Single-agent trabectedin as second-line therapy of persistent or recurrent endometrial cancer: results of a multicenter phase II study.

OBJECTIVE: To assess the efficacy and safety of single-agent trabectedin in women with persistent or recurrent endometrial cancer. METHODS: In this open-label, phase II multicenter trial, women with persistent or recurrent endometrial carcinoma were administered trabectedin as a 3-hour intravenous infusion every 21 days at a starting dose of 1.3 mg/m(2) with dexamethasone pretreatment. Clinical objective response was the primary efficacy endpoint. Secondary endpoints were time to progression (TTP), progression-free survival (PFS), overall survival (OS), and safety. RESULTS: The median age of the 50 women entering the study was 63 years (range, 22-87), with all having history of prior chemotherapy (92% combination regimens) and the majority having undergone surgery (92%) or radiation therapy (68%). Patients received trabectedin for a median duration of 6.8 weeks (range, 3-20). A median of 2 cycles (range, 1-6) was administered, with a median dose intensity of 0.4 mg/m(2) per week (range, 0.27-0.43) and a median relative dose intensity of 92% (range, 61.5-100.2%). One patient exhibited a complete response for an objective response rate of 2.2% (95% confidence interval [CI]: 0.1%, 11.5%). Median TTP and PFS were both 1.8 months (95% CI: 1.4, 2.9), and median OS was 6.7 months (95% CI: 5.2, 13.9). Most frequent adverse events were nausea (62%), asthenia (50%), vomiting (42%), and increased alanine aminotransferase (40%). CONCLUSION: Single-agent trabectedin displayed minimal antitumor activity in this pretreated population of women with persistent or recurrent endometrial cancer.

A phase I study of the safety and pharmacokinetics of trabectedin in combination with pegylated liposomal doxorubicin in patients with advanced malignancies.

BACKGROUND: To determine the maximum tolerated dose (MTD), safety, potential pharmacokinetic (PK) interactions, and effect on liver histology of trabectedin in combination with pegylated liposomal doxorubicin (PLD) for advanced malignancies. PATIENTS AND METHODS: Entry criteria for the 36 patients included normal liver function, prior doxorubicin exposure <250 mg/m(2), and normal cardiac function. A 1-h PLD (30 mg/m(2)) infusion was followed immediately by one of six trabectedin doses (0.4, 0.6, 0.75, 0.9, 1.1, and 1.3 mg/m(2)) infused over 3 h, repeated every 21 days until evidence of complete response (CR), disease progression, or unacceptable toxicity. Plasma samples were obtained to assess PK profiles. RESULTS: The MTD of trabectedin was 1.1 mg/m(2). Drug-related grade 3 and 4 toxic effects were neutropenia (31%) and elevated transaminases (31%). Six patients responded (one CR, five partial responses), with an overall response rate of 16.7%, and 14 had stable disease (less than a 50% reduction and less than a 25% increase in the sum of the products of two perpendicular diameters of all measured lesions and the appearance of no new lesions) >4 months (39%). Neither drug had its PK affected significantly by concomitant administration compared with trabectedin and PLD each given as a single agent. CONCLUSION: Trabectedin combined with PLD is generally well tolerated at therapeutic doses of both drugs in pretreated patients with diverse tumor types and appears to provide clinical benefit. These results support the need for additional studies of this combination in appropriate cancer types.

Chromosomal transformation in donor cells following allogeneic bone marrow transplantation.

We report here a monosomy 7 transformation of donor cells following matched-unrelated, same sex, allogeneic bone marrow transplantation in a patient with severe congenital aplastic anemia. A PCR technique was employed to amplify microsatellite markers on chromosome 7 to confirm donor/recipient identity. We found that the transformation of monosomy 7 occurred in previously genetically normal donor cells. This study suggests that the microenvironment of the bone marrow of our patient with severe congenital aplastic anemia may have played a critical role in the development of monosomy 7 of normal donor cells and we conclude that chromosomal microsatellite marker analysis can be a valuable tool for precise donor/recipient differentiation in engraftment monitoring.

Vasculopathy of small muscular arteries in pediatric patients after bone marrow transplantation.

Bone Marrow Transplant (BMT) is a critical therapeutic intervention for a variety of diseases occurring in the pediatric patient. Complications of allogeneic BMT include graft-versus-host disease (GVHD), infection, drug toxicity, thrombotic microangiopathy, and veno-occlusive disease. With solid organ transplantation, chronic vascular rejection has emerged as a major factor limiting long-term survival of the graft. We present a vasculopathy of small muscular arteries in 6 patients after allogeneic BMT. Cases include 4 boys and 2 girls ranging in age from 4 months to 13 years with full or partial human leukocyte antigen matching. Five of the 6 transplants were from related donors. The vasculopathy occurred 13 to 418 days after transplant and was noted in surgical specimens (2) and at autopsy (4). It was seen in the gastrointestinal tract and lung in 3 cases each. Vascular changes in small muscular arteries include concentric intimal or medial hyperplasia with luminal narrowing, prominent myxoid change, extravasated red blood cells, and presence of some foamy histiocytes with no evidence of thrombotic microangiopathy. Vasculopathy contributed to intestinal compromise requiring surgical intervention 3 times in 1 patient, and diffuse alveolar damage with hemorrhage in another. All 6 patients are dead. The cause of this unusual vasculopathy present in patients after BMT is likely to be multifactorial, involving effects of irradiation, chemotherapy, cyclosporine, and GVHD. Together these may create a negative synergy which produces an obliterative arteriopathy that should be recognized as a pathological entity and may be a harbinger of a poor prognosis.

Ex vivo treatment of bone marrow with phosphorothioate oligonucleotide OL(1)p53 for autologous transplantation in acute myelogenous leukemia and myelodysplastic syndrome.

Effective ex vivo purging techniques can decrease the likelihood of infusing bone marrow contaminated with leukemic cells during autologous transplantation. In preliminary studies, OL(1)p53, a 20-mer phosphorothioate oligonucleotide directed against p53 mRNA, decreased the number of acute myelogenous leukemia (AML) cells in vitro, suggesting a possible role for OL(1)p53 in purging bone marrow harvests of leukemia cells. To demonstrate that OL(1)p53 was nontoxic to hematopoietic progenitor cells, normal bone marrow cells were incubated with 10 microM OL(1)p53 for 36 h, and hematopoietic progenitor cell survival was determined by in vitro colony assays. OL(1)p53 had no toxic effect on the growth of either myeloid (CFU-GM) or erythroid (BFU-E) progenitor cells. OL(1)p53 was then used to ex vivo purge bone marrow harvests from nine patients with either AML or myelodysplastic syndrome (MDS). Bone marrow cells were incubated with 10 microM OL(1)p53 for 36 h before transplantation. The median times posttransplantation for the patient to recover an absolute neutrophil count greater than 0.5 x 10(9)/L and a platelet transfusion independence were 30 days and 56 days, respectively. Incubation of bone marrow cells with OL(1)p53 had no detrimental effect on the growth of hematopoietic progenitor cells, and transplantation of autologous bone marrow cells treated with the phosphorothioate oligonucleotide, OL(1)p53, resulted in successful recovery of circulating neutrophils following high-dose therapy in patients with AML or MDS. The data show that OL(1)p53 can be used safely to purge autologous bone marrow harvests from patients with leukemia.

Allogeneic bone marrow transplantation for children with acute leukemia: long-term follow-up of patients prepared with high-dose cytosine arabinoside and fractionated total body irradiation.

High-dose therapy and allogeneic matched sibling bone marrow transplantation (BMT) is considered to be the treatment of choice for children with relapsed acute lymphoblastic leukemia (ALL), or for children with acute myeloid leukemia (AML) in first remission. However, the rate of bone marrow relapse after transplant for either of these diseases remains high. In this study, we assessed the efficacy and toxicity of high-dose cytosine arabinoside and total body irradiation (TBI) followed by allogeneic BMT, for children with acute leukemia or myelodysplastic syndrome (MDS). Sixty-five pediatric patients underwent allogeneic related (n = 57) or unrelated (n = 8) BMT. Twenty-seven were transplanted for ALL in second remission (CR2), and 16 for AML in first remission (CR1). The other 22 were high risk patients: six were transplanted for ALL in third remission (CR3), two for AML in CR2, two for myelodysplastic syndrome (MDS) and 12 for acute leukemia in relapse. Patients were prepared with cytosine arabinoside 3000 mg/m2 per dose twice daily for 6 days followed by 12000 cGy TBI as 200 cGy fractions twice daily for 3 days. Minimum follow-up is 21 months. Five-year event-free survival (EFS) and the actuarial relapse rate is 59 and 14% for patients with ALL in second remission, and 38 and 14+% for patients with AML in first remission. Twelve patients have relapsed (three are alive in remission after testicular or marrow relapse) and 28 have died of other causes. Acute GVHD with or without infection was the cause of death in 11 patients. Ten of the 11 patients who died of acute GVHD were considered at 'high risk' for GVHD (inadequate GVHD prophylaxis, or mismatched family donor or a matched unrelated donor). Toxicities in the immediate post-BMT period included diarrhea, oropharyngeal mucositis and conjunctivitis. Significant late toxicities included short stature, avascular necrosis of bone, and poor school performance (most often in patients who had received prior cranial irradiation). Our conclusions are that high-dose Ara-C and TBI followed by allogeneic bone marrow transplantation is effective therapy for children in second complete remission of their acute leukemia. However, significant late toxicities occur, and it is clear that more effective, less toxic therapies are necessary for these patients.

A study of thiotepa, etoposide and fractionated total body irradiation as a preparative regimen prior to bone marrow transplantation for poor prognosis patients with neuroblastoma.

We report the toxicity and efficacy of a new conditioning regimen for bone marrow transplantation (BMT) in children with poor prognosis neuroblastoma (NBL). Twenty-seven patients with poor prognosis NBL were treated with teniposide (360 mg/m2) or etoposide (500 mg/m2), thiotepa (600-900 mg/m2), and 1200 cGy fractionated total body irradiation (fTBI) followed by autologous marrow rescue (n = 19) or allogeneic BMT from HLA-identical siblings (n = 8). The two patients who received teniposide, 600 mg/m2 thiotepa and fTBI had minimal toxicity but relapsed 4 and 12 months post-auto BMT. The next two patients received 750 mg/m2 thiotepa, 500 mg/m2 etoposide and TBI. They tolerated the conditioning regimen well and are alive and in remission 77 and 75 months post-BMT. At the next thiotepa dose level (900 mg/m2), the first two allograft recipients both experienced fatal regimen-related toxicity. All subsequent allograft recipients received 750 mg/m2 thiotepa and autograft recipients received 900 mg/m2 thiotepa. As of 1 April 1995, eight of the 19 patients who received autologous marrow are surviving disease-free 21 to 77 months post-BMT. Nine autograft recipients relapsed at 2 to 37 months following transplantation. One patient died of hepatic veno-occlusive disease 2 months after auto BMT, and one of pneumonia 6 months post-transplantation. Three allograft recipients have relapsed at 6, 10 and 39 months post-transplant and three are alive and in remission 75, 53 and 27 months post-BMT. Overall, 11/27 patients (41%) are alive and in remission 21-77 months (median 47 months) following BMT. A conditioning regimen consisting of 500 mg/m2 etoposide, thiotepa (750 mg/m2 for allograft recipients and 900 mg/m2 for autograft recipients) and 1200 cGy fTBI has acceptable toxicity and is at least as effective as melphalan-containing regimens in the treatment of high-risk NBL.

Phase I trial of an antisense oligonucleotide OL(1)p53 in hematologic malignancies.

PURPOSE: The phosphoprotein p53 is involved in transcriptional regulation and is detected in hematologic malignancies. In vitro incubation of acute myelogenous leukemia with OL(1)p53, a 20-mer phosphorothioate oligonucleotide complementary to p53 mRNA, results in leukemic cell death. A phase I dose-escalating trial was conducted to determine the toxicity of OL(1)p53 following systemic administration to patients with hematologic malignancies. PATIENTS AND METHODS: Sixteen patients with either refractory acute myelogenous leukemia (n = 6) or advanced myelodysplastic syndrome (n = 10) participated in the trial. Patients were given OL(1)p53 at doses of 0.05 to 0.25 mg/kg/h for 10 days by continuous intravenous infusion. RESULTS: No specific toxicity was directly related to the administration of OL(1)p53. One patient developed transient nonoliguric renal failure. One patient died of anthracycline-induced cardiac failure. Approximately 36% of the administered dose of OL(1)p53 was recovered intact in the urine. Plasma concentrations and area under the plasma concentration curves were linearly correlated with dose. Leukemic cell growth in vitro was inhibited as compared with pretreatment samples. There were no clinical complete responses. CONCLUSION: A phosphorothioate oligonucleotide, OL(1)p53, can be administered systemically without complications. This type of modified oligonucleotide can be administered without complete degradation, as it was recovered from the urine intact. This oligonucleotide may be useful in combination with currently available chemotherapy agents for the treatment of malignancies.

Continuous infusion of antisense phosphorothioate therapeutics.

Current therapy for acute myelogenous leukemia (AML) includes induction with Ara-C and an anthracycline, such as daunorubicin, idarubicin, or mitoxantrone. Unfortunately, most patients relapse from initial remission. Nearly one-fifth of early relapses experience treatment-related deaths. In addition, patients refractory to Ara-C die within months. Hence, new therapeutic agents must be identified capable of enhanced remission rates, diminished treatment-related mortality, or that can achieve remissions in refractory patients.

Fractionated total-body irradiation preceding high-dose cytosine arabinoside as a preparative regimen for bone marrow transplantation in children with acute leukemia.

Twenty children with acute leukemia between 3 and 19 years of age underwent allogeneic bone marrow transplantation from HLA-matched sibling donors after conditioning with total-body irradiation (1,200 cGy in six fractions of 200 cGy twice daily for 3 days) and high dose cytosine arabinoside (3 g/m2 given every 12 hours for 12 doses). Three patients died with acute toxicity. Six patients developed grade II acute graft versus host disease. With a median follow-up of 68 months (range 26-96 months), thirteen children (65%) are alive and in remission with Karnofsky scores of 90-100%. A patient with AML in resistant relapse went into remission but relapsed and died 5 months post-transplantation. Three other patients relapsed, 8, 12, and 16 months post BMT. Our results suggest that this conditioning regimen is associated with high but manageable acute toxicity and may be highly effective in controlling leukemia resistant to conventional chemotherapy.

Selective cytotoxicity to human leukemic myeloblasts produced by oligodeoxyribonucleotide phosphorothioates complementary to p53 nucleotide sequences.

Cells were treated in vitro with oligodeoxyribonucleotide phosphorothioates (ODNs) complementary to sites common to both wild-type and mutant p53 nucleotide sequences. Acute myelogenous leukemia (AML) blasts from peripheral blood were exposed to four different p53 ODNs and showed anti-leukemic effects in suspension culture. This effect continued after removal of the ODN from the medium. Blocking of self-renewal of the leukemic blast stem cells in secondary plating of cells from cloning assays by two of the p53 ODNs was also observed. Control ODNs had no effect on leukemic blasts. Treatment of normal bone marrow cells with the four p53 ODNs did not influence their growth, nor was there any effect by the p53 ODNs on the leukemic cell-line, HL60, that does not express p53. These data suggest that p53 ODNs are selectively toxic to primary myelogenous blasts and may be therapeutically useful in AML.

Oligonucleotides in the treatment of leukemia.

The use of synthetic oligonucleotides directed towards specific genes in the therapy of leukemias has evolved rapidly over the past 5 years to early clinical trials. Undoubtedly, use of these compounds for systemic therapy and bone marrow 'purging' will escalate. Such trials will be models for the treatment of many other malignant diseases and, indeed, non-malignant diseases which may be expected to respond to eradication of a specific gene function. We are currently at the threshold of a new era of therapy which holds the promise of totally transforming clinical medicine in the next decade.

Systemic administration of a phosphorothioate oligonucleotide with a sequence complementary to p53 for acute myelogenous leukemia and myelodysplastic syndrome: initial results of a phase I trial.

A synthetic phosphorothioate oligonucleotide was administered systemically to five patients with either relapsed or refractory acute myelogenous leukemia (AML), or myelodysplastic syndrome (MDS). Patients received a 10-day continuous intravenous infusion of this compound, which is complementary to p53 mRNA. No major toxicity attributable to a dose of 0.05 mg/kg/hr was observed. A range of approximately 9 to 18% of the administered dose was recovered in the urine as intact oligonucleotide. Evaluation of malignant cells recovered from bone marrow and peripheral blood at intervals before, during, and after treatment reveals no enhanced growth potential following oligonucleotide administration. Hence, a phosphorothioate oligonucleotide complementary to p53 mRNA can be administered at this dose level to humans without major toxicity. Higher doses need to be evaluated for toxicity and potential clinical efficacy.

Bone marrow transplantation for Niemann-Pick type IA disease.

Bone marrow transplantation has been undertaken with encouraging results as therapy for a wide variety of lysosomal storage diseases. We report a case of Niemann-Pick disease Type IA in which, despite the presence of only mild hypotonia with depressed reflexes, the clinical course of the disease appeared to be only slightly modified by this procedure, which was performed at the earliest practical opportunity. The patient was diagnosed early when asymptomatic, because of a family history of an affected sibling who died at 14 months. He received a bone marrow transplant from an HLA-identical, MLC non-reactive sibling donor, whose leukocyte sphingomyelinase activity was in the homozygote normal range. There was adequate engraftment as evidenced by persistently normal leukocyte sphingomyelinase activities, and there was no evidence of graft-versus-host disease. Visceral storage and neurological impairment were less rapidly progressive than in his untreated sibling but he eventually died at 30 months. Autopsy confirmed that this was essentially due to the effects of the underlying Niemann-Pick disease. We conclude that despite some success in other neurovisceral lysosomal storage disorders, bone marrow transplantation is not likely to be an adequate treatment for Niemann-Pick disease Type IA.

Hurler syndrome with special reference to histologic abnormalities of the growth plate.

Hurler syndrome is a mucopolysaccharide disorder resulting from an heritable deficiency in alpha-L-iduronidase, an enzyme required in the catabolism of heparan sulfate and dermatan sulfate glycosaminoglycan (GAGs). The resultant intracellular accumulation of GAG leads to disruption of the intracellular and extracellular environment and dysfunction of multiple organ systems. Among the most noted manifestations of this disease is disproportionate short trunk dwarfism, which develops during the first years of life. Histochemical and electron-microscopic observations on a 30-month-old child with Hurler syndrome showed marked irregularities in chondrocyte orientation within the growth plate, along with disruption of the normal columnar architecture. Vacuolization with enlargement of the cellular border was the characteristic ultrastructural finding. An heritable abnormality in the enzymatic degradation of structural glycosaminoglycans leads to profound disruption of the normal mechanisms of growth and development.

Retroviral transfer of genes into erythroid progenitors derived from human peripheral blood.

Human erythroblasts are a logical target for studies of expression of transferred globin genes because high-level expression is a prerequisite for gene therapy of hemoglobinopathies. Early erythroid progenitors (erythroid burst-forming units, BFU-E) are readily available from human peripheral blood and can be cultured to produce erythroblasts. However, conditions for efficient transfer into these normal progenitors have not been previously described. Here we demonstrate efficient transfer of the neomycin resistance gene into human peripheral blood BFU-E using the retrovirus vector, N2. We show that liquid culture of mononuclear cells from peripheral blood for 18-24 h prior to retroviral infection leads to increased transfer efficiency of N2 as determined by G418 resistance, and we are able to detect viral DNA by polymerase chain reaction (PCR) analysis. In addition, a second retrovirus, beta(gamma)-SVX, prepared with a human beta-globin gene containing a gamma-globin second exon to facilitate transcript detection and the 3'-enhancer sequence, was also used to determine whether similar results could be obtained when more than one gene is transferred. Using the beta(gamma)-SVX virus, increased transfer efficiency into BFU-E was similarly found after liquid culture for up to 4 days. Expression of the transferred globin gene was also detected by PCR analysis of cDNA made from erythroblast RNA. The human peripheral blood BFU-E system described should allow determination of sequences required for high-level expression of transferred globin and other erythroid genes.