Risk of cancer after assisted reproduction: a review of the available evidences and guidance to fertility counselors.

OBJECTIVE: Infertile women requiring ovarian stimulation and assisted reproduction techniques (ART) are faced with difficult issues. The fear that using hormones could increase their risk of cancer is the most significant. One of the main challenges for assessing cancer risk after ART is the difficulty to separate it from the underlying condition of infertility per se. The delay or the inability to achieve a pregnancy is an important risk factor for breast, endometrial and ovarian cancer. We analyzed the current literature on the topic. MATERIALS AND METHODS: The published literature in Medline and Cochrane was screened using the following keywords: ovulation induction, reproductive techniques, clomiphene, in vitro fertilization, fertility agents, female/adverse effects, female/toxicity gonadotropins/ adverse effects or gonadotropins/toxicity and "neoplasms or cancer". RESULTS: A total of 95 articles were evaluated. Limited evidence suggests that high doses or many cycles of clomiphene citrate could increase the risk of endometrial cancer, although the confounding factors of polycystic ovarian disease and overweight are not always considered. In some studies, ART modestly increased the risk of borderline ovarian cancer. Fertility treatments do not increase the risk of breast, cervical, endometrial and ovarian cancers, thyroid, melanoma and colon cancer. CONCLUSIONS: Women can be reassured that fertility drugs do not appear to significantly increase the risk of invasive ovarian, endometrial, breast or other cancers, while achieving a pregnancy at an earlier age is a significant protective factor.

Induction of thymidylate synthase as a 5-fluorouracil resistance mechanism.

Thymidylate synthase (TS) is a key enzyme in the de novo synthesis of 2'-deoxythymidine-5'-monophosphate (dTMP) from 2'-deoxyuridine-5'-monophosphate (dUMP), for which 5,10-methylene-tetrahydrofolate (CH(2)-THF) is the methyl donor. TS is an important target for chemotherapy; it is inhibited by folate and nucleotide analogs, such as by 5-fluoro-dUMP (FdUMP), the active metabolite of 5-fluorouracil (5FU). FdUMP forms a relatively stable ternary complex with TS and CH(2)THF, which is further stabilized by leucovorin (LV). 5FU treatment can induce TS expression, which might bypass dTMP depletion. An improved efficacy of 5FU might be achieved by increasing and prolonging TS inhibition, a prevention of dissociation of the ternary complex, and prevention of TS induction. In a panel of 17 colon cancer cells, including several variants with acquired resistance to 5FU, sensitivity was related to TS levels, but exclusion of the resistant variants abolished this relation. For antifolates, polyglutamylation was more important than the intrinsic TS level. Cells with low p53 levels were more sensitive to 5FU and the antifolate raltitrexed (RTX) than cells with high, mutated p53. Free TS protein down-regulates its own translation, but its transcription is regulated by E2F, a cell cycle checkpoint regulator. Together, this results in low TS levels in stationary phase cells. Although cells with a low TS might theoretically be more sensitive to 5FU, the low proliferation rate prevents induction of DNA damage and 5FU toxicity. TS levels were not related to polymorphisms of the TS promoter. Treatment with 5FU or RTX rapidly induced TS levels two- to five-fold. In animal models, 5FU treatment resulted in TS inhibition followed by a two- to three-fold TS induction. Both LV and a high dose of 5FU not only enhanced TS inhibition, but also prevented TS induction and increased the antitumor effect. In patients, TS levels as determined by enzyme activity assays, immunohistochemistry and mRNA expression, were related to a response to 5FU. 5FU treatment initially decreased TS levels, but this was followed by an induction, as seen with an increased ratio of TS protein over TS-mRNA. The clear retrospective relation between TS levels and response now forms the basis for a prospective study, in which TS levels are measured before treatment in order to determine the treatment protocol.

Modulation of 5-fluorouracil in mice using uridine diphosphoglucose.

Uridine diphosphoglucose (UDPG) is a precursor of uridine that can be used as a rescuing agent from 5-fluorouracil (5FU) toxicity. Four doses of UDPG (2000 mg/kg i.p. or p.o. at 2, 6, 24, and 30 h after 5FU bolus) allowed the escalation of a weekly bolus of 5FU from 100 mg/kg (5FU100) to 150 mg/kg (5FU150) in healthy and tumor-bearing BALB/c, C57/BI, and CD8F1 (BALB/c x DBA/8) mice. 5FU150 without rescuing agents is not tolerated by the animals. When followed by UDPG, on the contrary, it is possible to increase the dose of 5FU even when it is modulated by leucovorin. Toxicity was the same for 5FU100 and 5FU150 + UDPG, and the nadir values (expressed as a percentage of pretreatment values) were 83 and 85% for weight, 45 and 45% for hematocrit, and 45 and 61% for leukocytes, respectively. Platelets were not affected by treatment. A protective effect was also shown for the gastrointestinal tract. The enzymes thymidine kinase, maltase, and sucrase were measured in the intestinal mucosa at different times after 5FU treatment with or without UDPG rescue. Even if the nadir values in enzyme activities were similar in mice receiving or not receiving UDPG, the pattern of recovery showed that cell repopulation was more rapid in the group treated with UDPG. 5FU150 + UDPG had enhanced antitumor activity against CD8F1 mammary carcinoma and against the resistant tumor Colon 26 (tumor doubling time 1.9 days for controls, 8.5 days for 5FU100, 13.7 days for 5FU150 + UDPG, and 15.9 days for 5FU150 + leucovorin + UDPG). We demonstrated that UDPG administered at 2, 24, and 30 h after 5FU100 does not reduce the antitumor activity of 5FU in two sensitive tumors (Colon 38 and Colon 26-10). In conclusion, UDPG is a promising rescuing agent for 5FU; it reduces the toxic side effects and increases the therapeutic index.

Pharmacokinetics of bolus 5-fluorouracil: relationship between dose, plasma concentrations, area-under-the-curve and toxicity.

The pharmacokinetics of 5-fluorouracil (5FU) have been related to toxicity and antitumor activity, in particular for continuous infusion schedules, but to a lesser extent for frequently used bolus injections. The use of intensive sampling schedules limits the application of pharmacokinetics to optimize individual dosing or to define the ideal combination with other drugs. We therefore reanalyzed a pharmacokinetic study in order to develop a limited sampling schedule. Patients received escalating doses of 5FU at 500, 600 and 720 mg/m2 as a bolus until toxicity developed. Blood samples were analyzed until 24 h after administration. The area under the concentration time curve from 0-90 min (AUC(0-90)) was strongly correlated with dose and also with toxicity (p = 0.0009). The 5FU concentrations at 30 and 60 min were correlated to the AUC(30-240) and to that of the AUC(0-90) (r2 = 0.970). The use of limited sampling (30, 60, 90 min) in a patient given 353 mg/m2 5FU with severe toxicity at initial dosing at 500 mg/m2 revealed that the AUC(0-90) at 353 mg/m2 was higher than the normal AUC(0-90) for 500 mg/m2. This patient appeared to have an 8-fold lower activity of the 5FU degradation enzyme dihydropyrimidine dehydrogenase. Limited sampling will allow us to define potential aberrant kinetics of pharmacokinetic interaction of 5FU with other drugs being developed for treatment of colorectal cancer.

Thymidylate synthase and drug resistance.

Thymidylate synthase is an important target for both fluorinated pyrimidines and for new folate analogues. Resistance to 5-fluorouracil (5FU) can be related to insufficient inhibition of thymidylate synthase. The 5FU-nucleotide FdUMP induces inhibition of thymidylate synthase which is enhanced and retained for longer in the presence of increased folate pools, for which leucovorin is a precursor. In a murine model system, 5FU treatment caused a 4-fold induction of thymidylate synthase levels which may have contributed to resistance. Addition of leucovorin to this treatment prevented this induction and increased the antitumour effect 2-3-fold. In the clinical setting, 5FU administration to patients resulted in approximately 50% inhibition of TS after 48 h. The combination with leucovorin resulted in a more pronounced inhibition after 48 h (approximately 70%). A significant relationship was observed with outcome of treatment; when thymidylate synthase levels were high and inhibition was low, no response was observed. A separate study showed that low thymidylate synthase levels appeared to be an independent prognostic factor for adjuvant therapy.

Antitumour activity, toxicity and inhibition of thymidylate synthase of prolonged administration of 5-fluorouracil in mice.

Continuous infusions of 5-fluorouracil (5-FU) are increasingly used in the treatment of cancer. Their optimal use, however, has still to be determined since the availability of suitable animal models is limited. We studied continuous infusions in mice using subcutaneously implanted pellets that release 5-FU over a period of 3 weeks. At the maximum tolerated dose (MTD) (based on the systemic toxicity in healthy animals) we assessed the antitumour activity, haematological toxicity, inhibition of thymidylate synthase (TS) in tumours and the concentration of 5-FU in plasma during the 3-week period. We also studied the addition of leucovorin in different schedules. The dose-limiting toxicity was weight loss, and at the MTD of 10 mg of 5-FU released in 21 days per mouse myelosuppression was tolerable (nadir for leucocytes and thrombocytes was approximately 40% of pretreatment levels). In several independent experiments using the 5-FU-resistant Colon 26 tumour, a good antitumour activity was observed during the first part of the infusion, but thereafter the growth of the tumours resumed; the overall effect of continuous infusions was thus comparable to that of bolus injections. Coadministration of leucovorin did not enhance the therapeutic results; depending on the schedule used, it proved ineffective or only increased toxicity. Similar results were obtained with head and neck squamous cell carcinomas and with the 5-FU-sensitive tumour Colon 38. In Colon 26 tumours the TS activity (FdUMP-binding assay) initially decreased to 20-30% of controls and returned to normal after 11 days. In the catalytic TS assay a slight inhibition was observed for the continuous infusion, followed after 11 days by a marked (4-fold) increase in activity. 5-FU plasma levels varied from 0.1 to 1 microM following a circadian rhythm (with a peak at 6 h after light onset), and were maintained during the entire period. Subcutaneously implanted pellets represent a suitable model to study prolonged administration of 5-FU in mice and to evaluate the effect of modulating agents in laboratory animals before transferring data obtained in vitro to the clinic.

Intermittent continuous infusion of 5-fluorouracil and low dose oral leucovorin in patients with gastrointestinal cancer: relationship between plasma concentrations and clinical parameters.

Modulation of 5-fluorouracil (5-FU) by leucovorin and continuous infusion of 5-FU can both result in enhanced therapeutic efficacy. The main objective of this study was to determine the maximum tolerated dose (MTD) of oral leucovorin in combination with continuous infusion of 5-FU for 14 days every 4 weeks at a dose of 300 mg/m2/day in 30 patients with gastrointestinal cancer. The MTD of oral leucovorin was established at 10 mg/day. Dose-limiting toxicities were mucositis, diarrhoea and hand-foot syndrome. Plasma leucovorin concentrations were below the detection limit of the assay (< 0.5 microM). Plasma 5-FU concentrations varied considerably from 0.06 to 11.3 microM. A relation between toxicity, response and plasma concentration of 5-FU could not be established. Our data may indicate that even very low plasma concentrations of leucovorin are able to modulate 5-FU. In 17 patients with colorectal cancer the response rate was 24% (95% CI: 7-50%), which is comparable to other treatment schedules with leucovorin or to continuous infusion of 5-FU alone.

Elevated doses of carmustine and mitomycin C, with lonidamine enhancement and autologous bone marrow transplantation in the treatment of advanced colorectal cancer: results from a pilot study.

10 patients with advanced colorectal cancer were treated with elevated doses of carmustine and mitomycin C. The regimen was potentiated by lonidamine and supported by autologous bone marrow transplantation. The results of this pilot study were encouraging, with a response rate of 50% and a significantly better survival for responders versus non-responders. No appreciable toxicity of the therapy was observed. This aspect, together with the simplicity of the procedure, calls for further investigations to confirm the good therapeutic index of the treatment.

A new anthracycline regimen for prolymphocytic leukemia? Study of a case report with flow cytometric implications.

A case of prolymphocytic leukemia (PL) is reported, which showed a good response to a new antiblastic schedule (4-epidoxorubicin-asparaginase-dexamethasone) in spite of the resistance to other chemotherapy regimens. However during the course of the disease it was possible to observe the terminal appearance of a small aneuploid cell population in the peripheral blood of the patient and, in the same time, the clinical condition deteriorated considerably. The significance of this neoplastic progression and the pros and cons of aggressive chemotherapy regimens remain to be carefully evaluated in PL and related disorders.

Comparative in vitro and in vivo study of a nitroxyl derivative of 5-fluorouracil (magnizil) on human gastrointestinal tumors.

AIMS AND BACKGROUND: There is much interest in nitroxyl derivatives of cytotoxic agents. We evaluated the potential activity of magnizil, a derivative of 5-fluorouracil, on human gastrointestinal tumors in 3 different in vitro and in vivo experimental models. METHODS: The activities of magnizil and 5-fluorouracil were comparatively determined in vitro on the HT29 cell line by a clonogenic assay and on tumor clinical specimens by an antimetabolic assay. The activity of both the drugs against human tumors was also assessed in mice with the subrenal capsule assay. RESULTS: A similar cytotoxic activity was found for magnizil and 5-fluorouracil on the HT29 cell line. As regards human tumors, a lower activity was observed for the nitroxyl derivative than for 5-fluorouracil, with response rates of 25% and 50%, respectively, at comparable concentrations. Moreover, among the tumors transplanted in the subrenal capsule of mice, two were sensitive to magnizil and 3 to 5-fluorouracil. CONCLUSIONS: Even though experimental results on human tumors indicate a somewhat lower activity for magnizil than the parent compound, its low toxicity and the possibility to clinically use high doses suggest the opportunity to further investigate the potential of this new anticancer agent on larger series of colorectal cancers in experimental systems.

Anthracycline in colorectal carcinoma: an in vitro short-term assay to predict drug sensitivity. Preliminary results.

The chemosensitivity of 26 non-pretreated colorectal carcinomas (primary tumors and/or colorectal metastases) was studied by an in vitro antimetabolic assay, which evaluates the interference of drugs on the incorporation of 3H-thymidine and 3H-uridine in short-term cultures of human tumors. Our results correlate with the response rate obtained in clinical studies with monochemotherapy and justify the possibility of a future prospective study using individually tailored chemotherapy regimens. Doxorubicin-analogues, with an overall in vitro efficacy in 16.0% and 14.3% for 4-epidoxorubicin (epi-DX) and 4-deoxydoxorubicin (deo-DX), respectively, seem to deserve a modest role in the treatment of colorectal cancer, provided that a careful selection of patients is performed. Variability in anthracycline activity is indeed evident, also in our study, in relation to the different neoplastic picture of the various patients.

Intrarticular methotrexate in the therapy of rheumatoid arthritis.

Five patients with oligoarticular rheumatoid arthritis were treated with intra-articular injections of methotrexate and orgotein in the knee joints. The employed dose of the antimetabolite was very low and orgotein was simultaneously administered to prevent local tissues from cytolysis-related damage. Clinical results were fairly good and support the hypothesis that methotrexate may be used intra-articularly as an immunosuppressor rather than at the heavily toxic doses required for a cytostatic effect.

Selective protection by uridine of growth inhibition by 5-fluorouracil (5FU) mediated by 5FU incorporation into RNA, but not the thymidylate synthase mediated growth inhibition by 5FU-leucovorin.

Fluorouracil (5FU) acts by RNA-incorporation and inhibition of thymidylate synthase; the first action is counteracted by uridine, and the second is enhanced by leucovorin (LV). Growth inhibition of C26-10 colon cancer cells by 5FU was enhanced by LV and rescued by uridine, but 5FU-LV was only partially rescued by uridine. In WiDr cells, 5FU sensitivity was not enhanced by LV, while both 5FU and 5FU-LV were rescued by uridine. Intermediate trends were found in SW948 and HT29 cells. Uridine rescue in mice allowed 1.5-fold increase in 5FU dose, leading to 2-fold increase in the antitumor effect and thymidylate synthase inhibition in resistant Colon-26 tumors. In the sensitive Colon-26-10 tumor, uridine rescue decreased 5FU-RNA incorporation > 10-fold, without affecting the antitumor activity. The use of LV and uridine can differentiate between two mechanisms of action of 5FU.

5-fluorouracil modulation in the chemotherapy of colorectal cancer.

More than ten years after the introduction into the clinic of Folinic Acid associated with 5-FU in the treatment of colorectal cancer, it appears that in terms of objective responses, associations with modulating agents are more effective than 5-FU alone. An improvement in survival has been observed in some studies, but this remains a debated subject. Results can probably be further improved by multiple modulation also using Interferon and by a more careful evaluation of drug scheduling. The data on the activity of modulated 5-FU in the adjuvant treatment of colon cancer await the publication of confirmatory trials, but they are already sufficiently convincing to assert that adjuvant treatment should be part of the standard approach to patients with locally advanced colon carcinoma. The comparison of reports from different groups emphasizes the need for protocol standardization and a better description of patients based not only on pathological staging, but also on the biological characteristics of the tumour.

Cytosine arabinoside-induced differentiation and killing: unresolved questions.

A brief survey of the problems related to leukemic cell differentiation is proposed. The role of cytosine-arabinoside among the differentiation inducers and its potential clinical utility is discussed on the basis of the present literature. The authors report a case of myelomonocytic leukemia which displayed a differentiative response to cytarabine treatment.