15130 Background: PPX is a conjugate of paclitaxel to a polyglutamate polymer. Preclinically, PPX demonstrated a radiation enhancement factor (REF) >7.0, versus 1.5–2.0 for paclitaxel. (Milas et al Int J Rad Onc 55:2003, Li et al. Clin Cancer Res 6:2000). The maximally tolerated dose (MTD) of PPX was determined previously to be 70 mg/m2 /week with concurrent radiation. We initiated a phase I study of PPX, cisplatin, and concurrent radiation with patients with esophageal and gastric cancer. Methods: Patients with esophageal or gastric cancer receiving chemoradiation for locoregional control, adjuvant, or neoadjuvant treatment were eligible. All patients received radiation at a dose of 50.4 Gy delivered in 28 fractions (5 fractions per week for 5 1/2 weeks), and cisplatin (25 mg/m2) on days 1, 8, 15, 22, 29, and 36. PPX was given as a 10 minute infusion in escalating dosages prior to each cisplatin dose. Dose limiting toxicities (DLTs) were defined as grade 4 hematologic toxicity, esophagitis, nausea/vomiting, or dehydration, or any other grade 3/4 non-hematologic toxicity. Patients were enrolled in successive cohorts of three. The MTD was defined as the dose level at which no more than 2 of 6 patients have DLTs. Results: Eleven patients have been entered over 2 dose levels of PPX: 50 mg/m2 (six patients, dose level 1), and 60mg/m2 (5 patients, dose level 2). Five patients had esophageal cancer and six had gastric cancer. All histologies were adenocarcinomas. One of six patients treated at dose level had a DLT (esophagitis). Three of five patients had DLTs at dose level 2, including esophagitis, nausea, vomiting, and dehydration. Conclusions: PPX is a novel radiation sensitizer for patients with esophageal and gastric cancer. The MTD for PPX is 50 mg/m2 /week in combination with cisplatin 25mg/ m2 /week for 6 weeks, and 50.4 Gy concurrent radiation for patients with esophagogastric cancer. A phase II study of PPX/cisplatin and radiation will be initiated. No significant financial relationships to disclose.

7039 Background: Women with NSCLC are younger and are more likely to be non-smokers than the overall NSCLC population. Estrogen seems to contribute to these differences in lung cancer biology, but its effect on treatment efficacy has not been well described. Methods: Two phase III trials in chemo-naïve PS2 patients with advanced NSCLC compared PPX to either gemcitabine or vinorelbine (STELLAR 4), or PPX/carboplatin to paclitaxel/carboplatin (STELLAR 3). Eligibility criteria were identical and pts were stratified for gender and age. Analysis of overall survival (OS), the primary study endpoint, showed similar survival between treatment arms. A trend to improvement with PPX for females but not males in both studies prompted an exploratory combined analysis of the 198 women in STELLAR 3 (93/400 pts) and STELLAR 4 (105/381 pts) using univariate and multivariate Cox regression analysis of OS. Results: Combined log-rank analysis of STELLAR 3 and 4 shows improved OS for females receiving PPX vs control (9.5 mo vs 7.8 mo; HR=0.70; p=0.03) and no difference in males (7.3 mo vs 6.9 mo; HR=1.06; p=0.53). Cox multivariate analysis identified treatment with PPX (HR=0.64; p=0.019), smoking (HR=1.50; p=0.037), and presence of extra-thoracic metastases (HR=1.76; p=0.003) as independent prognostic factors in these women. In the combined analysis, median survival advantage for PPX-treated pts was greater in women <55 years old (10.0 vs 5.2 mo, p=0.038) compared to women 55 or older (8.9 vs 8.6 mo, p=0.134). Pretreatment estrogen levels were available for 86 pts in STELLAR 3; pts with estrogen >30 pg/dl had a significant survival benefit when receiving PPX compared to paclitaxel (10.2 vs 5.5 mo; p=0.039). Conclusions: While the efficacy of PPX is comparable to other treatment options in NSCLC, PPX may be more effective female pts, particularly premenopausal women, compared to females in the control arms. Preclinical data suggest that estrogen enhances PPX distribution to lung tissue and upregulates the rate-limiting metabolic enzyme cathepsin B in NSCLC. To prospectively test the effect of estrogen on OS, a phase III trial (PIONEER) has been initiated to compare PPX to paclitaxel in chemo-naïve PS2 females with NSCLC. [Table: see text]

We have shown previously with in vivo and in vitro animal models that the lens epithelium, in contrast to the nucleus, is remarkably resistant to hyperoxia. The main purpose of this study was to investigate the mRNA response of cultured human lens epithelial cells (LECs) to challenge by a high level of hyperbaric oxygen. Cells were treated for 3 hr with 50 atm of 99% O2, and then cultured normally for various times up to 11 days. Although the cells appeared normal immediately after the O2-treatment, they failed to grow and suffered 50% cell loss, as well as significant mitochondrial damage, during normal post-culture. Growth of the cells resumed after 3 days and by day 11, the number of O2-treated cells was the same as the controls. Remarkably, the 3 hr O2-treatment produced no immediate effects on either the cellular level of GSH, or on the activities of a number of antioxidant enzymes including glyceraldehyde-3-phosphate dehydrogenase, which is generally regarded as being highly sensitive to oxidation. In contrast, the activity of thioredoxin reductase (TrxR) was severely affected by the O2, decreasing by 51% after the 3 hr exposure. O2-induced death of the cells appeared to be caused by loss of ATP since a 31% decrease in ATP level occurred immediately after the O2-treatment, in spite of a 46% increase in lactate production. Analysis with real-time PCR showed a maximum 3–6-fold increase in mRNA levels 9 hr after the 3 hr O2-exposure for the enzymes heme oxygenase-1 (HO-1), MnSOD and TrxR1 (the cytoplasmic form of TrxR). These results were confirmed with the use of one-step RT-PCR and Northern blotting. Initial upregulation of message for HO-1 occurred a few hours before any upregulation of MnSOD could be detected, suggesting that release of free iron from the degradation of heme by HO-1 may have played a role in the upregulation of the dismutase. No significant changes in mRNA levels were observed for the antioxidant enzymes catalase, CuZnSOD, glutathione reductase and glutathione peroxidase, or for the antioxidant protein thioredoxin. Recovery of TrxR activity over a 4-day period appeared to parallel the return of the cells to a normal rate of growth. The results indicate that damaging effects of hyperoxia on cultured LECs occur primarily in the mitochondria, rather than in the cytoplasm. Cells avoid O2-induced cell death, and return to a normal rate of proliferation by upregulating mRNA levels for HO-1, MnSOD and TrxR1. It appears that full activity of TrxR1, an enzyme required for the production of deoxyribonucletides for DNA synthesis, is essential for the normal growth of O2-challenged LECs.

7230 Background: PPX (XYOTAX), a macromolecular drug conjugate that links paclitaxel with biodegradable polymer, poly-L-glutamic acid, is water soluble and can be administered as a 10 minute infusion. Eliminating the need for CremophorEL reduces the risk of hypersensitivity reactions, even without premedication. Preclinical and clinical findings show that PPX is relatively stable in plasma; more than 95% of paclitaxel in circulation is present as the inactive conjugate, reducing systemic exposure to high concentrations of free paclitaxel. The antitumor activity of single agent PPX in chemotherapy naïve high-risk patients with NSCLC has been previously reported. In addition, PPX has been successfully combined with standard doses of cisplatin and carb. This multicenter, open label study evaluates the safety and efficacy of PPX in a standard doublet regimen for 1st line treatment of NSCLC. Methods: PPX (210 mg/m2) was administered as a short (10–20 minute) IV infusion followed carb (AUC 6) q3w to chemotherapy-naïve patients (pts) with performance status (PS) 0–2. Pts were treated for up to 6 cycles. Safety was assessed using NCI CTC (v 2). Efficacy assessments were done after every 2nd cycle using RECIST. Plasma samples were collected for pharmacokinetic (PK) assessment of PPX. Results: 74 pts were treated. The median age was 64 (range 32–85). 53% of pts were male. 14% of pts had PS2. Of the 60 pts with efficacy assessment following cycle 2, 9 pts (15%) had partial response and 24 pts (40%) had stable disease as the best response. Adverse event (AE) data are available for 40 pts (see table). No hypersensitivity reactions were observed. Detailed PK data will be presented. Conclusions: Preliminary data suggest that PPX (210 mg/m2) in combination with carb (AUC 6) is active as 1st line treatment for NSCLC. PPX demonstrates easily manageable toxicities and is generally well tolerated. Author Disclosure Employment or Leadership Consultant or Advisory Role Stock Ownership Honoraria Research Funding Expert Testimony Other Remuneration Cell Therapeutics Cell Therapeutics Cell Therapeutics

Bone marrow cells expressing the surface antigen CD34 comprise approximately 1% of harvested marrow and are highly enriched for marrow progenitor cells, including the cells believed to be responsible for long-term engraftment following bone marrow transplantation (BMT). Selection of CD34-expressing cells was applied in allogeneic BMT (alloBMT) to decrease the number of T lymphocytes in the infused marrow in an attempt to prevent severe graft-versus-host disease (GVHD). We report 14 patients who underwent HLA-identical sibling-matched alloBMT with marrow-enriched for CD34 cells using the Isolex 300 SA device. Patients received total body irradiation, thiotepa, cyclophosphamide, antithymocyte globulin and methylprednisolone prior to marrow infusion. No post-transplantation immunosuppressive therapy was given except for a 5-week course of steroids. The purity of the infused marrow was 64.9 ± 6.0% (mean ± s.e.m.) CD34-positive cells and patients received a mean of 1.24 ± 0.21 × 10 6 CD34 cells/kg. A mean of 9.4 ± 1.7 × 10 4 CD3 T cells/kg were present in the CD34-enriched product, representing a 2.7 ± 0.1 log depletion. There were no graft rejections and patients achieved a sustained absolute granulocyte count of >500 in a median of 10.5 days and a sustained platelet engraftment of >20000 untransfused in a median of 27 days. Patients were discharged a median of 21.5 days after marrow infusion. There were no instances of grade III or IV graft-versus-host disease (GVHD) and no unexpected adverse events during the transplant hospitalization. With a median follow-up of 12 months, the estimated 100 day survival is 86 ± 9%. CD34 selection in alloBMT permits rapid engraftment without unanticipated toxicities.

Seventy-one patients with poor-prognosis breast cancer were enrolled after informed consent in a multicentre randomized study to evaluate the use of selected peripheral blood CD34+ cells to support haematopoietic recovery following high-dose chemotherapy. Patients who responded to conventional chemotherapy were mobilized with chemotherapy (mainly high-dose cyclophosphamide) and/or recombinant human granulocyte colony-stimulating factor (rhG-CSF). Patients who reached the threshold of 20 CD34+ cells per microl of peripheral blood underwent apheresis and were randomized at that time to receive either unmanipulated mobilized blood cells or selected CD34+ cells. For patients in the study arm, CD34+ cells were selected from aphereses using the Isolex300 device. Fifteen patients failed to mobilize peripheral blood progenitors and nine other patients were excluded for various reasons. Forty-seven eligible patients were randomized into two comparable groups. CD34+ cells were selected from aphereses in the study group. Haematopoietic recovery occurred at similar times in both groups. No side-effect related to the infusion of selected cells was observed. The frequency of epithelial tumour cells in aphereses was low (8 out of 42 evaluated patients), as determined by immunocytochemistry. We conclude that selected CD34+ cells safely support haematopoietic recovery following high-dose chemotherapy in patients with poor-prognosis breast cancer.

PURPOSE The objective of this study was to characterize CD34+ cell grafts, obtained using a novel technique, from children undergoing autologous bone marrow transplantation (BMT) for cancer therapy. In particular, we wanted to determine if the CD34+ marrow cell grafts generated hematopoietic reconstitution, since a positive result would motivate further development and use of this methodology. PATIENTS AND METHODS This pilot feasibility clinical trial involved 13 patients < or = 25 years of age with advanced solid tumors, including seven children with neuroblastoma. Harvested bone marrow underwent immunomagnetic CD34+ selection. RESULTS In three of 13 enrolled patients, low purities of the CD34+ preparations disqualified the use of the CD34+ marrow grafts. Ten patients received myeloablative chemotherapy with etoposide, carboplatin, and cyclophosphamide, then were transplanted with CD34+ marrow grafts. In the 10 patients transplanted with CD34(+)-selected cells, the CD34+ cell purity (nucleated RBCs excluded) in the cell graft preparation was 91% total cell recovery from the starting light-density cells 2.2%, CD34+ cell recovery 38%, colony-forming unit-granulocyte-macrophage (CFU-GM) recovery 23%, and estimated tumor-cell depletion 2.6 logs (medians). The CD34+ marrow grafts administered to these patients contained a median of 2.3 x 10(6) nucleated cells, 1.4 x 10(6) CD34+ cells, and 1.3 x 10(4) CFU-GM per kilogram patient weight. Most patients experienced only the toxicities previously observed with this myeloblative chemotherapy regimen, although two unusual toxicities were observed. All 10 patients transplanted with CD34+ cell grafts engrafted. CONCLUSION The CD34+ purified grafts were enriched in stem/progenitor cells, with five of these 10 preparations containing > or = 94% CD34+ cells. Engraftment with CD34(+)-purified cell grafts as pure as 99% confirms that autologous CD34+ cells, alone, are sufficient to provide hematopoietic rescue for myeloablated patients. The best purification results were obtained on small marrow harvests from patients with neuroblastoma. The engraftment of highly purified CD34+ cells obtained by this technology and the antitumor effect of the transplant, by which two of 10 poor prognosis patients remain clinically free of tumor, have stimulated further clinical trials.

BACKGROUND: After the collection of granulocyte‐colony‐stimulating factor (G‐CSF)‐mobilized peripheral blood stem cells from healthy donors, the donor platelet counts fall. However, the magnitude and duration of this decrease are not known. STUDY DESIGN AND METHODS: Sixty healthy people were given G‐CSF (5, 7.5, or 10 micrograms/kg/day) for 5 days (Days 1–5), and 1 peripheral blood stem cell component was collected on Day 6. The platelet count, white cell count, absolute neutrophil count, hematocrit, and red cell count were measured before administration of G‐CSF (Day 0), before collection of peripheral blood stem cells on Day 6, and on Days 8, 10, 13, 16, and 20.RESULTS: The platelet count fell from 261 +/− 47 × 10(9) cells per L on Day 0 to 159 +/− 30 × 10(9) cells per L on Day 8 (p < 0.0001) and reached its lowest level on Day 10 (146 +/− 30 × 10(9)/L; p < 0.001). Compared to Day 0 levels, the platelet count was lower on Day 13 (185 +/− 49 × 10(9)/L, p < 0.001), was the same on Day 16 (270 +/− 53 × 10(9)/L), and was greater on Day 20 (333 +/− 60 × 10(9)/L, p < 0.0001). The white cell count returned to pretreatment values on Day 13, and the absolute neutrophil count returned to pretreatment values on Day 10 (Day 0 white cell count = 6.05 +/− 1.59 × 10(9)/L and Day 0 absolute neutrophil count = 3.97 +/− 1.52 × 10(9)/L). On Day 20, both were less than pretreatment values (white cell count = 5.14 +/− 1.24 × 10(9)/L, p = 0.0007 and absolute neutrophil count = 3.20 +/− 1.24 × 10(9)/L, p = 0.0036). The red cell counts on Day 16 (4.52 +/− 0.41 × 10(12)/L) and Day 20 (4.42 +/− 0.39 × 10(12)/L) were less than Day 0 values (4.73 +/− 0.43 × 10(12)/L, p = 0.008 and p < 0.0001, respectively). The hematocrit on Day 20 (39.2 +/− 3.2%) was also less than that on Day 0 (41.2 +/− 4.8%; p = 0.01). The changes in these blood counts were not affected by the dose of the G‐CSF.CONCLUSION: After stimulation with granulocyte‐colony‐stimulating factor and the collection or peripheral blood stem cells, the platelet counts in normal donors were decreased for at least 7 days (Days 6–13). Two weeks after collection of peripheral blood stem cells (Day 20), platelet production was increased, but the production of neutrophils and red cells was decreased. If two or more peripheral blood stem cell components are collected, then the platelet count should be measured after the second and subsequent collections. Further studies on the long‐term effect of G‐CSF on blood counts are needed.