γ-Glutamyl hydrolase modulation and folate influence chemosensitivity of cancer cells to 5-fluorouracil and methotrexate.

BACKGROUND: γ-Glutamyl hydrolase (GGH) regulates intracellular folate and antifolates for optimal nucleotide biosynthesis and antifolate-induced cytotoxicity, respectively. The modulation of GGH may therefore affect chemosensitivity of cancer cells, and exogenous folate levels may further modify this effect. METHODS: We generated a novel model of GGH modulation in human HCT116 and MDA-MB-435 cancer cells and investigated the effect of GGH modulation on chemosensitivity to 5-fluorouracil (5FU) and methotrexate (MTX) at different folate concentrations in vitro and in vivo. RESULTS: Overexpression of GGH significantly decreased chemosensitivity of MDA-MB-435 cells to 5FU and MTX at all folate concentrations as expected. In contrast, in HCT116 cells this predicted effect was observed only at very high folate concentration, and as the folate concentration decreased this effect became null or paradoxically increased. This in vitro observation was confirmed in vivo. Inhibition of GGH significantly increased chemosensitivity of both cancer cells to 5FU at all folate concentrations. Unexpectedly, GGH inhibition significantly decreased chemosensitivity of both cancer cells to MTX at all folate concentrations. In both GGH modulation systems and cell lines, the magnitude of chemosensitivity effect incrementally increased as folate concentration increased. CONCLUSION: Modulation of GGH affects chemosensitivity of cancer cells to 5FU and MTX, and exogenous folate levels can further modify the effects.

Farletuzumab, an anti-folate receptor α antibody, does not block binding of folate or anti-folates to receptor nor does it alter the potency of anti-folates in vitro.

PURPOSE: Folate is a cofactor in the synthesis of purines and pyrimidines; folate analogs are potent cytotoxic drugs. Folate receptor alpha (FRα), a protein-mediating cellular accumulation of folate (and anti-folates), has limited expression in normal tissues and is overexpressed by numerous carcinomas. Limited distribution and high affinity for folic acid have resulted in the development of antibodies or the use of folic acid coupled to toxins or radionuclides as therapeutic and imaging agents. Farletuzumab is an anti-FRα antibody in clinical trials for ovarian and non-small cell lung cancers. Our goal was to evaluate the effect of farletuzumab on binding and uptake of folates and anti-folates and the potency of anti-folates in vitro. METHODS: Direct binding and uptake of radiolabeled folates and anti-folates and the assessments of drug concentration of drug that inhibited cell growth 50 % (IC(50)) in vitro in the presence or absence of antibody. RESULTS: Farletuzumab did not block membrane binding of radiolabeled folic acid, 5-methyltetrahydrofolate, pemetrexed, and other anti-folates; folic acid blocked >95 %. Farletuzumab had a minimal effect on the cytoplasmic accumulation of 5-methyltetrahydrofolate or pemetrexed; folic acid had a considerable but variable effect on the different cell lines. As a single agent, farletuzumab did not affect cell viability or the IC(50) of pemetrexed and other anti-folates in vitro. CONCLUSIONS: Farletuzumab does not block FRα binding of folates and anti-folates, minimally retards folate delivery via FRα-mediated transport, and minimally retards the growth of cells in vitro. Concomitant use of farletuzumab and pemetrexed is not contraindicated.

Effects of overexpression of gamma-Glutamyl hydrolase on methotrexate metabolism and resistance.

Intracellular metabolism of methotrexate (MTX) to MTX-polyglutamates (MTXPG) is one determinant of cytotoxicity. Steady-state accumulation of MTXPG seems to depend on the activity of two enzymes: folylpolyglutamate synthetase (FPGS), which adds glutamate residues, and gamma-glutamyl hydrolase (GGH), which removes them. Overexpression of GGH would be expected to decrease intracellular MTXPG, thereby increasing efflux of MTX and decreasing cytotoxicity. Increased expression of GGH has been shown to be associated with resistance to MTX in human sarcoma cell lines and a rat hepatoma cell line. To clarify the specific role of GGH in determining MTX sensitivity, we investigated the phenotype produced by forced GGH overexpression in two cell types. Furthermore, because MTX and folic acid share metabolic pathways, we measured the effects of GGH overexpression on folic acid metabolism. The full-length cDNA for GGH, subcloned into a constitutive expression vector, was transfected into a human fibrosarcoma (HT-1080) and a human breast carcinoma (MCF-7) cell line. Compared with the clones containing an empty vector, the GGH-overexpressing cells express 15- to 30-fold more GGH mRNA, more GGH protein, and 15- to 90-fold more GGH enzyme activity. GGH overexpression altered MTX accumulation and metabolism to long-chain polyglutamates. In contrast to expectations, however, GGH overexpression did not confer resistance to short MTX exposures in either cell line. Changes in MTX metabolism were found to be balanced by alterations in accumulation and metabolism of folic acid. The ratio of MTX:folate accumulation may be a better predictor of MTX cytotoxicity than the accumulation of either alone. We conclude that, at least for these two cell lines, GGH overexpression alone is insufficient to produce clinical resistance to MTX.

Fractionated cyclophosphamide and etoposide for children with advanced or refractory solid tumors: a phase II window study.

PURPOSE: Cyclophosphamide (CPA) has a broad spectrum of activity against solid tumors. Hepatic self-induction of the active metabolite 4-hydroxycyclophosphamide occurs after repeated administration. We evaluated the clinical efficacy of a window regimen that administers fractionated CPA in conjunction with etoposide (VP16) in children with advanced or refractory solid tumors. PATIENTS AND METHODS: Seventeen children with advanced (n = 12) or refractory (n = 5) solid tumors were entered onto this phase II window study. The treatment regimen consisted of intravenous (IV) CPA 500 mg/m(2)/d and IV VP16 100 mg/m(2)/d. Both drugs were administered daily by short infusions for 5 consecutive days. RESULTS: A total of 34 courses were administered, with a median of two courses per patient. The median interval between chemotherapy courses was 21 days (range, 17 to 35 days). Thirty-three courses were assessable for toxicity, and all patients were assessable for response. No life-threatening toxicities were observed. The incidence of grade 3 or 4 neutropenia was 94% and of fever and neutropenia 38%. Fever and neutropenia occurred after 12 of 26 courses without recombinant human granulocyte colony-stimulating factor (rhG-CSF) and after one of eight courses with rhG-CSF (P =. 09). Grade 3 or 4 thrombocytopenia occurred after 10 courses (29%). There were no positive blood cultures. One heavily pretreated patient developed a localized perirectal abscess that required drainage. There were 10 patients (59%) with partial responses, four (23.5%) with stable disease, and three with progressive disease. CONCLUSION: Fractionated IV CPA and VP16 over 5 days can be safely administered in children with advanced or refractory solid tumors and has notable antineoplastic activity.

Ratio of methotrexate to folate uptake by lymphoblasts in children with B-lineage acute lymphoblastic leukemia: a pilot study.

PURPOSE: Methotrexate (MTX) remains one of the most effective drugs for the treatment of children with acute lymphoblastic leukemia (ALL). Because MTX and 5-methyltetrahydrofolate (5CH3THF) share uptake and metabolic pathways, the efficacy of MTX is likely to depend not only on its metabolism but also on how well folate is accumulated by lymphoblasts. The authors' goal was to compare in vitro folate and antifolate uptake in B-lineage lymphoblasts from patients who remained in continuous complete remission (CCR) and those in whom relapse occurred. PATIENTS AND METHODS: Twenty-four children with B-lineage ALL were studied at diagnosis (n = 20) or relapse (n = 4). Lymphoblasts obtained by bone marrow aspiration were incubated for 24 hours in vitro with 0.05 microM 5CH3[3H]THF or 1 microM [3H]MTX. RESULTS: As of July 1999, 16 patients studied at diagnosis remained in CCR at a median follow-up of 45 months after achieving remission. Two of the patients studied at relapse are in second CCR; the remaining two died from progressive disease. The median uptake of neither [3H]MTX nor 5CH3[3H]THF differed significantly between the 16 patients in first CCR studied at diagnosis and the 4 patients studied at relapse. However, the median ratio of [3H]MTX:5CH3[3H]THF uptake differed significantly for patients who remained in first CCR versus patients studied at relapse. CONCLUSIONS: The uptake of [3H]MTX in relation to 5CH3[3H]THF by leukemic lymphoblasts in vitro may correlate positively with treatment outcome in children with B-lineage ALL. A larger study of homogeneously treated patients is necessary to confirm these results.

Glutathione S-transferase genotypes in children who develop treatment-related acute myeloid malignancies.

Epipodophyllotoxin-associated secondary myeloid leukemia is a devastating complication of acute lymphoblastic leukemia (ALL) therapy. The risk factors for treatment-related myeloid leukemia remain incompletely defined. Genetic deficiencies in glutathione S-transferase (GST) activities have been linked to higher frequencies of a number of human malignancies. Our objective was to determine whether the null genotype for GSTM1, GSTT1, or both, was more frequent in children with ALL who developed treatment-related myeloid malignancies as compared to those who did not. A PCR technique was used to assay for the null genotype for GSTM1 and GSTT1 in 302 children with ALL, 57 of whom also subsequently developed treatment-related acute myeloid leukemia or myelodysplastic syndrome. Among children with ALL who did not develop treatment-related myeloid malignancies, the frequencies of GSTM1 and GSTT1 wild-type, GSTM1 null-GSTT1 wild-type, GSTM1 wild-type-GSTT1 null, and GSTM1 and GSTT1 null genotypes were 40%, 42%, 9% and 9%, respectively. The corresponding frequencies for patients who developed acute myeloid malignancies were 42%, 32%, 11% and 16%, respectively (P = 0.26). A statistically significant increase in the frequency of the GST null genotype was observed in male patients who developed myeloid malignancies as compared to male ALL control patients (P = 0.036), but was not observed in female patients (P = 0.51). Moreover, a logistic regression analysis of possible predictors for myeloid malignancies, controlling for gender and race, did not reveal an association of GSTM1 or GSTT1 null genotypes (P = 0.62 and 0.11, respectively) with treatment-related malignancies. Our data suggest that GSTM1 and GSTT1 null genotypes may not predispose to epipodophyllotoxin-associated myeloid malignancies.

Mice lacking the folic acid-binding protein Folbp1 are defective in early embryonic development.

Periconceptional folic acid supplementation reduces the occurrence of several human congenital malformations, including craniofacial, heart and neural tube defects. Although the underlying mechanism is unknown, there may be a maternal-to-fetal folate-transport defect or an inherent fetal biochemical disorder that is neutralized by supplementation. Previous experiments have identified a folate-binding protein (Folbp1) that functions as a membrane receptor to mediate the high-affinity internalization and delivery of folate to the cytoplasm of the cell. In vitro, this receptor facilitates the accumulation of cellular folate a thousand-fold relative to the media, suggesting that it may be essential in cytoplasmic folate delivery in vivo. The importance of an adequate intracellular folate pool for normal embryogenesis has long been recognized in humans and experimental animals. To determine whether Folbp1 is involved in maternal-to-fetal folate transport, we inactivated Folbp1 in mice. We also produced mice lacking Folbp2, another member of the folate receptor family that is GPI anchored but binds folate poorly. Folbp2-/- embryos developed normally, but Folbp1-/- embryos had severe morphogenetic abnormalities and died in utero by embryonic day (E) 10. Supplementing pregnant Folbp1+/- dams with folinic acid reversed this phenotype in nullizygous pups. Our results suggest that Folbp1 has a critical role in folate homeostasis during development, and that functional defects in the human homologue (FOLR1) of Folbp1 may contribute to similar defects in humans.

Parameningeal alveolar rhabdomyosarcoma with an isolated pancreatic metastasis.

A 15-year-old girl with alveolar rhabdomyosarcoma of the paranasal sinuses was found to have a solitary visceral metastatic focus in the pancreas. This unusual occurrence is presented and discussed.

Plasma homocysteine levels and folate status in children with sickle cell anemia.

PURPOSE: A sensitive inverse relationship between plasma homocysteine concentration and folate status has been demonstrated. Although children with sickle cell anemia (SCA) are at potential risk for folate deficiency, plasma homocysteine levels have not been reported in such patients. Therefore, a study was designed to assess plasma homocysteine levels as a marker of folate status. DESIGN: Plasma homocysteine concentrations were measured in 120 children with SCA (102 in steady state and 18 during an acute complication) who had never received supplemental folic acid. Folate status was directly assessed in 34 of these patients. RESULTS: Plasma homocysteine levels in the patients with SCA and control subjects were similar. The mean value +/- 1 SD was 5.8+/-2.5 micromol/L (range, 1.6 to 14.1 micromol/L) in the patients with SCA and 6.1+/-2.7 micromol/L (range, 1.7 to 15.3 micromol/L) in 73 pediatric control subjects. In a subpopulation of the study group (34 children), simultaneous serum folate, red cell folate, and total homocysteine concentrations were also measured. Their serum folate and red cell folate concentrations were normal: 12.4+/-10.0 nmol/L (range, 1 to 42 nmol/L) and 604+/-374.7 nmol/L (range, 205 to 1741 nmol/L), respectively. There was no correlation of plasma homocysteine concentration with various clinical or laboratory measures or with red cell folate concentration. CONCLUSION: Folate stores in children with SCA not receiving folic acid supplements are adequate despite an underlying hemolytic anemia.

Carboplatin for the treatment of children with newly diagnosed optic chiasm gliomas: a phase II study.

Management of low grade optic glioma in children and adolescents remains controversial. Treatment with chemotherapy may delay or eliminate the need for radiation therapy. Children with newly diagnosed optic chiasm glioma were eligible for enrollment in this phase II trial and received intravenous carboplatin (CBDCA) (560 mg/m2) every four weeks. Patients were monitored closely for toxicity and tumor status. Twelve children were enrolled. Six patients had stable disease, four a partial response and two progressed on therapy. Overall progression free survival was 83 +/- 11%. The median duration of follow-up was 38.6 months (range 18-63 months). No deaths were noted in our series. Thrombocytopenia was the major toxicity, and two patients required platelet transfusions. One child developed an urticarial reaction requiring discontinuation of therapy. Another child developed unilateral high frequency hearing loss. No renal toxicity was encountered. We have demonstrated that carboplatin can eliminate or delay radiation therapy in children and adolescents with low grade optic glioma. CBDCA deserves further investigation in larger clinical trials as a treatment for children with optic chiasm glioma.

Delayed methotrexate clearance in a patient with sickle cell anemia and osteosarcoma.

A 15-year-old girl with homozygous sickle cell anemia (HbSS) and osteosarcoma is described. Delayed clearance of methotrexate (MTX) after the second course of high-dose MTX (HDMTX) led to the development of renal and hepatic toxicities. Rescue was accomplished with high-dose leucovorin, intravenous carboxypeptidase G2, and thymidine. Although the renal and hepatic abnormalities resolved, focal tonic-clonic seizures developed, accompanied by abnormal brain imaging. Four weeks after this episode, all clinical and biochemical abnormalities resolved. Preexistent end-organ damage associated with HbSS may compromise the ability to deliver high-dose chemotherapy with curative intent in patients with malignant disease.

Acute lymphoblastic leukemia complicated by choroid plexus carcinoma.

A 14-year-old boy with acute lymphoblastic leukemia in remission developed headaches characteristic of migraines. His neurologic examination was normal, serial studies of cerebrospinal fluid were unremarkable, and the headaches responded promptly to antimigraine therapy. Ultimately, radiographic and pathologic examinations demonstrated a choroid plexus carcinoma.

High-dose trimetrexate and minimal-dose leucovorin: a case for selective protection?

Our objective was to find the minimum dose of leucovorin (LV; 5-formyltetrahydrofolate) needed to potentially provide selective protection of normal tissue in patients with tumors resistant to methotrexate (MTX) by virtue of transport during prolonged therapy with high-dose trimetrexate (TMTX). Based upon the known daily requirement for folate, that tumors are often resistant to methotrexate via a transport-based mechanism, and that large doses of trimetrexate can be given with large doses of leucovorin for the treatment of patients with Pneumocystis carinii, a protocol was designed to find the minimum LV dose required to allow the administration of large doses of TMTX. Patients were treated in 28-day cycles consisting of 14 consecutive days of oral TMTX (45 mg/m2 every 12 h), followed by 14 days of rest. The dose of concurrent LV was started at 5 mg/m2 twice daily. Cohorts of patients received successive half doses of LV so long as three consecutive patients had less than or equal to grade 3 toxicity. Ten patients received 29 courses of therapy. The most common toxicities encountered were thrombocytopenia (38%), mucositis (14%), and neutropenia (10%). At a LV dose of 2.5 mg/m2, toxicities were consistently limited to less than or equal to grade 3 and only one episode of grade 4 hematological toxicity. Although there was marked interpatient variability, the minimally effective LV dose for selective protection seems to be 2.5 mg/m2. If tumors are resistant to methotrexate because of decreased transport of drug (and also folate), then the same pharmacological principle used to develop TMTX/LV for the treatment of P. carinii may be applied to treatment of some patients with cancer.

Receptor-mediated folate uptake is positively regulated by disruption of the actin cytoskeleton.

Receptor-mediated folate uptake is initiated by binding of ligand to a glycosyl phosphatidylinositol-anchored protein, folate receptor alpha (FR alpha). This receptor is expressed in a limited number of normal tissues but is overexpressed in a large number of epithelial malignancies. FR alpha synthesis, at least in part, is regulated by endogenous folate and by hormones in some cells, but much less is known about the control of function. Recently, we showed that phorbol 12-myristate 13-acetate increases the rate of receptor cycling, increases the rate of folate delivery, and causes the majority of the receptor to reside on the cell surface in nonmalignant cells in vitro (C. M. Lewis et al., Biochim. Biophys. Acta, 1401: 157-169, 1998). However, based upon effects (or lack of effects) of specific inhibitors of protein kinase C, the mechanism of action of phorbol 12-myristate 13-acetate is not likely via protein kinase C. Because exo- and endocytosis are controlled by the actin cytoskeleton, we tested cytochalasin D and latrunculin B, actin-disrupting agents, on FR alpha-mediated folate uptake. Disruption of the actin cytoskeleton reversibly increases the proportion of receptors on the cell surface and increases the rate of 5-methyltetrahydrofolate delivery. Disrupting microtubules with nocodazole had no effect. The increased rate of folate delivery caused by cytochalasin D is not observed in FR-negative cell lines. Although we have not yet identified the upstream effectors, likely candidates include small G-proteins such as rho, which are known to cause actin polymerization. In addition to identifying the machinery for receptor-mediated folate uptake, it may be important to integrate this new data into studies of FR alpha as a tumor antigen for imaging or delivering molecules via anti-FR antibodies or compounds coupled to folic acid.