Phase II trial of high-dose carboplatin and etoposide with autologous bone marrow transplantation in first-line therapy for patients with poor-risk germ cell tumors.

BACKGROUND: Between 20% and 30% of patients with advanced germ cell tumors (GCTs) fail to have durable complete response to conventional cisplatin-based induction chemotherapy. However, third-line therapy with high-dose carboplatin and etoposide plus autologous bone marrow transplantation (AuBMT) has induced durable complete response in 10%-20% of patients with cisplatin-resistant GCT. PURPOSE: We conducted a phase II trial of first-line therapy that included high-dose carboplatin and etoposide plus AuBMT in untreated men with advanced GCTs and unfavorable prognosis (i.e., "poor-risk" patients). METHODS: Twenty-eight patients were treated with a conventional-dose, cisplatin-containing regimen (VAB-6 [cisplatin, vinblastine, bleomycin, cyclophosphamide, dactinomycin]) with or without high-dose carboplatin (1500 mg/m2) and etoposide (1200 mg/m2) plus AuBMT. Twenty-two of these patients were selected for treatment with two cycles of high-dose carboplatin and etoposide plus AuBMT when reduced clearance of serum tumor markers (alpha-fetoprotein [AFP] or human chorionic gonadotropin [HCG]), as evidenced by prolonged half-life (> 7 days for AFP and > 3 days for HCG), was observed after two cycles of conventional treatment. RESULTS: Fifteen (56%) of 27 patients considered assessable for response achieved a complete response (12 treated with high-dose chemotherapy plus AuBMT). Sixteen (57%) are alive; 13 (46%) are free of disease at a median follow-up of 31.2 months. For 36 cycles of high-dose chemotherapy, the median duration from bone marrow infusion until a granulocyte count of 0.5/mm3 and a platelet count of 50,000/mm3 was 16 days (range, 7-41 days and 8-30 days, respectively). Analysis showed a trend toward improved survival (P = .07) in patients treated with high-dose chemotherapy in this study, compared with 68 poor-risk patients with GCT treated with conventional-dose therapy alone in two earlier studies. Toxicity was not cumulative, and recovery of blood counts after AuBMT was generally rapid. CONCLUSIONS: Inclusion of high-dose carboplatin-containing chemotherapy in treatment of poor-risk GCT patients is feasible when serum tumor marker half-life is used to predict resistance to standard cisplatin-based therapy. High-dose therapy in this setting was well tolerated. IMPLICATIONS: Early use of a dose-intensive regimen may increase survival compared with conventional-dose therapy alone. Further studies with standard induction therapy and intensive high-dose therapy using hematopoietic growth factor support are warranted, followed by a randomized trial comparing this strategy with standard therapy.

Comparison of the cytotoxic effects of merocyanine-540 on leukemic cells and normal human bone marrow.

Various chemical compounds have been described to induce photosensitization of tumor cells resulting in cell death. We studied the effect of merocyanine-540 (MC-540) on both leukemic and normal bone marrow (BM) cells. Acute promyelocytic leukemia (HL-60) and common acute lymphoblastic leukemia antigen-positive non-T, non-B acute lymphoblastic leukemia (Reh) cell lines were incubated with MC-540 and simultaneously exposed to white light. Normal human BM and mixtures of leukemic cells with BM cells were treated under similar conditions. At constant illumination rates of 50,000 lx, significant (at least 4 to 5 logs) tumor cell destruction was obtained with MC-540 concentrations of 20 micrograms/ml or more for HL-60, and 10 micrograms/ml or more for Reh cells. Incubation of BM under equivalent conditions preserved 18.0% of granulocyte-macrophage colony-forming units and 14.2% of erythroid burst-forming units. Similar results were obtained when tumor cells were mixed with irradiated BM and then treated with MC-540. In summary, cell photosensitization with MC-540 has a selective cytotoxic effect towards leukemic cells and therefore may be useful for purging tumor cells from autologous BM.

Discordant maturation as the primary biological defect in chronic myelogenous leukemia.

Comparative studies of the in vitro growth characteristics of normal and chronic myelogenous leukemic (CML) progenitor cells have provided further evidence that discordant maturation is the primary biological defect in CML. The in vitro growth of total normal and CML granulocyte/macrophage colony forming unit (CFU-GM) populations were compared with early and intermediate (HLA-DR positive) CFU-GM derived from the same marrows. The absolute number of total CML CFU-GM exceeded the number generated by normal marrow through 7 days of culture due entirely to an excess of CML CFU-GM with limited proliferative capacity. Unlike normal colonies, relatively few of the leukemic colonies grew to a large size; the early and intermediate (HLA-DR positive) CML progenitors also exhibited limited proliferative capacity compared to normal. Highly enriched progenitor populations were prepared, and it was observed that the primitive (small) CML CFU-GM also had greatly reduced proliferative potential compared to primitive normal progenitors, but rather behaved similarly to normal mature (large) CFU-GM. Similarly, CML erythroid burst forming units were at a more advanced stage of maturation than normal erythroid burst forming units as evidenced by their reduced proliferative capacity, the observation that a reduced proportion required burst promoting activity to enable them to respond to erythropoietin and the observation that a larger fraction than normal could sustain a limited period of erythropoietin deprivation in the absence of burst promoting activity. Based on these findings and supporting evidence from our previous studies and those reported by other investigators, it is concluded that the dominance of the leukemic population is not due to unregulated proliferation but rather to discordant maturation resulting in expansion in the later maturational compartments which are not under strict regulatory control.

Phase I trial with pharmacokinetic analyses of high-dose carboplatin, etoposide, and cyclophosphamide with autologous bone marrow transplantation in patients with refractory germ cell tumors.

Thirty patients with cisplatin-refractory germ cell tumor were treated with high-dose carboplatin, etoposide, and cyclophosphamide and autologous bone marrow transplantation. The total dose of carboplatin was 1500 mg/m2, etoposide 1200 mg/m2, and cyclophosphamide was increased by increments from 60 to 150 mg/kg. Twenty-five cycles of treatment, given to 17 patients, did not include granulocyte-colony stimulating factor (G-CSF). Nineteen cycles of high-dose chemotherapy, given to 13 patients at the 2 highest dose levels of cyclophosphamide, included G-CSF. The dose of cyclophosphamide was escalated to 150 mg/kg/cycle without prohibitive toxicity. The use of G-CSF resulted in a shorter duration of neutropenia (P = 0.07); the median number of days until the recovery of an absolute granulocyte count > 0.5 was 25 without G-CSF and 14 with G-CSF. The most frequent nonhematological toxicity was hepatic, and there were 2 (7%) treatment-related deaths. Thirteen (43%) patients achieved a complete response, and 8 are alive and free of disease (27%); 7 are in continuous complete response (23%), and 1 after resection of a solitary site of disease following a relapse after high-dose chemotherapy. Five patients had pharmacology studies performed to determine the area under the curve (AUC) of free and total platinum, carboplatin, etoposide, cyclophosphamide, and phosphoramide mustard. There was a decrease in the AUC of cyclophosphamide and an increase in the AUC of phosphoramide mustard, the "active" metabolite, with successive days of treatment. The interpatient variability of the AUC of cyclophosphamide/phosphoramide mustard that was demonstrated was most likely a result of each individual's metabolic capacity. The measured AUC of carboplatin and/or free platinum closely approximated the predicted AUC of carboplatin calculated by renal function in 3 of the 5 patients. In summary, cyclophosphamide administered at a dose of 50 mg/kg x 3 days was achieved with acceptable toxicity, and no further dose escalation is planned. High-dose carboplatin, etoposide, and cyclophosphamide achieved a 23% continuous complete response proportion (27% alive, free of disease) when used as third-line therapy in germ cell tumor patients refractory to cisplatin + ifosfamide-based chemotherapy. Ongoing studies are addressing the role of high-dose carboplatin-containing chemotherapy in previously untreated patients with poor prognostic features or as a part of first-line salvage.

Synergistic effect of 4-hydroperoxycyclophosphamide and etoposide on a human promyelocytic leukemia cell line (HL-60) demonstrated by computer analysis.

Autologous stem cell transplantation using cryopreserved bone marrow offers the opportunity to rescue patients from hematopoietic toxicity caused by intensive chemotherapy. This approach is potentially useful for high-risk leukemias as well as for other cancers. The development of suitable methods for purging malignant cells from the bone marrow will offer a better chance of success for autologous stem cell transplantation. In this paper, we describe our efforts at purging myeloid cells. HL-60, a promyelocytic leukemia cell line, was used as a model. 4-Hydroperoxycyclophosphamide (4-HC) and VP-16-213 (VP-16) (either alone or in combination) were used to treat HL-60 cells and normal bone marrow. The cytotoxic effect of 4-HC (29.2 micrograms/ml; 100 microM) upon the HL-60 cell line was 99.8 +/- 0.12% (SE), and the colony-forming units-granulocyte, macrophage (CFU-cs) of normal bone marrow was inhibited by 82.5%. VP-16, at a concentration of 25 micrograms/ml (42.5 microM), can kill 99% of HL-60 cells and inhibit 72.7% of the CFU-cs. A drug mixture containing 4-HC (29.2 micrograms/ml) and VP-16 (10 micrograms/ml) (combination ratio, 1:0.342) reduces HL-60 cells to an undetectable number, and the CFU-cs were inhibited by 87.2%. The laboratory data were further analyzed for the synergistic effect of these two drugs by quantitative determination of the median effect plot and the multiple drug equation recently described by Chou and Talalay (Adv. Enz. Regul., 22: 27-55, 1984). Interactions of two drugs at different effect levels and at different combination ratios were then determined by computer simulation. At high effect levels, 4-HC and VP-16 in combination gave a synergistic cytocidal effect on HL-60 leukemic cells and gave an antagonistic inhibitory effect on normal bone marrow CFU-cs. This combination greatly increases the safety margin. Computer simulation of a dose effect relationship has also shown that the 4-HC:VP-16 combination ratio of 1:0.342 yields a better selective effect than a ratio of 1:0.856. This quantitative analysis suggests that the combination of these two drugs at the selected dose level offers a good method for purging nonlymphoblastic leukemia cells.

Pregnancy after bone marrow transplantation.

PURPOSE: To evaluate the occurrence of pregnancy after bone marrow transplantation (BMT). DESIGN: Medline literature review of reported pregnancies in the BMT population published in the English language. RESULTS: Multiple case reports and a few series studies showed more than 250 offspring from BMT recipients. CONCLUSION: BMT patients receive high-dose chemotherapy and often radiation, as well. These agents are associated with gonadal dysfunction and the fertility of patients after BMT is of concern because BMT patients are often young people who wish to resume a normal quality of life, which for many patients involves the desire to have children. Our experience with the successful pregnancy of one of our BMT patients led to the investigation of reported cases that showed numerous other births. The issue of counseling BMT patients about fertility, pregnancy complications, and potential birth defects is becoming increasingly complex and warrants further investigation.

Harvesting bone marrow in an outpatient setting using newer anesthetic agents.

PURPOSE: To evaluate the results of outpatient bone marrow harvest (BMH). PATIENTS AND METHODS: Seventy-two adult patients with various malignancies had 79 BMH procedures performed for future autologous bone marrow transplantation (BMT) in our institution's outpatient surgical facility. All patients were evaluated and educated before the procedure. Newer anesthetic agents specifically developed to have shorter half-lives, more rapid recovery from general anesthesia, and fewer unpleasant side effects were chosen. Propofol was used for induction of anesthesia in 76 patients, the other three were induced with sodium pentothal. The blood volume removed was replaced by colloid (6% hydroxyethyl starch). Also, a new parenteral nonnarcotic pain medication, ketoroloc, was used during the last part of general anesthesia to help with expected postoperative pain in 76 patients. RESULTS: BMH took 111 +/- 24 minutes and patients were in postanesthesia care unit (PACU) for 220 +/- 72 minutes before being sent home with a companion and Tylenol with codeine (acetaminophen with codeine; McNeil Pharmaceutical, Spring House, PA). PACU complications were minor and included transient mild dizziness (7.6%), vomiting (3.8%), and fever (2.6%). No life-threatening complication was observed. Only one patient was hospitalized for observation (fever) and then sent home. Seventy-five patients (94.9%) were contacted at home by the hospital nursing staff the day following the procedure. Five (6.7%) complained of nausea or vomiting, and four (5.3%) developed fever at home (temperature, 37.2 to 38.3 degrees C). Only 36% of patients actually took oral narcotic pain medication at home. CONCLUSION: Autologous BMH (AuBMH) is a safe outpatient procedure with minimal side effects when newer anesthetic agents are used.

Autologous bone marrow transplantation for patients with poor-prognosis lymphoma.

Review of prognostic factors at Memorial Hospital in New York City has shown that adult patients with large-cell lymphoma (diffuse histiocytic lymphoma by Rappaport classification) who have high lactic dehydrogenase (LDH) and/or bulky mediastinal or abdominal disease are destined to do poorly with conventional combination chemotherapy, with a 2-year disease-free survival of about 20%. Patients who relapse after conventional combination chemotherapy have a similar poor prognosis. Thirty-one such patients with lymphoma were studied to evaluate the efficacy of intensive radiotherapy (hyperfractionated total body irradiation [TBI] [1,320 rad]), and cyclophosphamide (60 mg/kg/d for two days) followed by autologous bone marrow transplantation (ABMT). Our results show a disease-free survival advantage (P = .002) for 14 patients who underwent ABMT immediately after induction of remission with 79% surviving at a median follow-up 49.2+ months, compared with a median survival of 5.2 months for 17 patients administered ABMT while in relapse and/or after failing conventional treatment. Our results support the use of aggressive therapy as early treatment for patients with poor prognostic features.

Accelerated hyperfractionated total-lymphoid irradiation, high-dose chemotherapy, and autologous bone marrow transplantation for refractory and relapsing patients with Hodgkin's disease.

PURPOSE: To evaluate the feasibility and therapeutic effect of accelerated hyperfractionated total-lymphoid irradiation (TLI), high-dose chemotherapy, and autologous bone marrow transplantation (AuBMT) in patients with relapsing or chemotherapy-resistant Hodgkin's disease (HD). PATIENTS AND METHODS: Forty-seven patients with HD who either relapsed after chemotherapy (n = 19), or failed to respond (n = 28) to at least two regimens of combination chemotherapy were studied. No patient received prior radiation therapy (RT). Treatment started with reinduction with standard-dose chemotherapy, followed by involved-field irradiation (15 Gy) to areas of relapsed or persistent disease and TLI (20.04 Gy given in 1.67 Gy fractions three times per day for 4 days). Subsequently, patients received etoposide and high-dose cyclophosphamide, followed by infusion of unpurged autologous bone marrow. All surviving patients had a minimum follow-up duration of 1 year. The median follow-up duration for survivors was 40+ months, and the maximum follow-up duration was 80+ months. RESULTS: Of the 47 patients treated, eight (17%) died of toxicity during the peritransplant period. Twenty-nine of the remaining 39 assessable patients (74%) attained a complete response (CR), while 10 remained with residual disease and progressed early after AuBMT. Four of the CR patients (14%) relapsed and 25 patients remained alive and free of disease. The actuarial disease-free survival (DFS) rate for the entire group at 6.5 years was 50%. Patients who received the protocol for relapsing HD had a significantly better DFS rate (79%) compared with patients treated for continuous refractory disease (DFS, 33%; P < .03). CONCLUSION: Previously unirradiated patients with relapsing or chemotherapy-resistant HD who have exhausted conventional chemotherapy may still respond to an aggressive therapeutic approach consisting of accelerated hyperfractionated TLI, high-dose chemotherapy, and AuBMT rescue. This program offers a potential for long-term DFS to approximately one half of patients who would otherwise have a dismal prognosis with standard-dose salvage therapy.

Myeloablative combination chemotherapy without total body irradiation for neuroblastoma.

Myeloablative treatment intensification in 25 patients diagnosed when older than 12 months of age with stage IV neuroblastoma included sequential delivery of cisplatin 120 mg/m2 x 1, hyperfractionated radiation (2,100 cGy) to the primary site and adjacent lymph nodes, carmustine (BCNU) 200 mg/m2 x 1, melphalan 60 mg/m2/d x 3 (n = 13) or thiotepa 300 mg/m2/d x 3. (n = 12), and etoposide (VP 16) 300 mg/m2/d x 3. Seventy-two hours after the last dose of VP 16, histologically tumor-free and 4-hydroperoxycyclophosphamide (4-HC; 100 mumol/L)-purged autologous bone marrow (ABMT) was infused. Acute toxicities included grade 3 to 4 oral mucositis, grade 1 to 2 diarrhea, and fevers. No patient required infusion of unpurged reserve autografts. At ABMT, 16 patients (group I) were progression-free 6.5 months to 14 months (median, 9 months) from diagnosis: seven remain progression-free 20 months to 46 months (median, 39 months) off therapy, six relapsed 4 months to 17 months post-ABMT, and three died of toxicity (candidiasis, metabolic derangement, and venoocclusive disease [VOD]). The event-free survival of group I patients is 44% at 24 months post-ABMT. Nine patients (group II) were in second remission at ABMT, including three who had relapsed after other transplant procedures: two are progression-free 24 months and 41 months off therapy, four relapsed 3 months to 12 months post-ABMT, and three died of toxicity (aspergillosis, hemorrhagic cystitis, VOD). Only one of 10 relapses involved a primary site, suggesting a beneficial effect of local radiation. In terms of survival or toxicity, an advantage for melphalan or thiotepa was not evident. Regimens such as this may prolong the survival of selected patients with poor-risk neuroblastoma, but concerns over late relapses and toxicity mandate continuing efforts to devise alternative, less risky, and more clearly beneficial approaches for definitive ablation of neuroblastoma.

High-dose induction chemoradiotherapy followed by autologous bone marrow transplantation as consolidation therapy in rhabdomyosarcoma, extraosseous Ewing's sarcoma, and undifferentiated sarcoma.

PURPOSE: To improve response and survival rates in patients with high-risk rhabdomyosarcoma (RMS), extraosseous Ewing's sarcoma, and undifferentiated sarcoma, we used a short course of induction with multi-agent chemotherapy, hyperfractionated radiotherapy, and surgery when possible. Consolidation was with intensive chemotherapy and autologous bone marrow transplantation (ABMT). PATIENTS AND METHODS: Twenty-six patients (21 with RMS, three with undifferentiated sarcoma, and two with extraosseous Ewing's sarcoma) were entered onto the protocol between June 1990 and March 1994. Induction consisted of ifosfamide, etoposide, doxorubicin, dactinomycin, cyclophosphomide, and vincristine, and a split course of hyperfractionated radiotherapy. Patients who attained a complete response (CR) or good partial response (GPR) received consolidation with high-dose melphalan and etoposide followed by ABMT. RESULTS: Of 26 previously untreated patients 19 (73%) achieved a CR (n=13) or GPR (n=6) at the completion of induction and underwent ABMT. Two-year overall survival (OS) was 56% (95% confidence interval [CI], 36% to 76%) and progression-free survival (PFS) was 53% for the whole group (95% CI, 33% to 73%). CONCLUSION: Consolidation of response by myeloablative chemotherapy was well tolerated. Split-course hyperfractionated radiotherapy did not increase the rate of local control. The results of this short-course therapy were comparable to previous therapies of 1 to 2 years' duration. Induction and consolidation chemotherapy, as well as radiation dose, could be further intensified, since no death due to toxicity occurred among these patients.

Proliferative response of human acute myeloid leukemia cells and normal marrow enriched progenitor cells to human recombinant growth factors IL-3, GM-CSF and G-CSF alone and in combination.

The effects of human recombinant colony-stimulating factors (r-CSFs), interleukin 3 (IL-3), granulocyte-macrophage colony-stimulating factor (GM-CSF) and granulocyte colony-stimulating factor (G-CSF) on inducing the growth of colonies derived from patients with acute myeloid leukemia (AML) (CFU-L) were investigated and compared to the proliferative response of CFU-GM derived from highly enriched normal blast cell populations. The effects of GM-CSF and IL-3 alone were similar. Both only minimally stimulated normal colonies derived from CFU-GM when compared to stimulation with MoCM (a mean of 28% of the total colonies and 17% of the colonies greater than 100 cells obtained with MoCM). Similarly, the number of leukemic colonies was substantially less than with MoCM (less than 30% of MoCM) in all but 3/10 AML patients and both were only able to significantly stimulate CFU-L derived colonies greater than 50 cells from 2/10 patients. G-CSF alone stimulated some CFU-L derived colony growth in 9/10 patients but the number stimulated was minimal relative to MoCM in five of the patients and significant stimulation of colonies greater than 50 cells occurred in only one patient. The mean number of normal CFU-GM derived colonies stimulated by G-CSF was 41% of the total colonies and 34% of the colonies greater than 100 cells generated by MoCM. The combination of G-CSF with GM-CSF and G-CSF with IL-3 resulted in a synergistic or additive increase in the number of CFU-L in 5/10 and 7/10 patients, respectively, and a synergistic increase in the size of CFU-L in 5/10. The same combinations resulted in a significant synergistic effect on size of normal CFU-GM derived colonies. There was no evidence of a synergistic increase in the number or size of CFU-L and CFU-GM derived colonies stimulated with GM-CSF in combination with IL-3. In addition, a combination of all three (G-CSF + GM-CSF + IL-3) did not enhance the effect of G-CSF + GM-CSF or G-CSF + IL-3. These results suggest that there is significant heterogeneity among AML patients in the pattern of responsiveness of the leukemic cells to the recombinant growth factors. In addition, their responsiveness does not significantly differ from that of normal progenitors. In view of the current clinical trials with r-CSFs and cytotoxic drugs in AML patients, this issue is important and worthy of further investigation.(ABSTRACT TRUNCATED AT 400 WORDS)

Combination of hematopoietic growth factors containing IL-3 induce acute myeloid leukemia cell sensitization to cycle specific and cycle non-specific drugs.

Laboratory studies have suggested that hematopoietic growth factors (GF), combined with cytosine-arabinoside (Ara-C) can enhance cytotoxic effects of this agent against acute myeloid leukemia (AML) cells. While clinical trials based on this growth factor/chemotherapy combination (GF/CT) are progressing with discordant results, further information regarding the underlying mechanisms have been reported supporting this rationale and requiring additional investigation. To assess the role of cytokinetic changes in the GF/CT strategy and to evaluate if chemotherapeutic agents regimens other than Ara-C, when combined with GF, can enhance their cytotoxic effects, we have primed AML blasts with two cytokine combinations and then exposed these cells to the S-phase specific agent Ara-C as well as to the phase non-specific drug daunorubicin (DNR) and to the alkylating agent 4-hydroperoxycyclophosphamide (4-HC). The two cytokine combinations used for priming AML blasts were: (i) interleukin-3 (IL-3) + granulocyte-macrophage colony-stimulating factor (GM-CSF) + granulocyte colony-stimulating factor (G-CSF); and (ii) GM + G-CSF. Cytokinetic analysis in ten AML samples and clonogenic growth of leukemic colonies (CFU-L) in methylcellulose were used to detect proliferative and cytotoxic effects on AML samples. We report that in AML clonogenic cell growth can be stimulated by cytokines in 50% of the samples (4/8), and that Ara-C sensitization clearly occurs in two out of these four samples. Among the different cytokine combinations tested, the one containing IL-3 was the most effective through a cytokinetic mechanism consistent with recruitment (averaged G0 decrease p = 0.04; S-phase increase p = 0.005). Furthermore we observed increased cytotoxicity also to the phase non-specific drugs DNR and 4-HC, which may be mediated by other mechanisms recently described. We conclude that GF/CT combinations may also be beneficial in regimens containing drugs other than Ara-C, used for AML treatment, including bone marrow transplantation conditioning regimens.

In vitro cytotoxicity of VP-16-213 and nitrogen mustard: agonistic on tumor cells but not on normal human bone marrow progenitors.

The possible presence of tumor cells in remission bone marrow (BM) is one of the major problems for the success of autologous BM transplantation (ABMT), because the reinfusion of viable malignant cells may result in relapse. In this study we attempted the purging of the malignant cells by the use of VP-16-213 (VP-16) and nitrogen mustard (NM) either alone or in combination. Four cell lines from various hematological malignancies were utilized: SK-DHL-2 was established from a B-cell diffuse histiocytic lymphoma; RAJI was from an Epstein-Barr virus (EBV)-infected B-cell lymphoma cell line; K-562 were from a chronic myelogenous leukemia (CML) blastic crisis; and HL-60, derived from a human promyelocytic leukemia, were used in exponential growth phase. Four logs of tumor cell-elimination were observed after 1-h incubation of RAJI cells with 25 micrograms/ml of VP-16. K-562 and SK-DHL-2 cells showed a greater than 4 logs reduction after 1-h exposure to 75 micrograms/ml of VP-16, and HL-60 cell line growth was inhibited by 3.2 logs. Under the same conditions (i.e., the treatment with 75 micrograms/ml), we observed a mean recovery of 2.7% of BM granulocyte-macrophage colonies (granulocyte-macrophage colony-forming units, CFU-GM), 3.2% of erythroid (erythroid burst-forming units, BFU-E), and 2.5% of pluripotent (granulocyte erythrocyte macrophage megakaryocyte colony-forming units, CFU-GEMM) progenitors, respectively. More than 3 logs reduction of leukemia and lymphoma cell lines were reached following 1-h treatment with 1 micrograms/ml of NM. After exposure to the same concentration of the drug we obtained 2.5% CFU-GM, 1.2% BFU-E, and 2% CFU-GEMM recovery. A drug mixture containing constant doses of VP-16 (10 and 20 micrograms/ml) and NM (1 micrograms/ml) reduced HL-60 and SK-DHL-2 cell growth to undetectable levels (i.e., 4 and 5 logs elimination) in the presence of an excess of irradiated BM cells, whereas it did not further affect the recovery of the BM precursors as compared to the single drugs used alone. These results suggest that the combination of these two drugs at the selected dose level could provide a better therapeutic index (i.e., higher tumor cell killing coupled with no additional cytotoxic effect on normal BM cells) than the same chemotherapeutic agent used alone and that this mixture may be useful for the "ex vivo" treatment of BM grafts.

TGF-beta 3 protects normal human hematopoietic progenitor cells treated with 4-hydroperoxycyclophosphamide in vitro.

In this study we have investigated the ability of transforming growth factor-beta 3 (TGF-beta 3, 1000 pM) to protect hematopoietic bone marrow (BM) progenitor cells from the cytotoxic activity of 4-hydroperoxycyclophosphamide (4-HC, 100 microM) in vitro. Hematopoietic progenitors were purified by negative depletion of accessory and maturing cells or enriched by positive (CD 34+ cells) selection. For comparison the same treatment was tested on three different lymphoid cell lines CEM, SK-DHL-2, and LY-16. The experimental protocol was designed to mimic ex vivo purging conditions. Therefore, tumor cells and enriched hematopoietic precursors were mixed with irradiated BM cells. Our results demonstrated that preincubation of enriched progenitor cells with TGF-beta 3 for up to 72 h followed by 4-HC treatment resulted in an increased survival of colonies derived from granulocyte-macrophage (CFU-GM) and erythroid (BFU-E) colony-forming cells, whereas a substantially lower number of colonies was observed in the control group. Similar results were observed when BM cells were first treated with 4-HC followed by TGF-beta 3 incubation for 24 or 48 h. In contrast, TGF-beta 3 provided no protection to the 4-HC cytotoxicity toward the lymphoma and leukemia cell lines. Three to four log of tumor cell killing was induced by 4-HC in the presence or absence of preincubation with TGF-beta 3. These data suggest that TGF-beta 3 is able to protect normal BM progenitors from the cytotoxic activity of an alkylating agent (4-HC) in vitro, whereas it does not offer any protection to lymphoma cell lines. These findings will have important implications for developing better purging conditions for autologous GM transplantation.

Proliferation of human hematopoietic progenitors in long-term bone marrow cultures in gas-permeable plastic bags is enhanced by colony-stimulating factors.

We compared the recovery of human hematopoietic progenitors in long-term bone marrow culture (LTBMC) initiated in tissue culture (TC) flasks to that in "Lifecell" bags, which are gas-permeable plastic bags in which feeder-layer cells cannot adhere. Our results showed that granulocyte-macrophage colony-forming unit (CFU-GM) and erythroid burst-forming unit (BFU-E) cumulative recovery in cultures from normal donor marrow, expressed as a percent of the initial inoculum, was not statistically different in the two culture systems up to week 8, when the cultures were terminated: 31.5 +/- 19 (flask) vs 30 +/- 14 (bag) and 15.5 +/- 12 (flask) vs 11.5 +/- 8 (bag), respectively. The effects of weekly addition of recombinant human (r-hu)-interleukin 1 (IL1) and r-hu-interleukin 3 (IL3) were then studied, alone and combined, at two different concentrations. Addition of IL1, either alone or combined with IL3, in LTBMC established in flasks induced an increase of hematopoietic progenitors for the first week, but BFU-E and CFU-GM were no longer detectable at weeks 4 and 6, respectively. Analysis of adherent layer cells showed a decreased cellularity, no adipogenesis, and early disappearance of bone marrow (BM) progenitors, whereas the cycling rate of myeloid precursors, by cytosine arabinoside (Ara-C) suicide assay, was similar to that of untreated cultures. Conversely, IL3 alone (5 ng/ml) resulted in 3.6- and 5.4-fold peak increases for CFU-GM and BFU-E, respectively, at week 1 (adherent plus nonadherent cells), and the recovery of BM cells was still higher than that of control flasks at week 8. By comparison, stimulation with colony-stimulating factors (CSFs) of BM cells grown in bags never affected the longevity of the culture. Addition of IL3 (5 ng/ml) induced a higher recovery of total cells, CFU-GM (range: 1.6- to 15-fold peak increase during the culture), and BFU-E (1.2- to 3-fold) compared to the untreated controls. Bags treated with IL1 alone demonstrated only transient beneficial effects, and the number of hematopoietic precursors fell below the level of control bags during the culture. IL1 and IL3 induced 1.8- and 5.3-fold peak increases in BFU-E and CFU-GM at weeks 1 and 4, respectively. Simultaneous flow cytometric analysis of CD34+/CD33+ cells and DNA content showed increased numbers and proliferation of the committed BM progenitors when CSFs were added to the bag.(ABSTRACT TRUNCATED AT 400 WORDS)

Fusarium infections in patients with hematologic malignancies.

Two cases of Fusarium infection in patients with refractory hematologic malignancies are reported. In one patient septicemia progressed to death in septic shock. Miconazole showed some effect in clearing the lesions. There is some evidence that mycotoxins are related with Fusarium infections since severe myositis occurred in our patient. The other patient had a T-cell lymphoma, undergoing allogeneic bone marrow transplantation. The course was also complicated by Fusarium infection of the skin. This patient died of multiorgan failure. Recent literature on Fusarium is reviewed.

Excretion of Ascaris lumbricoides during total body irradiation.

We describe the excretion of Ascaris lumbicoides, an intestinal roundworm, in the emesis of an asymptomatic patient undergoing total body irradiation. This suggests that Ascaris is sensitive to irradiation.

Positive selection of hematopoietic CD34+ stem cells provides 'indirect purging' of CD34- lymphoid cells and the purging efficiency is increased by anti-CD2 and anti-CD30 immunotoxins.

Human CD34+ hematopoietic cells were purified using the avidin-biotin immunoabsorption technique. The selected population showed 78.6 +/- 3% CD34+ cells and the overall recovery of CD34+ cells, CFU-GM and BFU-E from the starting population was 34 +/- 5%, 71 +/- 4% and 67 +/- 2%, respectively. Hematopoietic progenitor cell purification also resulted in 3 log of normal T cell depletion from the bone marrow (BM) by immunofluorescence analysis. Moreover, when unseparated BM cells were mixed with the CD34- lymphoma cell lines D430B and Raji, the removal of greater than 3 log of tumor cells from the enriched CD34+ cell fraction was demonstrated. To increase the neoplastic cell purging, several immunotoxins (IT) containing the ribosome-inactivating protein (RIP) saporin and directed toward the lymphoid-associated antigens CD30 and CD2 were prepared. Our experiments showed only a minimal toxicity of the immunoconjugates on colony-forming cells (CFC) derived from purified CD34+ cells. Conversely, all the ITs were very effective in inhibiting protein synthesis and growth of normal and neoplastic lymphoid cells. Further experiments demonstrated that the sequential combination of CD34+ cells purification and IT treatment resulted in 5 or more log of tumor cell purging with no additional loss of BM progenitor cells.

GM-CSF accelerates neutrophil recovery after autologous hematopoietic stem cell transplantation.

Patients with non-myeloid hematologic malignancies (including Hodgkin's and non-Hodgkin's lymphomas, myeloma and acute lymphoid leukemia) or solid tumors underwent cytoreductive conditioning regimens followed by either autologous bone marrow transplantation (ABMT) (n = 343) or transplantation of peripheral blood stem cells (PBSC) with (n = 44) or without bone marrow (BM) (n = 16). In a randomized double-blind phase III multi-center trial, patients received either granulocyte-macrophage colony-stimulating factor (GM-CSF, 10 micrograms/kg/day) or placebo by daily i.v. infusion beginning 24 h after bone marrow infusion and continuing until the absolute neutrophil count (ANC) had recovered to > or = 1000/mm3, or for a maximum of 30 days. Median time to neutrophil recovery was significantly shorter in the GM-CSF group (18 vs 27 days, P < 0.001), and more GM-CSF patients had neutrophil recovery by day 30 (70 vs 48%). Median duration of hospitalization was significantly shorter in the GM-CSF group (29 vs 32 days, P = 0.02). GM-CSF significantly reduced the median time to neutrophil recovery in patients receiving bone marrow only (19 vs 27 days, P < 0.001) or PBSC with or without bone marrow (14 vs 21 days, P < 0.001). The overall incidence of adverse events was comparable in the two groups, although more patients in the GM-CSF group discontinued treatment due to adverse events (17 vs 9%, P < 0.001). No difference was noted in infection incidence or time to platelet independence. GM-CSF had no negative impact on time to relapse or long-term survival. These data indicate the positive influence of GM-CSF on neutrophil recovery and hospital stay in patients receiving ABMT for a variety of clinical indications.