Anthony D. Elias

GRANULOCYTE-MACROPHAGE COLONY STIMULATING FACTOR EXPANDS THE CIRCULATING HAEMOPOIETIC PROGENITOR CELL COMPARTMENT IN MAN

The effect of granulocyte-macrophage colony-stimulating factor (GM-CSF) on bone-marrow and peripheral-blood progenitor cells was investigated in a three-phase study in 13 patients with sarcoma. In the first phase patients were given GM-CSF alone. In phase II, which started a week after completion of phase I, patients received a course of cytotoxic chemotherapy, then a course of GM-CSF. Phase III consisted only of cytotoxic chemotherapy. GM-CSF (phase I) alone produced an 18-fold increase in peripheral blood granulocyte-macrophage colony-forming units (CFU-GM) and an 8-fold increase in erythroid burst-forming units (BFU-E) in the peripheral blood. GM-CSF had no effect on bone-marrow CFU-GM and BFU-E in the group as a whole. Three patients were investigated after phases II and III. GM-CSF increased the absolute number of peripheral blood CFU-GM by approximately 60-fold compared with the pretreatment baseline. These effects of GM-CSF may be of clinical importance with regard to facilitating the harvest of peripheral blood progenitor cells for autotransplantation.

Elevated serum periostin levels in patients with bone metastases from breast but not lung cancer.

Periostin is a recently identified gene that is preferentially expressed in periosteum, indicating a potential role in bone formation and maintenance of structure. We independently identified and isolated periostin from cancer tissue, using the palindromic PCR-driven cDNA Differential Display technique. For the present work, we developed a novel sandwich chemiluminescence assay to detect serum periostin level using newly developed monoclonal and polyclonal antibodies. We investigated serum periostin levels in breast cancer and small cell lung cancer patients, especially in patients with bone metastasis. The study included 58 breast cancer and 44 small cell lung cancer patients. Serum periostin levels were elevated in breast cancer patients presenting with bone metastases (92.0 ± 28.6 ng/ml) compared to similar breast cancer patients without evidence of bone metastasis (55.0 ± 16.6 ng/ml, p = 0.04). No correlation was found between the serum periostin level and any other prognostic factors, such as clinical stage and lymph node metastasis in breast cancer. Serum periostin levels thus appear to serve as a marker of bone metastasis from breast cancer. In contrast, serum periostin levels were similar in samples from patients with small cell lung cancer who did or did not have bone metastasis. However, increasing T-stage and N-stage of patients with small cell lung cancer were correlated with higher periostin levels (T4, 126.5 ± 29.7 ng/ml v.s. T2, 64.9 ± 16.1 ng/ml, p = 0.03; and T4 v.s. T1, 36.3 ± 7.5 ng/ml, p = 0.01; N3, 108.7 ± 17.3 ng/ml v.s. N2, 49.7 ± 10.9 ng/ml, p = 0.01). Periostin has a substantial homology with the insect cell adhesion molecule, fasciclin I. Thus, expression of periostin may facilitate tumor cell adhesion to the bone surface. In fact, we found by in situ RNA hybridization, that the periostin gene was highly expressed in the stromal cells immediately surrounding the tumor, but not within the breast cancer cells themselves.

High-dose ifosfamide with mesna uroprotection: a phase I study.

Phase II trials of ifosfamide have been performed with standard doses of 5 to 8 g/m2/course. In this phase I study, 29 patients were treated with a 4-day continuous infusion ifosfamide to determine the maximum-tolerated dose and the nonhematologic dose-limiting toxicity. Autologous bone marrow support was to have been used for the subsequent dose level if granulocytes were more than 500/microL for more than 14 days in two of two to five patients at a given dose level. Doses were escalated from 8 to 18 g/m2 ifosfamide. Mesna was given at an equivalent dose by continuous infusion for 5 days. At the 18 g/m2 dose level, dose-limiting renal insufficiency and a median of 11 days (range, 8 to 18 days) of granulocytopenia (less than 500/microL) were observed. Thus, autologous bone marrow reinfusion ws not used. The duration of myelosuppression, the frequency and severity of mucositis, and renal tubular acidosis were all dose-dependent. Mild to moderate CNS toxicity also appeared to be related to dose; however, severe CNS toxicity (transient confusion, hallucinations, and somnolence) was observed sporadically at both low- and high-dose levels. Transient hematuria (greater than 50 red blood cells [RBCs]/high power field) occurred once but did not affect treatment. There were nine responses (two complete) in 27 heavily pretreated assessable patients including seven responses in 20 patients with advanced refractory sarcoma. Ifosfamide with mesna uroprotection can undergo considerable dose escalation over the usual prescribed doses before nonhematologic dose-limiting toxicity is encountered. Ifosfamide has broad cytotoxicity against solid tumors and may prove to be an important addition to high-dose combination chemotherapy regimens.

Phase II study of topotecan in metastatic non-small-cell lung cancer.

PURPOSE Topotecan is an inhibitor of topoisomerase I that has shown preclinical activity against non-small-cell lung cancer (NSCLC). This phase II study was designed to determine the clinical activity and toxicity spectrum of topotecan in untreated patients with metastatic NSCLC. PATIENTS AND METHODS Twenty previously untreated patients received topotecan every 21 days at the dose of 2 mg/m2/d intravenously (IV) for 5 days for two cycles, at which point response was assessed. Patients with either clinical response or stable disease (SD) received additional cycles of the drug until toxicity developed or disease progression (PRG) occurred. RESULTS This study was designed to enter 30 patients. However, because no clinical responses were seen in the first 20 patients entered onto the study, the early-stopping rule was invoked and patient accrual was halted. Eleven patients (55%) had SD on topotecan, and nine (45%) had PRG. Toxicity included neutropenia and rash. The median survival duration for all patients was 7.6 months. CONCLUSION We observed no objective clinical responses despite producing high-grade neutropenia. Phase II trials of topotecan using different schedules or higher doses supported by growth factors may clarify the role of topotecan in the treatment of NSCLC. The combination of topotecan with cisplatin and topoisomerase II inhibitors such as etoposide should be explored. Finally, the median survival duration of 7.6 months for 20 patients treated with an agent that failed to produce any obvious clinical responses compares favorably to the survival obtained with combinations of existing agents. This supports the further study of novel compounds in this clinical setting.

Neoadjuvant therapy for surgically staged IIIA N2 non-small cell lung cancer (NSCLC)

INTRODUCTION Neoadjuvant therapy in patients with Stage IIIA NSCLC is associated with a 50-70% resection rate and a 3-5 year survival of 20-32%, but few trials have required meticulous staging of the mediastinum to ensure homogeneity of the study population. Continuous infusion cisplatin 25 mg/m2/day 1-5, 5-fluorouracil 800 mg/m2/day 2-5, and high-dose leukovorin 500 mg/m2/day 1-5 (PFL) given every 4 weeks achieved a 41% response rate in metastatic NSCLC (Lynch TJ, Kalish LA, Kass F, Strauss G, Elias A, Skarin A, Shulman L, Sugarbaker D, Frei E. Continuous infusion cisplatin, 5-fluorouracil, and leukovorin for advanced non-small cell lung cancer. Cancer 1994; 73: 1171-1176). The regimen was therefore evaluated in 34 patients with pathologic Stage IIIA N2 disease between 3/91 and 10/92. METHODS Staging consisted of chest, liver, brain computerized tomography and bone scan, bronchoscopy and surgical mediastinal node mapping. Patients received PFL for 3 cycles, followed by thoracotomy and thoracic radiotherapy (TRT) to 54-60 Gy. RESULTS Median age was 57 (42-68) years. Demographic factors included: male 56%; adenocarcinoma 59%, squamous cell carcinoma 24%; Stage T3N2 26%, T2N2 56%, and T1N2 18%. No treatment related deaths occurred. Radiographically defined response to PFL was 65% (6% complete). Thoracotomy was performed in 28 patients (82%) (6 had no attempt due to disease progression). Complete resection was achieved in 21 (75%) and seven were unresectable. Pathologic complete response was observed in five patients (15%) and an additional unresectable patient had fibrosis-only documented at thoracotomy for an overall clinicopathologic response rate of 76% (18% pathologic CR). Another ten patients had residual primary with or without hilar disease with resolution of previously documented mediastinal involvement. Six (18%) patients remain alive and disease-free with a median follow-up of 46 (33-50) months, four of whom had achieved pathologic complete response at time of surgery. CONCLUSIONS Long-term event-free survival was associated with complete surgical resection which in turn was associated with clinical response to chemotherapy. There was a possible trend associating pathologic downstaging (absent residual disease in mediastinal nodes), particularly pathologic complete response observed in patients with non-bulky mediastinal disease, with improved event-free survival. Pathologic downstaging might therefore be a useful surrogate endpoint in trials evaluating the preoperative activity of new chemotherapy regimens. While radiographic response generally correlated with findings at surgery, response as determined by histologic examination of resected tissue was generally more extensive and may more accurately reflect the systemic impact of the chemotherapy regimen.

Immunotoxin therapy of small-cell lung cancer: a phase I study of N901-blocked ricin.

PURPOSE Immunotoxins could improve outcome in small-cell lung cancer (SCLC) by targeting tumor cells that are resistant to chemotherapy and radiation. N901 is a murine monoclonal antibody that binds to the CD56 (neural cell adhesion molecule [NCAM]) antigen found on cells of neuroendocrine origin, including SCLC. N901-bR is an immunoconjugate of N901 antibody with blocked ricin (bR) as the cytotoxic effector moiety. N901-bR has more than 700-fold greater selectivity in vitro for killing the CD56+ SCLC cell line SW-2 than for an antigen-negative lymphoma cell line. Preclinical studies suggested the potential for clinically significant cardiac and neurologic toxicity. We present a phase I study of N901-bR in relapsed SCLC. PATIENTS AND METHODS Twenty-one patients (18 relapsed, three primary refractory) with SCLC were entered onto this study. Successive cohorts of at least three patients were treated at doses from 5 to 40 microg/kg/d for 7 days. The initial three cohorts received the first days dose (one seventh of planned dose) as a bolus infusion before they began the continuous infusion on the second day to observe acute toxicity and determine bolus pharmacokinetics. Toxicity assessment included nerve-conduction studies (NCS) and radionuclide assessment of left ventricular ejection fraction (LVEF) before and after N901-bR administration to fully assess potential neurologic and cardiac toxicity. RESULTS The dose-limiting toxicity (DLT) of N901-bR given by 7-day continuous infusion is capillary leak syndrome, which occurred in two of three patients at the dose of 40 microg/kg (lean body weight [LBW])/d. Detectable serum drug levels equivalent to effective in vitro drug levels were achieved at the 20-, 30-, and 40-microg/kg(LBW)/d dose levels. Specific binding of the immunotoxin to tumor cells in bone marrow, liver, and lung was observed. Cardiac function remained normal in 15 of 16 patients. No patient developed clinically significant neuropathy. However, a trend was noted for amplitude decline in serial NCS of both sensory and motor neurons. One patient with refractory SCLC achieved a partial response. CONCLUSION N901-bR is an immunotoxin with potential clinical activity in SCLC. N901-bR is well tolerated when given by 7-day continuous infusion at the dose of 30 microg/kg(LBW)/d. Neurologic and cardiac toxicity were acceptable when given to patients with refractory SCLC. A second study to evaluate this agent after induction chemoradiotherapy in both limited- and extensive-stage disease was started following completion of this study.

Sources and sequelae of bacterial contamination of hematopoietic stem cell components: implications for the safety of hematotherapy and graft engineering

Background: It is important to compare the incidence of bacterial contamination of components collected from the peripheral blood or bone marrow (BM), as well as of components processed with or without cell selection or depletion, and to evaluate the sequelae of such contamination.

High-dose multimodality therapy with autologous stem-cell support for stage IIIB breast carcinoma.

PURPOSE Women with locally unresectable and inflammatory breast carcinoma (IBC) have an approximately 30% 5-year disease-free survival (DFS) rate with conventional multimodality therapy. A short but dose-intensive multimodality phase II trial was designed in an attempt to improve outcome in stage IIIB disease. Mastectomy was performed after high-dose therapy to evaluate pathologic response to treatment. METHODS Women with newly diagnosed disease received four 2-week cycles of doxorubicin 90 mg/m2 with granulocyte colony-stimulating factor (G-CSF), followed by cyclophosphamide 6,000 mg/m2, thiotepa 500 mg/m2, and carboplatin 800 mg/m2 (CTCb) with marrow and peripheral-blood progenitor cell (PBPC) support. Local therapy consisted of mastectomy and radiotherapy. Tamoxifen (5 years) was begun if the patient was estrogen receptor-positive (ER+). RESULTS Fifty women (46 stage IIIB [91% IBC], four stage IIIA) entered the study and 47 are assessable. Ten had mastectomy before any systemic therapy (seven with pathologic IBC, three with residual tumor after mastectomy). Eighty percent received full-dose doxorubicin with 60% on schedule. Clinical response rates to induction were 15% complete response (CR), 5% very good partial response (VGPR), 59% partial response (PR), and 21% minor response (MR)/stable disease (SD). Mastectomy after CTCb in 37 patients showed a 14% pathologic CR rate, 29% microscopic foci in breast and/or axilla, and 57% gross tumor. Fifteen (32%) patients have relapsed (median, 17 months post-CTCb). The 30-month DFS is estimated at 64%. For those in pathologic CR, with microscopic, or with gross disease remaining after CTCb, the 30-month DFS is estimated at 100%, 70%, and 38%, respectively. Those with zero, one to three, or > or = four positive nodes at axillary dissection had a median DFS of 31, 18, and 13 months, respectively. CONCLUSION This short but dose-intensive multimodality approach for stage IIIB breast carcinoma is feasible with encouraging results to date.

Selective transgene expression for detection and elimination of contaminating carcinoma cells in hematopoietic stem cell sources.

Tumor contamination of bone marrow (BM) and peripheral blood (PB) may affect the outcome of patients receiving high dose chemotherapy with autologous transplantation of hematopoietic stem cell products. In this report, we demonstrate that replication defective adenoviral vectors containing the cytomegalovirus (CMV) or DF3/MUC1 carcinoma-selective promoter can be used to selectively transduce contaminating carcinoma cells. Adenoviral-mediated reporter gene expression in breast cancer cells was five orders of magnitude higher than that found in BM, PB, and CD34+ cells. Our results demonstrate that CD34+ cells have low to undetectable levels of integrins responsible for adenoviral internalization. We show that adenoviral-mediated transduction of a reporter gene can detect one breast cancer cell in 5 x 10(5) BM or PB cells with a vector containing the DF3/MUC1 promoter. We also show that transduction of the HSV-tk gene for selective killing by ganciclovir can be exploited for purging cancer cells from hematopoietic stem cell populations. The selective expression of TK followed by ganciclovir treatment resulted in the elimination of 6-logs of contaminating cancer cells. By contrast, there was little effect on CFU-GM and BFU-E formulation or on long term culture initiating cells. These results indicate that adenoviral vectors with a tumor-selective promoter provide a highly efficient and effective approach for the detection and purging of carcinoma cells in hematopoietic stem cell preparations.

Double dose-intensive chemotherapy with autologous stem-cell support for metastatic breast cancer: no improvement in progression-free survival by the sequence of high-dose melphalan followed by cyclophosphamide, thiotepa, and carboplatin.

PURPOSE Twenty-one percent of responding metastatic breast cancer patients remain progression-free a median 50 months following one intensification cycle of cyclophosphamide (6,000 mg/m2), thiotepa (500 mg/ m2), and carboplatin (800 mg/m2) (CTCb) with autologous bone marrow transplantation (ABMT). This trial studied whether the sequence of high-dose melphalan followed by CTCb resulted in improved disease response and duration. METHODS Women with at least partial responses (PRS) to induction received melphalan (140 or 180 mg/ m2) with peripheral-blood progenitor cell (PBPC) and granulocyte colony-stimulating factor (G-CSF) support. They were monitored as outpatients. After recovery, patients were hospitalized for CTCb with marrow, PBPC, and G-CSF support. RESULTS Data on 67 women, at a median of 25 months from CTCb, were examined. After melphalan, 49 (73%) required admission for fever (89%), mucositis (35%), or infection (15%) (median stay, 8 days). All received CTCb. For the first 33 patients, the median days from start of melphalan to CTCb was 24. After liver toxicity (one death from venoocclusive disease [VOD]) developed in 11 patients during CTCb, the interval between intensifications was increased to 35 days without incident. Twenty-three patients (34%) are progression-free a median of 16 months post-CTCb. The median progression-free survival (PFS) and survival times for the whole group are estimated at 11 and 20 months, respectively. CONCLUSION Treatment with this sequence of high-dose melphalan followed by CTCb has not resulted in superior PFS to date, when compared with single-intensification CTCb. This report discusses factors related to patient selection, the role of induced drug resistance, and the schedule of administration of alkylating agenting that may adversely influence outcome.