Calibrating from Within: Multipoint Internal Calibration of a Quantitative Mass Spectrometric Assay of Serum Methotrexate.

BACKGROUND: Clinical LC-MS/MS assays traditionally require that samples be run in batches with calibration curves in each batch. This approach is inefficient and presents a barrier to random access analysis. We developed an alternative approach called multipoint internal calibration (MPIC) that eliminated the need for batch-mode analysis. METHODS: The new approach used 4 variants of 13C-labeled methotrexate (0.026-10.3 µM) as an internal calibration curve within each sample. One site carried out a comprehensive validation, which included an evaluation of interferences and matrix effects, lower limit of quantification (LLOQ), and 20-day precision. Three sites evaluated assay precision and linearity. MPIC was also compared with traditional LC-MS/MS and an immunoassay. RESULTS: Recovery of spiked analyte was 93%-102%. The LLOQ was validated to be 0.017 µM. Total variability, determined in a 20-day experiment, was 11.5%CV. In a 5-day variability study performed at each site, total imprecision was 3.4 to 16.8%CV. Linearity was validated throughout the calibrator range (r2 > 0.995, slopes = 0.996-1.01). In comparing 40 samples run in each laboratory, the median interlaboratory imprecision was 6.55%CV. MPIC quantification was comparable to both traditional LC-MS/MS and immunoassay (r2 = 0.96-0.98, slopes = 1.04-1.06). Bland-Altman analysis of all comparisons showed biases rarely exceeding 20% when MTX concentrations were >0.4 µM. CONCLUSION: The MPIC method for serum methotrexate quantification was validated in a multisite proof-of-concept study and represents a big step toward random-access LC-MS/MS analysis, which could change the paradigm of mass spectrometry in the clinical laboratory.

Ectopic Cervical Pregnancy: Treatment Route.

Background and objectives: Cervical pregnancy (CP) is a rare form of ectopic pregnancy (EP) in which the embryo implants and grows inside the endocervical canal. Early diagnosis is essential in order to allow conservative medical and surgical treatments. Although many treatment approaches are disponible, the most effective is still unclear. The aim of this study is to evaluate the efficacy of hysteroscopic management in early CP in order to preserve future fertility. Materials and Methods: This is a retrospective observational case series. Five patients with a diagnosis of CP, hemodynamically stables and managed conservatively between 2014 and 2019 at the Institute of Child and Maternal Health Burlo Garofolo in Trieste, Italy, were included. Four patients, with ßhCG levels >5000 mUi/mL were managed by hysteroscopy, with or without a previous systemic Methotrexate (MTX). One case with ßhCG levels <5000 mUi/mL was treated using MTX combined to Mifepristone and Misoprostol. Results: In one patient treated by hysteroscopy alone it occurred a profuse vaginal bleeding with necessity for blood transfusion. Haemorrhage was controlled by a second hysteroscopic procedure. No complications, such as vaginal bleeding, were recorded in the other cases. Serum ß-hCG levels become undetectable in a range of 15-40 days after hysteroscopic management; after medical treatment it become undetectable after 35 days. Serum ßhCG levels had a faster drop the day after hysteroscopy than post medical management. The onset of a spontaneous pregnancy at the normal implantation site occurred after five months in one case treated by hysteroscopy. Conclusions: Many therapeutic approaches are effective for CP treatment. Hysteroscopy, alone or in combination with MTX, may provide a greater effect on the descent of ßhCG, leading to a reduction of the hospitalization stay, decreasing costs and period for attempt pregnancy. Further prospective studies on larger samples are needed to define therapeutic protocols for CP management.

Is there uniformity in definitions and treatment of gestational trophoblastic disease in Europe?

OBJECTIVES: Because gestational trophoblastic disease is rare, little evidence is available from randomized controlled trials on optimal treatment and follow-up. Treatment protocols vary within Europe, and even between different centers within countries. One of the goals of the European Organization for Treatment of Trophoblastic Diseases (EOTTD) is to harmonize treatment in Europe. To provide a basis for international standardization of definitions, treatment and follow-up protocols in gestational trophoblastic disease, we evaluated differences and similarities between protocols in EOTTD countries. METHODS: Members from each EOTTD country were asked to complete an online structured questionnaire comprising multiple-choice and multiple-answer questions. The following themes were discussed: incidence of gestational trophoblastic disease and gestational trophoblastic neoplasia, definitions, guidelines, classification system, treatment, recurrence, and follow-up. RESULTS: Forty-four respondents from 17 countries participated in this study. Guidelines were present in 80% of the countries and the FIGO (Fédération Internationale de Gynécologie et d'Obstétrique) staging and risk classification was often used to estimate risks. Agreement about when to start chemotherapy for post-molar gestational trophoblastic neoplasia was present among 66% of the respondents. Preferred first-line treatments in low- and high-risk gestational trophoblastic neoplasia were methotrexate (81%) and EMA-CO (etoposide, methotrexate, actinomycin D, cyclophosphamide, vincristine) (93%), respectively. The definition of human chorionic gonadotropin normalization after hydatidiform mole evacuation was two consecutive normal values for nine countries. The FIGO definition of post-molar gestational trophoblastic neoplasia based on human chorionic gonadotropin plateau or rise was agreed on by 69% of respondents, and only 69% and 74% defined low-risk and high-risk disease, respectively, using FIGO criteria. There were major differences in definitions of recurrence, chemotherapy resistance and follow-up protocols among countries, despite EOTTD consensus statements. CONCLUSIONS: This questionnaire provides a good overview of current clinical practices in different countries. Based on the survey results, it is clear that there are several gestationaltrophoblastic disease-related topics that need urgent attention within the EOTTD community to create more uniformity and to aid the development of uniform guidelines in Europe.

[Standardization of methotrexate administration in routine rheumatological practice].

The review is devoted to administration of methotrexate--a basic anti-inflammatory drug--in rheumatoid arthritis. Some issues of practical use of this drug are still disputable. Recommendations of international experts and information on standardization for patients are considered.

Novel gemcitabine-containing triplets in the management of urothelial cancer.

Chemotherapy has been the cornerstone of treatment of advanced urothelial cancer. For a decade, the combination regimen of methotrexate/vinblastine/doxorubicin/cisplatin has been considered the standard for these patients. The need for improved efficacy and reduced toxicity of a predominantly palliative therapy has propelled efforts for new drug development. Of the newly identified agents with documented activity, both gemcitabine and paclitaxel have been evaluated with a platinum and have been incorporated into multiagent chemotherapy combinations. Phase II data from two gemcitabine-based triplets are currently available. Combination gemcitabine/paclitaxel/cisplatin and gemcitabine/paclitaxel/carboplatin have high levels of activity with overall and complete response rates of 76% and 26%, respectively, for the former and 68% and 32%, respectively, for the latter combination. The role of gemcitabine-based multiagent combinations compared with standard therapy awaits evaluation in prospectively randomized trials.

Current and future perspectives in advanced bladder cancer: is there a new standard?

The methotrexate/vinblastine/doxorubicin/cisplatin (MVAC) regimen has been the standard treatment in patients with locally advanced and metastatic urothelial cancer for the past 15 years. The minimal or moderate survival benefit-depending on prognostic features-and the severe toxicity associated with the MVAC regimen have made the search for new drugs and drug combinations of utmost importance to increase efficacy and/or decrease toxicity. In this respect, the taxanes and gemcitabine are promising new drugs. Paclitaxel and docetaxel as single agents have yielded overall response rates of 7% to 56%, depending on whether the patients have received prior chemotherapy for metastatic disease. The combination of paclitaxel and cisplatin has been explored in three studies with a total of 104 evaluable patients, a pooled overall response (OR) rate of 61%, and a complete response (CR) rate of 20%. There are two studies of docetaxel and cisplatin with a total of 91 evaluable patients, an OR rate of 54%, and a CR rate of 16%. The OR rate for paclitaxel and carboplatin in six studies was 43%, with a CR rate of 13%; however, the reported median survival was only 8.5 to 9.5 months. The OR rate for single-agent gemcitabine based on five studies was 26%, with a CR rate of 9%, which was apparently independent of whether the patients had received prior chemotherapy. The OR rate for gemcitabine and cisplatin in four phase II studies ranged from 41% to 57%, with a CR rate of 15% to 22% and a median survival of 12.5 to 14.3 months. Based on the encouraging results for the combination of gemcitabine and cisplatin (GC), a randomized phase III trial comparing GC and MVAC was begun in late 1996. This study of 405 randomized patients showed that the two regimens were associated with similar response rates, time to progression, and overall survival, whereas GC was associated with less toxicity than MVAC. On the basis of this superior risk-benefit ratio, the GC regimen should be favored as a new standard treatment in patients with locally advanced and metastatic urothelial cancer. Other promising combinations include gemcitabine and paclitaxel, with or without cisplatin, and the combination of ifosfamide, paclitaxel, and cisplatin. The triple combination of gemcitabine, paclitaxel, and cisplatin has yielded an OR rate of 78%, a CR rate of 28%, and a median survival of 24 months. An international phase III trial comparing this triple combination with GC in patients with locally advanced and metastatic urothelial cancer has now been initiated.

Low-dose methotrexate in rheumatic diseases--efficacy, side effects, and risk factors for side effects.

Controlled trials and observational studies have shown low-dose methotrexate (MTX) to be a second-line agent of high potency with a favorable profile of safety and tolerability in the treatment of rheumatoid arthritis (RA). Its risk-benefit ratio in psoriatic arthritis is less well documented. Preliminary reports on its beneficial effects in other disorders, including the systemic manifestations of RA, other spondyloarthritides, and collagen vascular diseases, merit more detailed examination. Gastrointestinal intolerance and hepatic enzyme elevation are the most frequent side effects of MTX; life-threatening events such as severe hemocytopenia and MTX pneumonitis are rare and amenable to prevention by recognizing risk factors and premonitory signs. Hepatotoxicity does not appear to be a major limiting factor in RA patients over the first 2 to 3 years of MTX therapy; its impact on long-term tolerance remains to be clarified.

Cyclosporine combined with methylprednisolone or methotrexate in prophylaxis of moderate to severe acute graft-versus-host disease.

To evaluate the efficacy of cyclosporine (CYA) regimens in preventing moderate to severe acute graft-versus-host disease (GVHD), 25 patients received immunosuppressive therapy consisting of either CYA and methylprednisolone or CYA and methotrexate (MTX) and the incidence and severity of acute GVHD was compared. These patients had leukemia or myelodysplastic syndrome (MDS) and received bone marrow transplants (BMT) from genotypically HLA-identical siblings. The incidence of grade I-IV acute GVHD in patients on the CYA/methylprednisolone regimen was 64% (7 of 11) compared with 50% (7 of 14) in those on the CYA/MTX regimen. Five of 11 patients with the CYA/methylprednisolone regimen developed moderate to severe acute GVHD (grade II-IV), fatal in 3 cases. No patient on the CYA/MTX regimen developed moderate to severe acute GVHD. Engraftment was faster in the CYA/methylprednisolone group than in the CYA/MTX group. The incidence of toxicity observed soon after BMT was comparable between groups. The CYA/MTX regimen may be superior to the CYA/methylprednisolone regimen for preventing moderate to severe acute GVHD.

Evaluation of methotrexate in the treatment of juvenile chronic arthritis according to the subtype.

Methotrexate therapy was evaluated in 30 children with juvenile chronic arthritis according to the type of onset. The systemic form seemed less responsive than the ANA positive form with a polyarticular course or polyarticular onset. The clinical improvement, particularly in the ANA positive polyarticular course was confirmed by a significant decrease in the values of the ESR. Side effects occurred in 12 patients and consisted of gastrointestinal upset, mouth ulcers, slight leucopenia and elevated transaminases. They led to discontinuation of the treatment in only one child. Concomitant therapy could be stopped in 50% of the patients with an ANA positive polyarticular course, but remained necessary in the two other groups. These results indicate a differential effect of MTX therapy according to the type of JCA.

Purity profiles of pteroylglutamate reference substances by high-performance liquid chromatography.

High-performance liquid chromatography (HPLC) in the reversed-phase mode was used for the purity analysis of three pteroylglutamic acid-type reference substances (folic acid, leucovorin calcium, and methotrexate). The influence of the pH of the mobile phase on the separation of an artificial mixture of six pteroylglutamic acid derivatives and three potential impurities was studied. Results of purity analysis of current lots of USP reference standards are reported. A better separation of methotrexate from its major impurities was achieved by using a standard buffer, rather than an ion-pairing mobile phase. A separation of methotrexate and its biologically inactive 7-isomer is reported.

Methotrexate and misoprostol for abortion at 57-63 days gestation.

Methotrexate and misoprostol have been shown in preliminary studies to be effective for abortion at < or = 56 days gestation with minimal side effects. RU-486 is used in combination with prostaglandin for abortion < or = 49 days gestation in France but < or = 63 days gestation in England. This pilot study was performed to evaluate if methotrexate and misoprsotol may also be effective up to 63 days gestation. Ten pregnant women between 57-63 days gestation were treated with methotrexate 50 mg/m2 intramuscularly followed 3 days later by misoprostol 800 micrograms vaginally. Abortion occurred in 6 women; abortion occurred in one of the women after a repeat dose of misoprostol 24 hours after the first dose. The remaining 4 women all had a surgical abortion. In the successfully treated women, vaginal bleeding lasted 14 +/- 4 (mean +/- standard deviation) days and serum beta-hCG was < or = 25 IU/L by 32 +/- 5 days after the methotrexate injection. No methotrexate side effects occurred. Methotrexate and misoprostol do not appear to be as effective for medical abortion between 57 and 63 days gestation as compared to < or = 56 days gestation.

Revised FIGO staging system for gestational trophoblastic tumors. Recommendations regarding therapy.

OBJECTIVE: To evaluate the revised International Federation of Gynecology and Obstetrics (FIGO) staging system for gestational trophoblastic tumors (GTT) and to recommend therapy. STUDY DESIGN: Review of the literature regarding the development of the FIGO staging system, the World Health Organization (WHO) prognostic scoring system and Hammond's clinical classification for GTT plus analysis of response to single-agent chemotherapy in 546 patients treated at the New England Trophoblastic Disease Center. RESULTS: The revised FIGO staging system appears to successfully combine anatomic staging and a prognostic clinical classification. The revised FIGO staging system reliably predicts treatment outcome and therefore can be used to help select optimal treatment protocols. CONCLUSION: The revised FIGO staging system is capable of predicting patients who respond poorly to single-agent chemotherapy, appears to reliably predict outcome and therefore can be used to help select appropriate treatment protocols.

A radioimmunoassay for methotrexate adapted to the Centria System 2.

An automated radioimmunoassay for methotrexate using an iodinated tracer has been applied to the centrifugal analyser, Centria System 2. Results obtained for serum samples correlated closely with those using a manual radioimmunoassay method. A major advantage of the assay is its potential for processing large numbers of samples rapidly, making it highly suitable for routine clinical use.

[Stability of clinically used cytostatics (author's transl)]. / Stabilität klinisch gebräuchlicher Zytostatika

This paper concerns with the stability of aqueous solutions of clinically used cytostatics. The investigation was carried out with in vitro-activated cyclophosphamide from urine of patients, also with trenimon, vinblastine and methotrexate. The stability was tested by biological determination of tumor cell cultures, NBP-reaction and spectrophotometric investigations at different temperature of storage. The activated cyclophosphamide and the active metabolites from urine too are unstable in contrast to the active metabolites in lyophylisied urine. While vinblastine in our results are stable for ten days and more and methotrexate for fourteen days in the refrigerater in physiological solution, trenimon decomposed relative rapidly.

Treatment of rheumatoid arthritis with methotrexate: a prospective open longterm study of 191 cases.

One hundred and ninety-one patients with severe rheumatoid arthritis (RA) were included in a prospective open longterm study of the safety, efficacy and maintenance of methotrexate (MTX) treatment. The mean duration of MTX treatment was 19 +/- 13.2 (3-58) months; the mean weekly dose of MTX was 10.2 +/- 0.2 mg. Analysis of the 191 patients in an intent-to-treat manner showed a significant improvement of all the clinical variables and a decrease of erythrocyte sedimentation rate with a steroid sparing effect. The probability of continuing MTX therapy for up to 2 years was 65% and for up to 5 years was 46%. Adverse effects of MTX occurred in 37.1% of the patients, but only 15.7% discontinued MTX permanently.

Life table analysis of 879 treatment episodes with slow acting antirheumatic drugs in community rheumatology practice.

In 596 patients with RA managed over a decade in a community practice setting, 879 slow acting antirheumatic drug (SAARD) treatment episodes were analyzed using 5-year life tables. The probability of continuation of therapy was 50% by 9-24 months for all drugs except for methotrexate (MTX), which was 62% by 5 years [corrected]. MTX treatments were of significantly longer duration than those of all other SAARD (p less than 0.001); terminations for both inefficacy (p less than 0.001) and toxicity (NS) were less likely. These findings concur with recent evidence suggesting that MTX is a superior SAARD in this setting.

Radiographic evidence of disease progression in methotrexate treated and nonmethotrexate disease modifying antirheumatic drug treated rheumatoid arthritis patients: a meta-analysis.

Methotrexate (MTX) has proven to be efficacious in the treatment of rheumatoid (RA), but it remains to be proven whether it can slow disease progression, as determined radiographically, in comparison with other disease modifying antirheumatic drugs (DMARD). We performed a meta-analysis of the available data to answer this question. A literature search, including abstracts, was conducted and inclusion criteria developed (description of patients, accountability of patients, inclusion of a control group of patients, specified radiographic endpoint, and appropriate reading of the radiographs). Publications were scored on a scale of 0 to 5 with a score > or = 3 required for inclusion in the study. For abstracts selected, additional data were obtained directly from the investigators. Data for 353 MTX treated and 205 non-MTX-DMARD treated patients with RA were gathered. Not all publications used the same scoring system, so some assumptions were required to analyze the combined data. Only the erosion score was included since not all publications included a reading of the joint space. All scores were transformed into Sharp scores (Arthritis Rheum 1985;28:1449), including the important contributions of 3 Larsen scored publications. Finally a monthly rate of disease progression was computed. Several comparisons were made. Overall, the rates of disease progression were similar for MTX and non-MTX-DMARD treated patients with RA. The non-MTX-DMARD treated patients with RA were separated into a group treated with gold salts (oral or parenteral) and a group treated with azathioprine with each group compared to the MTX treated patients. MTX had slower rates of disease progression than azathioprine, (rates 0.004 vs 0.012) but not slower rates than gold salts (0.008 vs 0.008). Despite its efficacy, the possible role of MTX in slowing disease progression more than other DMARD, as determined radiographically, appears to be evident only when compared to azathioprine.