Relative sedimentation of hematopoietic progenitors in human cord blood, peripheral blood, and bone marrow as determined by counterflow centrifugal elutriation.

BACKGROUND: The current use of cord blood (CB) and peripheral blood (PB) stem cells as alternatives or adjunctives to bone marrow (BM) for hematopoietic reconstitution in the treatment of various diseases prompted an examination of the progenitors of these tissues by counterflow centrifugal elutriation (CCE). METHODS: The cells, obtained from normal donors not primed with colony-stimulating factors, were centrifuged at 3000 rpm in a Beckman Sanderson Chamber. Fractions (Frs.) were collected at (1) 18 ml/min, (2) 25 ml/min, (3) 32 ml/min, (4) 40 ml/min, and (5) the rotor-off fraction. RESULTS: Clonogenic assays revealed differences in the fraction localizations for CB and PB when compared to BM, i.e., recovery of the colony-forming units for CB and PB was greater in the small-medium cell size CCE fractions, and those from BM were found primarily among the medium-large cell size fractions. Thus, although colony-forming unit granulocyte/macrophage colonies were distributed throughout Frs. 2-5 of BM, CB and PB showed 80% of the total to be in Frs. 2 and 3. Further, although burst-forming unit erythroid colonies of BM were distributed equally in Frs. 2 and 3, greater than 70% of the total burst-forming unit erythroid colonies in CB and PB were found in Fr. 2. Distribution of the CD34 cells in the fractions correlated with the colony-forming units in that these were found primarily in Frs. 2 and 3 of CB and PB, whereas they were present in significant numbers throughout Frs. 1-5 of BM. CONCLUSIONS: We interpret these findings to indicate CB and PB to be qualitatively similar in their hematopoietic lineage development and to contain a greater proportion of early versus late progenitors relative to those found in BM.

Phase II study of Didemnin B in central nervous system tumors: a Southwest Oncology Group study.

Didemnin B 6.3 mg/m2 was administered intravenously to 48 patients with recurrent or progressive central nervous system tumors. One patient of 39 (2.9%, 95% confidence limits 0.1 to 13.5) eligible patients had a confirmed partial response utilizing standard solid tumor criteria which lasted 14 months. Toxicity was significant. Nausea and vomiting and lethargy were the most frequent toxicities, but multiple severe toxicities were seen. Further investigation of Didemnin B at this dose is not warranted in patients with central nervous system malignancies.

Evaluation of multimodality treatment of locoregional esophageal carcinoma by Southwest Oncology Group 9060.

BACKGROUND: Continuous infusion 5-fluorouracil (CI5-FU) has been utilized concurrently with radiotherapy to improve tumor control. In this pilot trial, cisplatin, CI5FU, and radiotherapy were utilized for the treatment of locoregional esophageal carcinoma. It was postulated that the combination would be well tolerated and associated with high response rate and survival duration. METHODS: Thirty-two eligible patients with locoregional squamous cell carcinoma and adenocarcinoma of the esophagus received a regimen consisting of the following: radiotherapy, 50 Gray (Gy) (30 Gy anteroposterior/posteroanterior regional with 20 Gy AP/LPO/RPO boost) over 5 weeks, with CI5-FU 250 mg/m2/d for the duration of radiotherapy and cisplatin 25 mg/m2/day on Days 1-3 during Weeks 1 and 4 of the radiotherapy cycle. Upon completion of radiotherapy, two additional course, of cisplatin 75 mg/m2 on Days 1 and 29 and CI5-FU 300 mg/m2/day on Days 1-21 and 29-50 were delivered. Following imaging and endoscopic reassessment, patients with no evidence of disease received more chemotherapy. Surgery was suggested only for patients with residual local disease. RESULTS: Complete response was demonstrated in 44% of patients, clinically in 12 patients, and during surgery in 2 others. The median survival was 20 months, and the 1-year survival rate was 59%. Toxicity was severe, comprised of esophagitis, infection, and gastrointestinal complications. Dose delays and reductions occurred in the majority of patients. Four early deaths were noted. CONCLUSIONS: The regimen that was the focus of this trial has been active in the treatment of esophageal carcinoma. However, compared with existing regimens, its complexity and toxicity preclude its future use without modifications.

Phase II trial and cost analysis of fazarabine in advanced non-small cell carcinoma of the lung: a Southwest Oncology Group study.

BACKGROUND: Fazarabine is a novel nucleoside with broad spectrum pre-clinical activity and was chosen for study in patients with incurable non-small cell carcinoma of the lung. The expenses associated with investigational treatment have been assumed to be more than what would occur with conventional therapy, however, data are limited. METHODS: Twenty-three patients with metastatic non-small cell lung cancer were treated with fazarabine. Fazarabine was administered as a 72 hour continuous infusion at 2.0 mg/M2/hour. A cost analysis of treatment was calculated for patients treated in Springfield, MO. RESULTS: There were no responses (0%, 95% confidence interval = 0-15%) and median survival was 8 months. An analysis of the cost of treatment in the 4 patients treated in Springfield, MO, was compared to the costs of treatment with 4 cycles of cisplatinum and etoposide. There were no significant differences in costs for patients treated with the investigational agent as compared with conventional chemotherapy. CONCLUSIONS: Fazarabine has no demonstrable activity in patients with metastatic non-small cell carcinoma of the lung. Treatment with this agent in an investigational setting was no more expensive than treatment with conventional chemotherapy.

A phase I/II trial of twice daily irradiation and concurrent chemotherapy for locally advanced squamous cell carcinoma of the head and neck.

PURPOSE: This study was designed to test the toxicity and efficacy of a regimen of twice daily irradiation and concurrent multiagent chemotherapy for patients with locally advanced squamous cell carcinoma of the head and neck. METHODS AND MATERIALS: This was a prospective Phase I/II trial. Patients received 125 cGy b.i.d. to 7000 cGy with a 6 hr interfraction interval. Chemotherapy was given during weeks 1 and 6 of irradiation and consisted of a 5 day infusion of 5-fluorouracil at 600 mg/M2/day and 5 daily injections of cisplatin at 12 mg/M2/day. Two additional cycles of chemotherapy were given after the completion of radiotherapy. RESULTS: Forty-six patients were evaluable: 28 had technically unresectable disease and 18 had resectable tumors. All had Stage III or IV disease: 84% had T3 or T4 primaries while 53% had > or = N2 neck disease. The primary acute toxicity, confluent mucositis, was seen in 74% of patients. Late side effects occurred in four patients. Median follow-up is 36 months (range 25-44 months). Kaplan-Meier estimates of 2-year disease-free survival and overall survival are 65% and 73%, respectively, while 2-year local regional control and distant disease-free survival are 72% and 88%, respectively. Multivariate analysis revealed that resectability and receiving > 2 cycles of chemotherapy significantly influenced local regional control while age < 60 significantly influenced disease-free survival. CONCLUSION: This form of treatment can be delivered safely. The encouraging results have led to the initiation of a Phase III trial comparing this regimen with b.i.d. radiation alone.

High-dose oral tamoxifen, a potential multidrug-resistance-reversal agent: phase I trial in combination with vinblastine.

BACKGROUND: P-glycoprotein mediates resistance to natural-product anti-neoplastic agents like vinblastine through an active transport process resulting in reduced intracellular concentration of these agents. The triphenylethylene antiestrogen tamoxifen and its major metabolite N-desmethyltamoxifen at concentrations of 4-6 microM enhance the intracellular concentration of natural-product antineoplastics and augment the cytotoxicity of such drugs three-fold to 10-fold in a variety of human and murine cell lines. PURPOSE: On the basis of these preclinical findings, we conducted a phase I clinical trial of high-dose, oral tamoxifen administered in conjunction with a 5-day continuous infusion of vinblastine. METHODS: We studied 53 patients with advanced epithelial tumors. Tamoxifen was given orally as a loading dose on day 1, followed by two doses a day on days 2-13. Vinblastine was given as a 120-hour continuous infusion (1.5 mg/m2 per day) on days 9-13 of each tamoxifen course. The starting dose of tamoxifen was 40 mg/m2 administered twice a day following a loading dose of 150 mg/m2. The maximum dose was 260 mg/m2 twice a day following a loading dose of 680 mg/m2. Treatment cycles were repeated every 28 days. RESULTS: The dose-limiting toxic effects of tamoxifen were neurologic and began within 3-5 days after the start of treatment. They consisted of tremor, hyperreflexia, dysmetria, unsteady gait, and dizziness. One patient experienced a grand mal seizure 24 hours after the last tamoxifen dose. Toxic effects were rapidly reversible. Asymptomatic prolongation of the QT interval on electrocardiogram occurred at doses of tamoxifen of 80 mg/m2 or higher given twice a day. No coagulation or ophthalmologic abnormalities occurred. Tamoxifen did not enhance the toxicity of vinblastine. Mean plasma concentrations of tamoxifen or N-desmethyltamoxifen at 260 mg/m2 tamoxifen given twice a day for 13 days were 6.04 and 6.56 microM, respectively. There was no relationship between plasma antiestrogen content and the development of neurotoxic effects. CONCLUSIONS: Tamoxifen at 150 mg/m2 given twice a day following a loading dose of 400 mg/m2 results in plasma levels of tamoxifen and N-desmethyltamoxifen of 4 and 6 microM, respectively, without dose-limiting toxicity. We recommend this dose for phase II trials of tamoxifen to modulate P-glycoprotein-mediated drug resistance. IMPLICATIONS: Our study demonstrates that high-dose tamoxifen can be safely administered and that plasma concentrations that may inhibit P-glycoprotein function can be achieved.

Paraneoplastic hepatopathy associated with soft tissue sarcoma.

Paraneoplastic syndromes associated with mesodermal tumors are relatively uncommon. An unusual case manifested by fever, anemia, thrombocytosis, coagulopathy, and idiopathic cholestatic liver dysfunction in association with soft tissue sarcoma is reported. A paraneoplastic syndrome is postulated in the absence of anatomic obstruction of bile flow, evidence of an infectious etiology, or neoplastic hepatic involvement.

Modulation of O6-alkylguanine-DNA alkyltransferase-mediated carmustine resistance using streptozotocin: a phase I trial.

1,3-Bis(2-chloroethyl)-1-nitrosourea (BCNU) resistance may be mediated by repair of chloroethylated guanine before stable cross-linking occurs. Guanine adducts may be repaired by the enzyme O6-alkylguanine-DNA alkyltransferase (O6-AGAT). Such repair irreversibly inactivates O6-AGAT. Streptozotocin (STZ) forms adducts at the O6 position of guanine; repair of these adducts consumes O6-AGAT. In vivo STZ potentiates BCNU cytotoxicity. The purpose of this trial was to determine the maximum tolerated dose of BCNU that can be administered together with STZ. The STZ dose was 500 mg/m2/day for 4 days and was not escalated. BCNU was given 4 h after the third dose of STZ at a starting dose of 75 mg/m2. A total of 43 patients were entered in the study. There were 4 dose escalations, reaching a maximum tolerated BCNU dose of 175 mg/m2. At this dose, thrombocytopenia was the dose-limiting toxicity (one patient, 25-49 x 10(9)/liter; 2 patients, less than 25 x 10(9)/liter); neutropenia was less severe (2 patients, 2.0-3.9 x 10(9)/liter, 1 patient, 1.0-1.9 x 10(9)/liter). Two other commonly seen toxicities were elevations in the serum alkaline phosphatase and mild elevations in the serum creatinine. Peripheral blood lymphocyte O6-AGAT levels decreased from a mean of 212 fmol/mg protein pretherapy to 8.2 fmol/mg protein on day 3 prior to BCNU (P = 0.03). Three partial responses were seen. There were no therapy-related fatalities, and toxicity was easily managed. This study established that 150 mg of BCNU can be administered safely together with STZ, 500 mg/m2/day for 4 days. Additional studies are required to determine whether O6-AGAT-mediated BCNU resistance is suppressed.

Polymorphism and altered methylation of the lactoferrin gene in normal leukocytes, leukemic cells, and breast cancer.

Human lactoferrin has been found to be decreased or absent in most breast cancer and leukemia cells. In order to examine the lactoferrin gene for both structural alterations and the degree of methylation, we isolated a 2117-kilobase complementary DNA from human breast tissue. This complementary DNA was used to probe DNA extracted from normal peripheral blood, leukemia cells from patients, leukemia cell lines, and breast cancer cell lines. Immunocytochemical staining of these cells confirmed the decreased production of lactoferrin in malignancy. MspI restriction enzyme fragment patterns demonstrated genetic polymorphism which occurred in DNA from both normal and malignant cells. Polymorphism was also noted with XbaI. In this case, there were two fragment patterns that were only found in DNA from malignant cells. The degree of DNA methylation was also evaluated. The methylation pattern of DNA extracted from malignant cells was highly variable and generally less methylated than DNA extracted from normal WBCs. It is possible that the decrease in lactoferrin associated with cancer is multifactorial and includes gene structural changes as well as altered regulation. Further study is needed to determine whether the changes found in this study are the result of the malignancy or contribute to its onset or maintenance.

Absence of phosphatidylinositol (PI)-linked proteins in a very early human multipotential haematopoietic marrow cell.

A very early human haematopoietic progenitor cell population which was negative for the major histocompatibility class II antigen (HLA-DR) and positive for the CD34 (MY10) antigen was separated into two subsets according to the expression of decay-accelerating factor (DAF) on the cell surface. Using immunoadherence, cell cycle analysis, and cell culture, we determined that there is a DAF- multipotential cell and a more differentiated DAF+ lineage specific progenitor cell existing in human bone marrow. The DAF- subset was highly enriched for CFU-GEMM, while the DAF+ subset contained only BFU-E and CFU-GM. The DAF- subset was approximately 0.03% and the DAF+ subset approximately 0.008% of the original bone marrow population. MIRL (membrane-inhibitory of reactive lysis), another PI-linked protein, was not expressed on the DAF- population but was expressed on the DAF+ cells. These observations indicate that PI-linked proteins are absent from the multipotential stem cell but are present on an early lineage specific cell. The absence of expression of PI-linked proteins can be used to further isolate and characterize a very early multipotential haematopoietic progenitor cell population.

Anti-human immunodeficiency virus synergism by zidovudine (3'-azidothymidine) and didanosine (dideoxyinosine) contrasts with their additive inhibition of normal human marrow progenitor cells.

The anti-human immunodeficiency virus (HIV) activity and hemopoietic toxicity of zidovudine (AZT) and didanosine (dideoxyinosine;ddI), alone and in combination, were assessed in a variety of cell types. AZT was more potent than ddI as an inhibitor of HIV in vitro. Synergistic inhibition of HIV by the combination of these agents was observed in MT4 cells, peripheral blood lymphocytes, and macrophages. Toxicity assessment in vitro by using progenitor (erythroid and granulocyte-macrophage) colony-forming assays with normal human bone marrow showed ddI to be less toxic than AZT. Addition of inhibitory concentrations of ddI to AZT resulted in additive inhibition of progenitor CFUs. These in vitro findings suggest that combinations of ddI and AZT at appropriately modified doses may provide an enhanced degree of selectivity in anti-HIV chemotherapy.

Effect of thimerosal in leukemia, in leukemic cell lines, and on normal hematopoiesis.

Anti-thymocyte globulin (ATG), a horse antiserum to human thymus tissue, has been shown to induce granulocytic differentiation of the HL-60 human leukemia cell line. In this paper we describe the effect of ATG on leukemic blasts and its effect on other human leukemia cell lines in vitro. The in vitro differentiation effect of ATG was observed in blasts from two patients with leukemia and the human leukemia cell line K562. The differentiation effect of ATG was attributable to its preservative, thimerosal, separable from ATG by high pressure liquid chromatography or dialysis. Subsequent studies with thimerosal alone showed it to induce differentiation in leukemic blasts from three patients and the human leukemia cell lines U937, K562, and KG-1. The differentiation effect of thimerosal is blocked by a sulfhydryl-protective agent, dithiothreitol, suggesting that the mechanism of differentiation may be mediated via a sulfhydryl group-dependent process.

Platelets acquire a secretion defect after high-dose chemotherapy.

Patients receiving high-dose chemotherapy (HDC) and autologous bone marrow transplantation (ABMT) may experience life-threatening hemorrhagic myocarditis. The authors investigated whether HDC was associated with an acquired platelet defect. Platelet aggregation and release were evaluated after HDC in ten patients with either metastatic breast carcinoma or melanoma. Platelets underwent shape change and a primary wave of aggregation. High-dose chemotherapy was associated with the inhibition of secondary aggregation of platelets induced by adenosine diphosphate (ADP), arachidonic acid, prostaglandin H2 (PGH2) analog (U44619), and collagen. Although electron microscopic study of the platelets revealed normal morphologic features with an adequate number of dense bodies and alpha-granules, release of adenosine triphosphate (ATP) from dense granules was less than 20% of normal. The acquired platelet defect occurred before development of thrombocytopenia. Aggregation of platelets from normal volunteers was not inhibited by either the addition of the chemotherapeutic agents, chemotherapy metabolites, or the patients' sera. In conclusion, HDC induces an acquired abnormality in platelet secretion and aggregation which may contribute to the development of hemorrhagic complications after ABMT.