Coverage Policy Manual
Policy #: 2011073
Category: Laboratory
Initiated: December 2011
Last Review: October 2018
  Biomarker, Methotrexate Polyglutamates to Predict Response to Methotrexate in Patients with Rheumatoid Arthritis (Avise PG)

Methotrexate is commonly prescribed for the treatment of rheumatoid arthritis. Despite its effectiveness, Methotrexate may cause serious side effects and the dosing levels and treatment response may vary considerably from one patient to another. Serious side effects from treatment with methotrexate may include: liver damage, bone marrow suppression, gastrointestinal disturbances, renal toxicity, severe and sometimes fatal dermatologic reactions, opportunistic infections and interstitial pneumonitis. Frequent blood tests are required to monitor for development of these and other side effects.
Measurement of methotrexate polyglutamate, the active metabolites of methotrexate, has been proposed as a method of monitoring methotrexate therapy (Dervieux, 2006). Avise PG, patented by Exagen Diagnostics, is a proprietary lab test used to measure methotrexate polyglutamates. Avise PG has not been approved by the U.S. Food and Drug Administration (FDA).
There is no specific CPT code for this test.  CPT 84999, Unlisted chemistry procedure, should be used when billing for Avise PG.

Testing for methotrexate polyglutamates with the Avise PG test to predict response in patients with rheumatoid arthritis does not meet member benefit certificate primary coverage criteria that there be scientific evidence of effectiveness in improving health outcomes because there is considerable disagreement in the medical literature over the effectiveness of this test.
For contracts without primary coverage criteria, testing for methotrexate polyglutamates with the Avise PG test to predict response in patients with rheumatoid arthritis is considered investigational.  Investigational services are specific contract exclusions in most member benefit certificates of coverage.

This policy was developed after requests were received for the use of Avise PG to monitor methotrexate treatment in patients with rheumatoid arthritis.  As a result of these requests, a literature search was conducted using the Medline database, focusing on randomized controlled trials, prospective trials and meta-analyses concerning the measurement of methotrexate glutamates in patients treated with methotrexate for a diagnosis of rheumatoid arthritis.
There were no randomized controlled trials evaluating the clinical utility of the Avise PG test used to monitor patients taking methotrexate for the treatment of rheumatoid arthritis.  
A series of studies by Dervieux et al., assessing the association between pharmacogenomic and metabolic markers were identified (Dervieux, 2004) (Dervieux, 2005) (Dervieux, 2006).  In 2004, Dervieux and colleagues published results of a cross-sectional study assessing the contribution of genetic polymorphisms and methotrexate polyglutamates in the treatment effect of patients being treated with methotrexate for rheumatoid arthritis.  A total of 108 patients undergoing methotrexate treatment for at least 3 months were enrolled. The newly developed analytical method of measuring the concentration of methotrexate polyglutamate (MTXPG) in red blood cells was studied. Methotrexate dosing was not associated with the number of swollen or painful joints; however, there was an association between higher doses of methotrexate and higher scores on physician and patient’s global assessment of disease activity. According to the authors, higher concentrations of RBC long-chain MTXPG were associated with lower numbers of tender and swollen joints as well as lower scores on the physicians global response assessments (Dervieux, 2004).
In another study, by the same authors (Dervieux et al., 2005), 226 adults were enrolled in a multicentre, 3 site, cross sectional observational study. Participants were all diagnosed with rheumatoid arthritis and had been on methotrexate therapy for more than 3 months. Red blood cell (RBC) MTXPG, folate polyglutamate (PG) and pharmacokinetic indices were measured on all participants. Attending physicians were blinded to all levels throughout the study. The authors report that lower (less than 60 nmol/L) levels of RBC MTXPG levels were associated with a poor methotrexate response. Although the authors conclude that measurement of RBC MTXPG levels may aid in the management of methotrexate treatment, they indicate that prospective studies are needed to assess the “predictive value” of this and other markers studied.
A third study (Dervieux, 2006) was conducted, at a single center, on forty-eight methotrexate naïve patients diagnosed with rheumatoid arthritis. Again, this study assessed the association of RBC MTXPG, folate PG and pharmacogenetic measurements on the effects of methotrexate in patients with rheumatoid arthritis. Measurements were taken at baseline and four subsequent visits every four to six weeks. At the month 6 visit, three patients were assessed to be nonresponders. These patients had corresponding lower RBC MTXPG levels than did the responders. In addition, less improvement was seen in these patients in the physician’s assessment of disease activity.  The authors conclude that this study shows the association between methotrexate PG concentrations and methotrexate effects in methotrexate naïve patients.
A cross-sectional study by Stamp and colleagues (Stamp, 2010) did not show a correlation between methotrexate glutamate concentration and reduced disease activity in patients receiving long-term treatment with methotrexate for a diagnosis of rheumatoid arthritis. A total of 192 patients with rheumatoid arthritis receiving methotrexate treatment were enrolled. The authors conclude that a long-term prospective study is needed to clarify the differences between this study and previous studies; and additionally to determine whether there is clinical benefit to increasing the dosage in patients that do not respond to methotrexate treatment.
A search of the website revealed two studies which are listed to have been completed (NCT01038349) (NCT00695188).  The results of NCT01038349 were reported in an abstract for the American College of Rheumatology/Association of Rheumatology Health Professionals Annual Scientific Meeting (Sunkureddi, 2010).  The results showed that in 45% of cases, the physicians modified the treatment plans in response to MTXPG results.  However, this study was an observational study and did not indicate whether the treatment modifications improved health outcomes.  The results have not been published in peer review literature.  
Currently, there is a lack of scientific evidence that using Avise PG testing to monitor methotrexate treatment response improves outcomes in individuals with rheumatoid arthritis.
2013 Update
A literature search was conducted using the MEDLINE database through September 2013. There was no new information identified that would prompt a change in the coverage statement.
2014 Update
A literature search was conducted using the MEDLINE database through September 2014. There was no new information identified that would prompt a change in the coverage statement.
2015 Update
A literature search conducted through September 2015 did not reveal any new information that would prompt a change in the coverage statement. The key identified literature is summarized below.
Van Dijkhuizen and Wulffraat conducted a systematic literature search was performed in PubMed, Embase and The Cochrane Library, and 1,331 articles were identified. These were selected based on their relevance to the topic and critically appraised according to pre-defined criteria. Predictors for MTX efficacy and adverse events were extracted from the literature and tabulated (van Dijhuizen, 2014). Twenty articles were selected. The overall quality of the studies was good. For MTX efficacy, candidate predictors were antinuclear antibody positivity, the childhood health assessment questionnaire score, the myeloid-related protein 8/14 level, long-chain MTX polyglutamates, bilateral wrist involvement and some single nucleotide polymorphisms (SNPs) in the adenosine triphosphate binding cassette and solute carrier transporter gene families. For MTX adverse events, potential predictors were alanine aminotransferase and thrombocyte level and two SNPs in the γ-glutamyl hydrolase and methylenetetrahydrofolate reductase genes. However, validation of most predictors in independent cohorts was still lacking. Interesting candidate predictors were found, especially for MTX efficacy. However, most of these were not validated. This should be the goal of future efforts. A clinically relevant way to validate the predictors is by means of creating a clinical prediction model.
Hawwa and colleagues published development and validation of a selective and sensitive LCMS method for the determination of methotrexate polyglutamate in dried blood spots (DBS) (Hawwa, 2014). DBS samples [spiked or patient samples] were prepared by applying blood to Guthrie cards which was then dried at room temperature. The method utilized 6-mm disks punched from the DBS samples (equivalent to approximately 12 µl of whole blood). The simple treatment procedure was based on protein precipitation using perchloric acid followed by solid phase extraction using MAX cartridges. The extracted sample was chromatographed using a reversed phase system involving an Atlantis T3-C18 column (3 µm, 2.1 × 150 mm) preceded by Atlantis guard column of matching chemistry. Analytes were subjected to LCMS analysis using positive electrospray ionization. The method was linear over the range 5-400 nmol/L. The limits of detection and quantification were 1.6 and 5 nmol/L for individual polyglutamate and 1.5 and 4.5 nmol/L for total polyglutamate, respectively. The method has been applied successfully to the determination of DBS finger-prick samples from 47 pediatric patients and results confirmed with concentrations measured in matched RBC samples using conventional HPLC-UV technique. The methodology has a potential for application in a range of clinical studies (e.g. pharmacokinetic evaluations or medication adherence assessment) since it is minimally invasive and easy to perform, potentially allowing parents to take blood samples at home. The feasibility of using DBS sampling can be of major value for future clinical trials or clinical care in pediatric rheumatology.
2016 Update
A literature search was conducted using the MEDLINE database through September 2016. There was no new information identified that would prompt a change in the coverage statement.
2017 Update
A literature search conducted through September 2017 did not reveal any new information that would prompt a change in the coverage statement.  
2018 Update
A literature search was conducted through September 2018.  There was no new information identified that would prompt a change in the coverage statement.  

84999Unlisted chemistry procedure

References: Danila M.I., Hughes L.B., Brown E. E., et al.(2010) Measurement of erythrocyte methotrexate polyglutamate levels: ready for clinical use in rheumatoid arthritis? Curr Rheumatol Rep. 2010; 12:342-347.

Dervieux T. Furst D. Lein D.O., et al.(2005) Pharmacogenetic and metabolite measurements are associated with clinical status in patinets with rheumatoid arthritis treated with methotrexate: results of a multicentred cross sectional observational study. Ann Rheum Dis 2005;64:1180-1185.

Dervieux T., Furst D., Lein D.O., et al.(2004) Polyglutamation of methotrexate with common polymorphisms in reduced folate carrier, aminoimidazole carboxamide ribonucleotide transformylase and thymidylate synthase are associated with methotrexate effects in rheumatoid arthritis. Arthritis & Rheumatism. 2004;(50) 9: 2766-2774.

Dervieux T., Greenstein N., & Kremer J.(2006) Pharmacogenomic and metabolic biomarkers in the folate pathway and their association with methotrexate effects during dosage escalation in rheumatoid arthritis. Arthritis Rheum. 2006; 54:3095-3103.

Goodman S.(2010) Measuring methotrexate polyglutamates. Clin Exp Rheumatol. 2010 Sep-Oct;28(5Suppl 61):S24-6.

Hawwa AF, Albawab A, Rooney M , et al.(2014) A novel dried blood spot-LCMS method for the quantification of methotrexate polyglutamates as a potential marker for methotrexate use in children. PLoS One. 2014 Feb 25;9(2):e89908.

Methotrexate polyglutamate levels as a marker for the clinical outcome in the treatment of rheumatoid arthritis. NCT00695188. Retrieved at

Murosaki T, Nagatani K, Sato T,(2017) Prediction of the therapeutic response to methotrexate at 24 weeks by methotrexate-polyglutamates concentration in erythrocytes at 8 weeks in patients with rheumatoid arthritis. Mod Rheumatol. 2017 May;27(3):411-416

Stamp L.K., O'Donnell J.L., Chapman P.T., et al.(2010) Methotrexate polyglutamate concentrations are not associated with disese control in rheumatoid arthritis patients receiving long-term methotrexate therapy. Arthritis Rheum. 2010; 62(2):359-368.

Sunkureddi P.R., Gendreau J.F., Zablocki R.., et al.(2010) Demographic, disease and treatment characteristics of the rheumatoid arthritis population enrolled in the AMORA study in evaluationg the Avise PGSM test. [abstract]. Arthritis Rheum 2010;62 Suppl 10:302.

van Dijkhuizen EH, Wulffraat NM.(2014) Prediction of methotrexate efficacy and adverse events in patients with juvenile idiopathic arthritis: a systematic literature review. Pediatr Rheumatol Online J. 2014 Dec 11;12:51

van Roon EN, van de Laar MA.(2010) Methotrexate bioavailablity. Clin Exp Rheumatol. 2010 Sep-Oct;28(5 Suppl 61):S27-32, Epub 2010 Oct 28.

Group specific policy will supersede this policy when applicable. This policy does not apply to the Wal-Mart Associates Group Health Plan participants or to the Tyson Group Health Plan participants.
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