reduced doses in cycle 1 experienced worse failure-free survival. We recommend that initial doses of CAF be computed according to actual body weight.

Evaluation of the combination of nelarabine and fludarabine in leukemias: clinical response, pharmacokinetics, and pharmacodynamics in leukemia cells.

PURPOSE: A pilot protocol was designed to evaluate the efficacy of fludarabine with nelarabine (the prodrug of arabinosylguanine [ara-G]) in patients with hematologic malignancies. The cellular pharmacokinetics was investigated to seek a relationship between response and accumulation of ara-G triphosphate (ara-GTP) in circulating leukemia cells and to evaluate biochemical modulation of cellular ara-GTP metabolism by fludarabine triphosphate. PATIENTS AND METHODS: Nine of the 13 total patients had indolent leukemias, including six whose disease failed prior fludarabine therapy. Two patients had T-acute lymphoblastic leukemia, one had chronic myelogenous leukemia, and one had mycosis fungoides. Nelarabine (1.2 g/m(2)) was infused on days 1, 3, and 5. On days 3 and 5, fludarabine (30 mg/m(2)) was administered 4 hours before the nelarabine infusion. Plasma and cellular pharmacokinetic measurements were conducted during the first 5 days. RESULTS: Seven patients had a partial or complete response, six of whom had indolent leukemias. The disease in four responders had failed prior fludarabine therapy. The median peak intracellular concentrations of ara-GTP were significantly different (P =.001) in responders (890 micromol/L, n = 6) and nonresponders (30 micromol/L, n = 6). Also, there was a direct relationship between the peak fludarabine triphosphate and ara-GTP in each patient (r = 0.85). The cellular elimination of ara-GTP was slow (median, 35 hours; range, 18 to > 48 hours). The ratio of ara-GTP to its normal counterpart, deoxyguanosine triphosphate, was higher in each patient (median, 42; range, 14 to 1,092) than that of fludarabine triphosphate to its normal counterpart, deoxyadenosine triphosphate (median, 2.2; range, 0.2 to 27). CONCLUSION: Fludarabine plus nelarabine is an effective, well-tolerated regimen against leukemias. Clinical responses suggest the need for further exploration of nelarabine against fludarabine-refractory diseases. Determination of ara-GTP levels in the target tumor population may provide a prognostic test for the activity of nelarabine.

Phase I and pharmacologic study of continuous infusion topotecan in combination with cisplatin in patients with advanced cancer: a Cancer and LeukemiaGroup B study.

PURPOSE: Preclinical and clinical data suggest that topotecan may be more effective, and perhaps less toxic, when administered as a continuous intravenous infusion (CIVI). A previous Cancer and Leukemia Group B (CALGB) trial of topotecan, given on a daily bolus schedule in combination with cisplatin, produced more hematologic toxicity than expected with either drug alone. Therefore, we designed this phase I trial to define the dose-limiting toxicities (DLTs) and the recommended phase II doses of cisplatin in combination with topotecan administered as a CIVI. Population pharmacodynamic models for the combination also were investigated. PATIENTS AND METHODS: Patients with advanced solid tumors and a maximum of one prior chemotherapy regimen for metastatic disease were eligible if they had a performance status of 0 to 1 and adequate renal, hepatic, and bone marrow function. Prior treatment with camptothecins or platinum compounds and prior pelvic irradiation were not allowed. The initial schedule consisted of a fixed dose of topotecan 0.4 mg/m2/d administered as a CIVI for 21 days and escalating doses of cisplatin administered on days 1, 8, and 15 of a 28-day schedule, until the maximum tolerated dose (MTD) was achieved. After severe hematologic toxicity was observed in the first two patients, the topotecan infusion was shortened to 14 days, and the total dose of cisplatin was administered on day 1 in all subsequent patients. After the MTD was defined, that cohort was expanded to include a total of 12 assessable patients. Hematopoietic growth factors were not allowed. For the pharmacologic studies, total topotecan plasma concentrations were measured by high-pressure liquid chromatography (HPLC) during infusion on days 3, 8, and 11 on the first cycle, and the median steady-state concentration (Tss) was determined. Platinum plasma concentrations on day 3 were measured by atomic absorption spectrometry. RESULTS: Of the 32 patients enrolled, 28 were assessable for toxicity and 24 for response. The primary toxicity was hematologic, with both neutropenia and thrombocytopenia being dose-limiting. The MTD of cisplatin was 75 mg/m2 on day 1 in combination with topotecan 0.4 mg/m2/d for 14 days. At this dose level, three of a total of 12 assessable patients had DLT. The pharmacodynamic relationship between Tss and the absolute neutrophil count at the nadir (ANCn) was described by the following equation: log10 (ANCn)=4.23 - 0.47 x Tss - 0.01 x cisplatin dose (P < .0001; R2=0.64). The substitution of platinum concentration for cisplatin dose in this model did not result in a significant improvement. Three patients had a partial response: one with duodenal carcinoma; a second with small-cell lung cancer; and a third with melanoma. CONCLUSION: Cisplatin can be given safely in combination with CIVI topotecan. However, toxicity was still substantial. Based on the current results and our previous trial of this combination, we conclude that, when combined with cisplatin, CIVI topotecan does not seem to be advantageous compared with the more traditional daily bolus schedule.

Phase I and pharmacokinetic trial of gemcitabine in patients with hepatic or renal dysfunction: Cancer and Leukemia Group B 9565.

PURPOSE: To ascertain if hepatic or renal dysfunction leads to increased toxicity at a given dose of gemcitabine and to characterize the pharmacokinetics of gemcitabine and its major metabolite in patients with such dysfunction. PATIENTS AND METHODS: Adults with tumors appropriate for gemcitabine therapy and who had abnormal liver or renal function tests were eligible. Patients were assigned to one of three treatment cohorts: I-AST level less than or equal to two times normal and bilirubin level less than 1.6 mg/dL; II-bilirubin level 1.6 to 7.0 mg/dL; and III-creatinine level 1.6 to 5.0 mg/dL with normal liver function. Doses were explored in at least three patients within each cohort. Gemcitabine and its metabolite were to be measured in the blood in all patients. RESULTS: Forty patients were assessable for toxicity. Transient transaminase elevations were observed in many patients but were not dose limiting. Patients with AST elevations tolerated gemcitabine without increased toxicity, but patients with elevated bilirubin levels had significant deterioration in liver function after gemcitabine therapy. Patients with elevated creatinine levels had significant toxicity even at reduced doses of gemcitabine, including two instances of severe skin toxicity. There were no apparent pharmacokinetic differences among the three groups or compared with historical controls. CONCLUSION: If gemcitabine is used for patients with elevations in AST level, no dose reduction is necessary. Patients with elevated bilirubin levels have an increased risk of hepatic toxicity, and a dose reduction is recommended. Patients with elevated creatinine levels seem to have increased sensitivity to gemcitabine, but the data are not adequate to support a specific dosing recommendation.

Phase I study of paclitaxel and topotecan in patients with advanced tumors: a cancer and leukemia group B study.

PURPOSE: To define the dose-limiting toxicities (DLTs) and the recommended phase II doses of paclitaxel combined with topotecan, without and with filgrastim support. PATIENTS AND METHODS: Patients with advanced solid tumors and a maximum of one prior chemotherapy regimen for metastatic disease were eligible if they had a performance status of 0 to 1 and normal renal, hepatic, and bone marrow function. Prior treatment with taxanes or comptothecin analogs, and prior pelvic irradiation were not allowed. Patients with a history of cardiac disease or on medications known to affect cardiac conduction were excluded. The dose of topotecan was fixed at 1.0 mg/m2/d for 5 days. The dose of paclitaxel was escalated until the maximum-tolerated dose (MTD), without and with filgrastim 5 micrograms/kg subcutaneously (SC) on days 6 to 14, was reached. Paclitaxel was administered over 3 hours on day 1 before topotecan. Treatment cycles were repeated every 21 days. RESULTS: Of 46 patients entered, 45 were assessable for toxicity and 34 for response. The principal toxicity was neutropenia. Without filgrastim, the MTD of paclitaxel was 80 mg/m2 on day 1 in combination with topotecan 1.0 mg/m2/d for 5 days. With filgrastim, the dose of paclitaxel was escalated to 230 mg/m2 in combination with the same dose of topotecan. At this dose level, one patient had hematologic DLT and a second patient developed neuromuscular DLT. Three patients had a partial response (PR): one with head and neck cancer, a second with non-small-cell lung cancer, and the third with colon cancer. CONCLUSION: We conclude that paclitaxel can be given at clinically relevant doses in combination with topotecan and filgrastim. The recommended dose for phase II studies is paclitaxel 230 mg/m2 on day 1 and topotecan 1.0 mg/m2/day for 5 days with filgrastim 5 micrograms/kg on days 6 to 14.

Phase I and pharmacokinetic trial of paclitaxel in patients with hepatic dysfunction: Cancer and Leukemia Group B 9264.

PURPOSE: To characterize the maximum-tolerated dose, dose-limiting toxicities (DLTs), and pharmacokinetics of paclitaxel in patients with abnormal liver function. PATIENTS AND METHODS: Adults with tumors appropriate for paclitaxel therapy who had abnormal liver function tests were eligible. Patients were assigned to one of three treatment cohorts: I, AST level twofold normal and bilirubin level less than 1.5 mg/dL; II, bilirubin level 1.6 to 3.0 mg/dL; and III, bilirubin level greater than 3.0 mg/dL. Doses were explored in at least three patients within each cohort. Although designed to assess a 24-hour infusion schedule, the trial was extended to also assess a 3-hour regimen. Pharmacokinetics were to be studied in all patients. RESULTS: Eighty-one patients were assessable for toxicity. Patients with bilirubin levels greater than 1.5 mg/dL had substantial toxicity at all doses explored, whereas the toxicity for patients with elevated AST levels occurred at doses that ranged from 50 to 175 mg/m2 administered over 24 hours. In most patients, the DLT was myelosuppression. The pharmacokinetic data were insufficient to adequately evaluate the relationship between pharmacokinetics and toxicity in patients who received 24-hour infusions but provided evidence of a longer exposure to paclitaxel than anticipated for the doses used in this study in the 3-hour infusion group. CONCLUSION: If paclitaxel is used for patients with elevated levels of AST or bilirubin, dose reductions are necessary, and an increase in toxicity can be anticipated. The increased myelosuppression observed is at least partially because of altered paclitaxel pharmacokinetics in such patients.

Modulation of vinblastine resistance in metastatic renal cell carcinoma with cyclosporine A or tamoxifen: a cancer and leukemia group B study.

Multidrug resistance mediated by P-glycoprotein may be an important cause of chemotherapy failure. Renal cell carcinoma is a disease known to demonstrate a high degree of intrinsic chemotherapy drug resistance, and this has been shown to be related to intrinsic overexpression of P-glycoprotein. Cyclosporine A and tamoxifen have been shown to reverse multidrug resistance in renal cell carcinoma cell lines in vitro. Phase I studies have defined appropriate doses of cyclosporine A and tamoxifen that can be combined with continuous-infusion vinblastine and safely achieve serum levels associated in vitro with resistance reversal. A randomized Phase II study was carried out by the Cancer and Leukemia Group B to evaluate the potential of high doses of cyclosporine A or tamoxifen to modulate clinical vinblastine resistance in patients with advanced renal cell carcinoma. Patients were treated initially with continuous-infusion vinblastine alone (1.2 mg/m2/day for 4 days or 1.5 mg/m2/day for 5 days); patients with stable or progressive disease were then treated with the same vinblastine regimen, combined with a high-dose regimen of either cyclosporine A (12.5 mg/kg/day for 5 days) or tamoxifen (400 mg/m2 as a loading dose and 300 mg/m2/day for 13 days). Sixty-three patients were randomized to each arm. Eighty patients on both arms were evaluable for response to vinblastine alone; of these, only one patient achieved a partial response. Thirty-three patients went on to be treated with vinblastine and high-dose cyclosporine A. No responses were observed, although four patients with progressive disease on prior vinblastine achieved stabilization of disease after cyclosporine A was added. Addition of cyclosporine resulted in more leukopenia (5% versus 25%) and in transient hyperbilirubinemia (24%) and neurocortical changes (11%). No significant azotemia was observed. Thirty-five patients received high-dose tamoxifen with continuous-infusion vinblastine. One complete remission was seen in a patient who had stable disease only with prior vinblastine alone; no other responses were observed. Leukopenia was not more severe with the addition of tamoxifen to vinblastine, nor was hyperbilirubinemia observed. However, 9% of patients developed transient ataxia with or without neurocortical changes as a result of high-dose tamoxifen therapy, and 11% developed phlebitis. We conclude that advanced renal cell carcinoma is a highly chemoresistant tumor, that continuous-infusion vinblastine has no appreciable activity in the therapy of this disease, and that addition of high doses of cyclosporine A or tamoxifen was not able to modulate this resistance in these patients. Suggestions regarding study design for future drug resistance modulation trials were made based on the design and conduct of this study.

Pharmacology of 21-day oral etoposide given in combination with i.v. cisplatin in patients with extensive-stage small cell lung cancer: a cancer and leukemia group B study (CALGB 9062).

This was a pharmacological companion study to a randomized Phase III trial comparing 21-day oral versus 3-day i.v. etoposide in combination with i.v. cisplatin in patients with extensive-stage small cell lung cancer. Etoposide plasma concentrations were measured in patients randomized to the 21-day schedule and correlated with toxicity and tumor response. Patients were treated with etoposide (50 mg/m2/day) orally for 21 days and cisplatin (33 mg/m2/day) i.v. for 3 consecutive days every 28 days for 6 courses. Plasma samples before the daily etoposide dose (trough concentrations) and complete blood counts were obtained weekly during treatment. The average of three etoposide concentrations (EC) per course was calculated. Of 158 patients registered to this schedule of the study, 150 were eligible. In 106 patients, etoposide samples were obtained at least in the first course in which the mean EC was 0.39 microgram/ml (SD = 0.29). In 102 patients (missing albumin values in 4 of 106 patients), the concentration of etoposide not bound to protein (Efree) was estimated based on the following equation: percentage unbound = (1.4 x total bilirubin) - (6.8 x albumin) + 34.4. Regression analysis revealed that increasing age was correlated with higher EC (r = 0.27; two-tailed P < 0.01) and Efree (r = 0.31; two-tailed P < 0.01). Higher EC and Efree values were associated with lower WBC counts and absolute neutrophil counts after the first treatment course in 83 patients with nadir counts. Using multiple linear regression, a pharmacodynamic model was developed that included EC or Efree, age, and alkaline phosphatase. An interaction with bone marrow results at diagnosis was found, indicating a sharper decline in nadir counts with increasing EC or Efree when the marrow was involved with small cell lung cancer. This model explained 29% of the variation for WBC nadirs (P < 0.001) and 31% of the variation for absolute neutrophil count nadirs (P < 0. 001). Neither EC nor Efree showed a significant correlation with tumor response. A pharmacokinetic relationship between EC or Efree and age was found. A pharmacodynamic model could be developed for toxicity but not for tumor response.

Patterns and variability of tumor oxygenation in human soft tissue sarcomas, cervical carcinomas, and lymph node metastases.

PURPOSE: The validity of tumor pO2 measurement as a predictive outcome assay depends upon demonstrating that intrapatient pO2 variation is less than interpatient variation. No consensus exists regarding the appropriate distance between individual measurements. This distance could affect the calculation of the hypoxic fraction (% pO2s < 5 mm Hg) and the assessment of intra/interpatient heterogeneity. This study was performed to evaluate tumor oxygenation and to assess the effects of two different measurement intervals on pO2 heterogeneity in three different sets of patients. MATERIALS AND METHODS: Fifteen patients with soft tissue sarcoma, nine patients with cervical carcinoma, and eight patients with squamous carcinoma metastatic to lymph nodes underwent pretreatment polarographic pO2 measurements. Two grossly distinct sites were studied in each tumor, and 2-3 linear tracks were measured at each site. Track lengths varied from 20-36 mm. Distance between measured points was either 0.7-0.8 mm or 0.4 mm. Mean pO2, median pO2, and hypoxic fraction were calculated for each track. Data for each patient were also averaged across all tracks obtained for that patient. Track-specific data were used to evaluate intrapatient variation. The range of average values for each patient was used to assess interpatient heterogeneity. The ratio of these measures provided an assessment of within- vs. between-patient heterogeneity. RESULTS: The median number of pO2 measurements/patient was 200 (range: 88-356). The average length of hypoxic regions varied from 4.5-5.6 mm. Median tumor pO2s for the cervix, lymph node, and sarcoma patients were 4.5 mm Hg, 12.6 mm Hg, and 18.0 mm Hg, respectively (p = 0.07). Median hypoxic fractions were 0.61, 0.36, and 0.31, respectively (p = 0.07). Intrapatient heterogeneity was less than interpatient heterogeneity for all parameters in all patients, except for mean pO2 for the cervix patients measured at 0.7-mm increments (1.51). Assessment of oxygenation was not affected by the distance between samples. CONCLUSIONS: Heterogeneity of tumor oxygenation within tumors is less than that between tumors. Both 0.4 mm and 0.7-0.8 mm sampling increments provide similar data. Longer term follow-up of large numbers of uniformly treated patients is required to define the value of tumor oxygen measurement as a predictor of treatment outcome.

Pretreatment oxygenation profiles of human soft tissue sarcomas.

PURPOSE: Tumor oxygenation is thought to influence the radiocurability of many malignancies. Advances in polarographic electrode technology have facilitated the in situ measurement of human tumor pO2. The optimal method of defining a "hypoxic" tumor is not known. Characterization of intra-tumor and intertumor pO2 heterogeneity could help with this process. This study was performed to evaluate pretreatment tumor oxygenation status and pO2 heterogeneity in patients with soft tissue sarcoma. METHODS AND MATERIALS: Nine patients with soft tissue sarcomas underwent pretreatment pO2 measurements with the Eppendorf pO2 histograph. Two grossly distinct anatomic sites within each tumor were measured in all but one patient; these were localized under computerized tomography guidance to ensure that all measurements were obtained from tumor tissue. Multiple probe tracks were studied at each site. Measurements were performed in resting, awake patients. RESULTS: A total of 1588 pO2 readings was obtained (mean = 176/patient). Measurement path lengths ranged from 22-36 mm. The average hypoxic fraction (pO2 < 5 mm Hg) was 29% (range 0-76%). Arterial pO2 was positively correlated with mean and median tumor pO2. Tumor hypoxic fraction increased with increasing tumor volume. Linear pO2 profiles and frequency histograms provided similar estimates of the extent of hypoxia in individual tumors. Marked variation in oxygenation existed both within and between individual tumors. The intertumor variation was greater than the intratumor variation. CONCLUSION: Radiobiologic hypoxia exists in human soft tissue sarcomas. The pO2 variation within individual tumors is less than the variation between tumors. Further study is necessary to identify the best parameter for defining tumor hypoxia and to discern the relationship between tumor pO2 and treatment outcome.

Distribution of the hypoxia marker CCI-103F in canine tumors.

PURPOSE: The purpose of this study was to identify the prevalence and distribution of hypoxic tumor cells in spontaneous canine tumors, and to relate these parameters to various tumor and patient characteristics, such as tumor volume, tumor type, or tumor location. METHODS AND MATERIALS: Hypoxic tumor cells were labeled in vivo in 32 primary malignant canine tumors by bioreductive binding of the nitroimidazole hypoxia marker CCI-103F. CCI-103F was given at 40 mg/kg i.v. Tumors were completely excised, and CCI-103F adducts were detected in histologic sections (mean, 138 sections-per-tumor) by peroxidase-antiperoxidase immunostaining. Area fraction (area labeled/total area examined) of labeled regions was measured via computer assisted image analysis. In tumors with a volume < 100 cm3, each cubic centimeter of tumor was examined; in larger tumors 100 randomly selected 1 cm3 samples were examined. RESULTS: There were 13 soft-tissue sarcomas, 11 mast-cell tumors, five carcinomas, two lymphosarcomas, and one melanoma. Tumors varied from < .001 to > 2000 cm3. Labeled cells were present in 31 of 32 canine tumors examined, and varied between 0 and 35%. Mean (+/- SD) % label was 12.2% +/- 16.7%; 13 of the 32 dogs had % labeled area < 5.0%. The area fraction was not related to tumor site, tumor type, tumor volume, presence and degree of necrosis or tumor grade. Dog characteristics such as sex, age, and body size did not affect the degree of labeling of tumors. CCI-103F adducts were randomly distributed grossly, and at the microscopic level were not found near blood vessels or regions containing mitoses. Labeling was seen in a variety of normal tissues; not all binding in normal tissues could be attributed to hypoxia. CONCLUSION: CCI-103F labeling of hypoxic regions in tumors provides a nonradioactive method of detecting nitroimidazole adducts at the cellular level, and allows concurrent histologic examination. The pattern of labeling is consistent with detection of hypoxic tumor cells arising from oxygen diffusion limitations. This method may have clinical applicability in the detection of tumor hypoxia.

Quantification of CCI-103F labeling heterogeneity in canine solid tumors.

PURPOSE: The purposes of this study were to assess sources of variation in the distribution of nitroimidazole-labeled hypoxic cells in canine tumors and to quantify the reliability of estimating overall nitroimidazole-labeled area fraction from biopsies. METHODS AND MATERIALS: Hypoxic cells were labeled in 24 canine tumors by immunostaining of the nitroimidazole hypoxia marker CCI-103F. In tumors with a volume < 100 cm3, each cubic centimeter of tumor was examined; in larger tumors 100 randomly selected 1 cm3 samples were examined. These data were used to estimate the overall CCI-103F-labeled area fraction in the tumor. A variance components model was used to quantify intertumoral, intratumoral, and within slide (residual) sources of variation. The ability to estimate intratumoral CCI-103F-labeled area fraction based on information obtained from biopsies was assessed by randomly selecting two or four samples from the dataset for each tumor and comparing the mean CCI-103F-labeled area fraction from this limited sample to the labeled area fraction based on each cubic centimeter; this simulation process was repeated 1000 times. RESULTS: Intratumoral (27% of total) and intertumoral (30% of total) variation in CCI-103F-labeled area fraction were similar. Residual variation (variation at the microscopic level) accounted for 43% of total variation in CCI-103F labeling. Intratumoral variation in labeling decreased as the intratumoral CCI-103F mean labeled area fraction decreased. The accuracy of estimating the intratumoral CCI-103F-labeled area fraction in a tumor from limited sampling increased as the number of samples increased or the intratumoral labeled area fraction decreased. When four random samples were used to estimate overall CCI-103F-labeled area fraction in the tumor, estimates from approximately 90% of the 1000 simulations were within 0.10 of the intratumoral CCI-103F-labeled area fraction. Classifying a minimally labeled tumor as unlabeled based on limited sampling was unlikely. CONCLUSION: Despite intratumor variation, acceptable estimates of nitroimidazole-labeled cells in a tumor may be obtained from a clinically feasible number of biopsies.

Post-mastectomy radiotherapy following adjuvant chemotherapy and autologous bone marrow transplantation for breast cancer patients with greater than or equal to 10 positive axillary lymph nodes. Cancer and Leukemia Group B.

Between 2/87 and 2/91, 49 women with operable breast cancer involving greater than or equal to 10 axillary nodes were treated following mastectomy, with four cycles of Cyclophosphamide, Adriamycin, 5FU, followed by high doses of Cyclophosphamide, Cisplatin, Carmustine (HDCT) with autologous bone marrow transplant support. Forty patients received local-regional radiotherapy (generally to the chest wall, internal mammary, supraclavicular, +/- axillary nodal areas; minimum 44-50 Gy, 1.8-2 Gy/fraction, +/- 10-15 Gy scar boost; standard radiation techniques). The first nine patients did not receive local-regional radiotherapy. Three developed a local-regional failure (6-12 months after HDCT); six are without evidence of disease. Local-regional radiotherapy (LR XRT) was delivered to the subsequent 40 patients following HDCT+autologous bone marrow transplant. Six received less than 44 Gy of the planned local-regional radiotherapy due to significant toxicity and one of these failed locally. Only one local failure was observed among the 34 patients who received greater than or equal to 44 Gy. Two additional patients developed distant metastases. None of these 40 patients have failed in the axilla despite the fact that the axilla was irradiated in only 18 cases. Overall, 36/40 (90%) of these patients are without evidence of disease 4-30 months following HDCT (approximately 10-36 months after mastectomy, median 22 months). Radiotherapy was interrupted or discontinued because of progressive dyspnea, thrombocytopenia, or neutropenia in nine patients. Further studies to determine the roles of local-regional radiotherapy and HDCT in the development of these toxicities are underway. These encouraging results suggest that HDCT + autologous bone marrow transplant+local-regional radiotherapy may improve the survival rate in these high risk patients. A national randomized study to test the efficacy of this HDCT regimen is currently underway (Cancer and Leukemia Group B#9082 and Southwest Oncology Group #9114).

Radiation plus local hyperthermia versus radiation plus the combination of local and whole-body hyperthermia in canine sarcomas.

PURPOSE: The purpose of this study was to assess the effect of increasing intratumoral temperatures by the combination of local hyperthermia (LH) and whole body hyperthermia (WBH) on the radiation response of canine sarcomas. METHODS AND MATERIALS: Dogs with spontaneous soft tissue sarcomas and no evidence of metastasis were randomized to be treated with radiation combined with either LH alone or LH + WBH. Dogs were accessioned for treatment at two institutions. The radiation dose was 56.25 Gy, given in 25 2.25 Gy daily fractions. Two hyperthermia treatments were given; one during the first and one during the last week of treatment. Dogs were evaluated after treatment for local recurrence, metastasis, and complications. RESULTS: Sixty-four dogs were treated between 1989 and 1993. The use of LH+WBH resulted in statistically significant increases in the low and middle regions of the temperature distributions. The largest increase was in the low temperatures with median CEM 43 T90 values of 4 vs. 49 min for LH vs. LH + WBH, respectively (p<0.001). There was no difference in duration of local tumor control between hyperthermia groups (p = 0.59). The time to metastasis was shorter for dogs receiving LH + WBH (p = 0.02); the hazard ratio for metastatic disease for dogs in the LH + WBH group was 2.4 (95% confidence interval, 1.2-5.4) with respect to dogs in the LH group. Complications were greater in larger tumors and in tumors treated with LH + WBH, CONCLUSION: The combination of LH + WBH with radiation therapy, as described herein, was not associated with an increase in local tumor control in comparison to use of LH with radiation therapy. The combination of LH + WBH also appeared to alter the biology of the metastatic process and was associated with more complications than LH. We identified no rationale for further study of LH + WBH in combination with radiation for treatment of solid tumors.

The treatment of high-grade soft tissue sarcomas with preoperative thermoradiotherapy.

PURPOSE: To explore the use of a novel program of preoperative radiation and hyperthermia in the management of high-grade soft tissue sarcomas (STS). METHODS AND MATERIALS: Eligible patients were adults over 18 with Grade 2 or 3 STS, surgically resectable without a local excision prior to referral to Duke University Medical Center and without distant metastases. Patients were staged generally with CT and/or MR imaging. The diagnosis was established with fine needle aspiration or incisional biopsy. Patients were then treated with 5000 to 5040 cGy, 180-200 cGy per fraction. Chemotherapy was usually not employed. Generally two hyperthermia treatments per week were given with a planned thermal dose of 10-100 CEM 43 degrees T90. Invasive thermometry and thermal mapping were done in all patients. Surgical resection was planned 4-6 weeks after the completion of radiation and hyperthermia. RESULTS: Ninety-seven patients were treated on study between 1984 and 1996. Follow-up ranged from 12 to 155 months (median 32). All tumors were high-grade in nature, 44 greater than 10 cm in size (maximum tumor diameter), 43 5-10 cm in size, 10 less than 5 cm. Seventy-eight of the 97 tumors were located in an extremity. Of the 97 patients, 48 remain alive and continually free of disease following initial therapy. Of the remaining 49 patients, 44 have relapsed (34 dead, 10 living with disease), 3 have died secondary to complications of therapy, and 2 have died of unrelated causes. Ten-year actuarial overall survival, cause-specific survival, and relapse-free survival are 50, 47, and 47% respectively. The predominant pattern of failure has been distant metastases with only 2 patients developing local failure alone. Ten-year actuarial local control for extremity tumors is 94%, 63% for the 19 patients with tumors at sites other than the extremity. Of the 78 patients with extremity lesions, 63 have had limb preservation and remain locally controlled. Overall 38 patients experienced 57 major complications. There were 3 deaths, one due to adriamycin cardiomyopathy and two secondary to wound infections. Four patients required amputation secondary to postoperative wound healing problems. Complications directly attributable to hyperthermia occurred in 15 patients with 11 instances of second- or third-degree burns and two instances of subcutaneous fat necrosis. The hyperthermia complications were generally not severe and either healed readily or were excised at the time of surgical resection of the primary tumor. CONCLUSIONS: For these aggressive high-grade soft tissue sarcomas, this treatment program of preoperative thermoradiotherapy provided excellent local regional control for extremity lesions (95%) and satisfactory local regional control (63%) of nonextremity sarcomas, but did not appear to influence the rate of distant metastases or survival. Complications were frequent but apart from the direct thermal burns, not too different from those reported for preoperative radiotherapy alone. More effective adjuvant systemic therapy is necessary to impact favorably on survival.

Hypoxia marker labeling in tumor biopsies: quantification of labeling variation and criteria for biopsy sectioning.

BACKGROUND AND PURPOSE: The error associated with using biopsy-based methods for assessing parameters reflective of the tumor microenvironment depends on the variability in distribution of the parameter throughout the tumor and the biopsy sample. Some attention has been given to intratumoral distribution of parameters, but little attention has been given to their intrabiopsy distribution. We evaluated the intrabiopsy distribution of CCI-103F, a 2-nitroimidazole hypoxia marker. MATERIALS AND METHODS: The hypoxia marker CCI-103F was studied in dogs bearing spontaneous solid tumors. Two biopsies were taken from each of seven tumors, for a total of 14 biopsies. Biopsies were serially sectioned and four to six contiguous slides from each 100-150 microm of the biopsy were used to formulate the best estimate of CCI-103F labeled area throughout the biopsy sample. One, two or four slides were then randomly selected from each biopsy and the labeled area, based on this limited sample, was compared to the estimate obtained from counting all available slides. Random sampling of slides was repeated 1000 times for each biopsy sample. RESULTS: CCI-103F labeling variance throughout the biopsy decreased as the estimated overall labeled area in the biopsy decreased. The error associated with estimating the overall labeled area in a biopsy from a randomly selected subset of slides decreased as the number of slides increased, and as the overall labeled area in the biopsy decreased. No minimally labeled biopsy was classified as unlabeled based on limited sampling. CONCLUSION: With regard to CCI-103F labeling, quantification of the labeled area in four randomly selected slides from a biopsy can provide, in most biopsies, an estimate of the labeled area in the biopsy within an absolute range of +/-0.05.