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MP 2.04.68 Laboratory and Genetic Testing for Use of 5-Flourouracil in Patients With Cancer


Medical Policy    
Original Policy Date
Last Review Status/Date
Reviewed with literature search/3:2014
  Return to Medical Policy Index


Our medical policies are designed for informational purposes only and are not an authorization, or an explanation of benefits, or a contract. Receipt of benefits is subject to satisfaction of all terms and conditions of the coverage. Medical technology is constantly changing, and we reserve the right to review and update our policies periodically. 


Variability in systemic exposure to 5-fluorouracil (5-FU) is thought to directly impact 5-FU tolerability and efficacy. Two approaches have been proposed for modifying use of 5-FU:

1. Dosing of 5-FU in cancer patients to a predetermined area under the curve (AUC) serum concentration target: Accurate AUC determination relies on sampling at pharmacokinetically appropriate times, as well as on accurate methods of 5-FU serum concentration measurement. Available measurement methods are complex, making them less amenable to routine clinical laboratory settings.

2. Genetic testing for mutations affecting 5-FU metabolism: Genetic mutations may affect activity of enzymes involved in 5-FU metabolism. Currently-available polymerase chain reaction (PCR) tests assess specific mutations in genes encoding dihydropyrimidine reductase (DPYD) and thymidylate synthase (TYMS), enzymes in the catabolic and anabolic pathways of 5-FU metabolism, respectively.


5-FU is a widely used antineoplastic chemotherapy drug that targets TYMS, an enzyme involved in DNA production. 5-FU has a narrow therapeutic index; doses recommended for effectiveness often are limited by hematologic and gastrointestinal toxicity. Moreover, patients administered the same fixed-dose, continuous-infusion regimen of 5-FU have wide intra- and interpatient variability in systemic drug exposure, as measured by plasma concentration or, more accurately, by AUC techniques. AUC is a measure of systemic drug exposure in an individual over a defined period of time.

In general, the incidence of grade 3 to 4 toxicity (mainly neutropenia, diarrhea, mucositis, and hand-foot syndrome) increases with higher systemic exposure to 5-FU. Several studies also have reported statistically significant positive associations between 5-FU exposure and tumor response. In current practice, however, 5-FU dose is reduced when symptoms of severe toxicity appear, but is seldom increased to promote efficacy.

Based on known 5-FU pharmacology, it is possible to determine a sampling scheme for AUC determination and to optimize an AUC target and dose adjustment algorithm for a particular 5-FU chemotherapy regimen and patient population. For each AUC value or range, the algorithm defines the dose adjustment during the next chemotherapy cycle most likely to achieve the target AUC without overshooting and causing severe toxicity.

In clinical research studies, 5-FU blood plasma levels most recently have been determined by high-performance liquid chromatography or liquid chromatography coupled with tandem mass spectrometry. Both methods require expertise to develop an in-house assay and may be less amenable to routine clinical laboratory settings. One commercially available alternative is Saladax Biomedical’s My5-FU™, an immunoassay designed to measure patients' exposure to 5-FU to help oncologists adjust and optimize 5-FU dosing. My5-FU™ was originally marketed in the U.S. by Myriad Genetics as OnDose® under patents licensed from Saladax Biomedical (Bethlehem, PA).(1) In June 2013, rights to the assay reverted to Saladax Biomedical.(2)

Metabolism of 5-Fluorouracil

5-FU is a pyrimidine antagonist, similar in structure to the normal pyrimidine building blocks of RNA (uracil) and DNA (thymine). More than 80% of administered 5-FU is inactivated and eliminated via the catabolic pathway; the remainder is metabolized via the anabolic pathway.

  • Catabolism of 5-FU is controlled by the activity of DPYD. Because DPYD is a saturable enzyme, the pharmacokinetics of 5-FU are strongly influenced by the dose and schedule of administration. (3) For example, 5-FU clearance is faster with continuous infusion compared with bolus administration, resulting in very different systemic exposure to 5-FU during the course of therapy. Genetic mutations in DPYD, located on chromosome 1, can lead to reduced 5-FU catabolism and increased toxicity. Many variants have been identified (eg, IVS14+1G>A [also known as DPYD*2A], 2846A>T [D949V]). DPYD deficiency is an autosomal codominantly inherited trait. (4)
  • The anabolic pathway metabolizes 5-FU to an active form that inhibits DNA and RNA synthesis by competitive inhibition of TYMS or by incorporation of cytotoxic metabolites into nascent DNA. (5) Genetic mutations in TYMS can cause tandem repeats in the TYMS enhancer region (TSER). One variant leads to 3 tandem repeats (TSER*3) and has been associated with 5-FU resistance due to increased tumor TYMS expression in comparison with the TSER*2 variant (2 tandem repeats) and wild-type forms.

Myriad Genetics has developed a PCR test, TheraGuide®, to assess certain mutations in DPYD and TYMS. The Myriad Genetics website estimates that “up to 25% of individuals have variations in the DPYD and/or TYMS genes that are associated with an increased risk of toxicity to 5-FU.” (6) ARUP Laboratories also offers DPYD and TYMS mutation testing. (5)

FDA Status

Currently, U.S. Food and Drug Administration (FDA)-approved tests for 5-FU AUC measurement and for DPYD/TYMS mutation testing are unavailable. My5-FU™ is offered by Saladax Biomedical as a laboratory-developed test; other clinical laboratories may offer in-house assays to measure 5-FU AUC. Similarly, TheraGuide® is offered by Myriad Genetics as a laboratory-developed test; other laboratories may offer in-house assays for DPYD and TYMS mutation testing (eg, ARUP Laboratories). Clinical laboratories may develop and validate tests in-house and market them as a laboratory service; laboratories offering such tests as a clinical service must meet general regulatory standards of the Clinical Laboratory Improvement Act (CLIA) and must be licensed by CLIA for high-complexity testing. Both Saladax Biomedical and Myriad Genetics are CLIA-licensed laboratories.


My5-FU™ testing or other types of assays for determining 5-fluorouracil area under the curve in order to adjust 5-FU dose for colorectal cancer patients or other cancer patients is considered investigational.

TheraGuide® testing for genetic mutations in dipyrimidine dehydrogenase (DPYD) or thymidylate synthase (TYMS) to guide 5-FU dosing and/or treatment choice in patients with cancer is considered investigational.

Policy Guidelines

There is no specific CPT coding for the My5-FU test. According to the company, the following code is used:

84999: Unlisted chemistry procedure.

Effective for 2011, there is a specific HCPCS “S” code for the My5-FU test –

S3722 – Dose optimization by area-under-the-curve (AUC) analysis for infusional 5-fluorouracil (5-FU)

The TheraGuide testing might be reported with the following codes:

CPT code 81400 which includes the following test –

DPYD (dihydropyrimidine dehydrogenase) (eg, 5-fluorouracil/5-FU and capecitabine drug metabolism),

IVS14+1G>A variant

And CPT code 81401 which includes the following test:

TYMS (thymidylate synthetase) (eg, 5-fluorouracil/5-FU drug metabolism), tandem repeat variant 

Benefit Application
BlueCard/National Account Issues

No applicable information. 


Literature Review

This policy was originally created in 2011 and was updated regularly with searches of the MEDLINE database. The most recent literature review was performed through February 17, 2014. Following is a summary of the key literature to date.

5-Fluorouracil and Clinical Use

5-Fluorouracil (5-FU) is a pyrimidine analog, antineoplastic antimetabolite; 5-FU has been used for many years to treat solid tumors, eg, colorectal adenocarcinoma. The FDA-approved indication of 5-FU is for “palliative management of carcinoma of the colon, rectum, breast, stomach, and pancreas.”(7)

Colon Cancer

Potentiated by leucovorin (LV), 5-FU is the basis for several standard treatment regimens currently recommended by the National Comprehensive Cancer Network (NCCN) for the treatment of colorectal cancer (CRC).(8) For stage II CRC, NCCN recommends adjuvant therapy primarily for disease with high-risk features, individualized for each patient; for stage III disease, oxaliplatin in combination with 5-FU/LV is the preferred standard of care.(8) Based on results from the 2009 European Multicenter International Study of Oxaliplatin/5-Fluorouracil/Leucovorin in the Adjuvant Treatment of Colon Cancer (MOSAIC) trial, (9) in which the addition of oxaliplatin to a regimen of LV and infusional 5-FU every 2 weeks (ie, a FOLFOX [leucovorin calcium, fluorouracil, oxaliplatin] regimen) significantly increased disease-free (DFS) and overall survival (OS), the FOLFOX regimen is recommended for patients with stage III colorectal cancer.(8) A FOLFOX regimen also improves progression-free survival (PFS) in patients with advanced (ie, metastatic) CRC who are able to tolerate intensive versus single-agent 5-FU therapy.(10,11), and FOLFOX may be considered for individual patients with high-risk stage II disease. Other 5-FU-based combination chemotherapy regimens are options in advanced disease.(8) In patients with advanced or metastatic colon cancer, bolus 5-FU regimens seem to be more toxic than infusional regimens and are considered inappropriate when coadministered with either irinotecan (a topoisomerase inhibitor) or oxaliplatin.(8)

Head and Neck Cancers

5-FU has for many years been a component, with cisplatin, of induction therapy for squamous cell carcinoma of the head and neck in patients with advanced locoregional disease, yielding high rates of overall and complete clinical response. The addition of docetaxel was shown to improve survival, and this 3-drug combination is now considered the standard of care for induction chemotherapy.(12,13) Typical 5-FU administration is by continuous infusion.(14) 5-FU also is a component of several combination chemotherapy regimens used for primary systemic therapy in conjunction with radiotherapy, and of 2 combination regimens for recurrent, unresectable, or metastatic disease.(12)

Measuring Exposure to 5-Fluorouracil

Patient exposure to 5-FU is most accurately described by estimating the area under the curve (AUC), the total drug exposure over a defined period of time. 5-FU exposure is influenced by method of administration, circadian variation, liver function, and the presence of inherited dihydropyrimidine reductase (DPYD)-inactivating genetic variants that can greatly reduce or abolish 5-FU catabolism. As a result, both inter- and intrapatient variability in 5-FU plasma concentration during the course of administration is high.

As noted, determination of 5-FU AUC requires complex technology and expertise that may not be readily available in a clinical laboratory setting. In the U.S., Saladax Biomedical offers a commercial immunoassay, My5-FU™, that quantifies plasma 5-FU concentration from a blood sample drawn during continuous infusion at steady state (18-44 hours after the start of infusion) and provides a dose adjustment algorithm to maintain plasma 5-FU AUC between 20 to 30 mg/h/L during the next cycle.(15) The dosing algorithm is based on that developed by Kaldate et al (2012) using OnDose® (now called My5-FU™) in patients with CRC treated with FOLFOX.(16) Technical specifications for OnDose® can still be found on the Myriad Genetics website, which describes the test as a “competitive, homogeneous, 2-reagent nanoparticle agglutination immunoassay.”(17) Although a search of large clinical laboratories did not find tests for 5-FU AUC listed, it is possible that other clinical laboratories measure 5-FU levels by methods other than the specific method used by Saladax Biomedical.

Modifying 5-Fluorouracil Exposure to Improve Outcomes

A 2009 TEC Special Report reviewed the evidence for 5-FU AUC measurement to help modify subsequent 5-FU treatment doses to improve response and reduce toxicity.(18) Early evidence from small, cohort studies showed that in general, the incidence of grade 3 to 4 toxicity (mainly neutropenia, diarrhea, mucositis, hand-foot syndrome) increased with higher systemic exposure to 5-FU. This association has been studied extensively in head and neck cancer and in CRC. In addition, most studies reported statistically significant positive associations between 5-FU exposure and tumor response.

Based on these early results, various strategies have been tried to reduce variability in 5-FU pharmacokinetics, improve treatment efficacy, and decrease toxicity. In particular, individual pharmacokinetic dose adaptation can be accomplished by monitoring plasma 5-FU AUC at steady state during each treatment cycle and adjusting administered 5-FU dose for the next treatment cycle to achieve a target AUC value established as maximally efficacious and minimally toxic. The hypothesis is that individual 5-FU dose modulation to a target AUC value that is just below the threshold for severe toxicity could minimize toxicity while improving response.

The results of single-arm trials of AUC-targeted 5-FU dose adjustment in advanced CRC patients suggested consistently improved tumor response.(19-21) Similar, although less compelling results were seen in single-arm trials of AUC targeted 5-FU dosing in head and neck cancer.(22,23) The best contemporary evidence in support of AUC-targeted dosing consists of 2 randomized controlled trials (RCTs), one enrolling patients with CRC and the other patients with head and neck cancer. No trials of any design were identified for 5-FU dose adjustment in other malignancies.

Gamelin et al (1998)(19) developed a chart for weekly dose adjustment based on the results of an earlier, similar single-arm study,(24) in which dose was increased by prespecified increments and intervals up to a maximum dose or the first signs of toxicity. In an RCT enrolling patients with metastatic colorectal cancer, Gamelin et al (2008)(25) reported significantly improved tumor response (33.6% vs 18.3%, respectively; p<0.001) and a trend toward improved survival (40.5% vs 29.6%, respectively; p=0.08) in the experimental arm using AUC-targeted dosing (by high-performance liquid chromatography) for single-agent 5-FU. However, the authors also reported 18% grade 3 to 4 diarrhea in the fixed-dose control arm, higher than reported in comparable arms of 2 other large chemotherapy trials (5%-7%).(9,10) In the latter 2 trials, delivery over a longer time period for both 5-FU (22 hours vs 8 hours) and LV (2 hours vs bolus), which is characteristic of currently recommended 5-FU treatment regimens, likely minimized toxicity. The administration schedule used in the Gamelin et al (2008) (25) trial is “rarely used in current practice in most countries” as described in an accompanying editorial by Walko and McLeod(26) and is absent from current guidelines. (8) Additional optimization studies would be needed in order to apply 5-FU exposure monitoring and AUC-targeted dose adjustment to a more standard single-agent 5-FU treatment regimen, with validation in a comparative trial versus a fixed-dose regimen.

In 2012, the same group conducted a retrospective analysis to compare their dose adjustment protocol in a FOLFOX regimen for patients with CRC (n=118) with patients treated with FOLFOX administered in standard fashion according to body surface area. (n=39).(27) In the dose-adjusted group, the therapeutic dose at 3 months was 110% of the theoretic dose. Grade 3/4 toxicity was 1.7% for diarrhea, 0.8% for mucositis, 18% for neutropenia, and 12% for thrombocytopenia; corresponding numbers were 12%, 15%, 25% and 10%, respectively, in the standard group. At 3 and 6 months, objective response in the dose-adjusted group was 70% and 56%, respectively; at 3 months, objective response in the standard group was 46%. Median OS and median PFS were 28 and 16 months, respectively, in the dose-adjusted group, and 22 and 10 months, respectively, in the standard group. As the authors note, this proof of principle study needs confirmation in a randomized trial.

Fety et al (1998), in an RCT in patients with locally advanced head and neck cancer, used a different method of dose adjustment and reported overall 5-FU exposures in head and neck cancer patients that were significantly reduced in the dose-adjustment arm compared with the fixed-dose arm.(28) This resulted in reduced toxicity but no improvement in clinical response. The dose adjustment method in this trial may have been too complex, because the 12 patients with protocol violations in this treatment arm (of 61 enrolled) all were related to 5-FU dose adjustment miscalculations. Because patients with protocol violations were removed from analysis, results did not reflect “real-world” results of the dose adjustment method. In addition, the induction therapy regimen used 2 drugs, not the current standard of 3 and, therefore, generalizability of results to current clinical practice is limited.

Test Performance


None of these studies used the OnDose® or My5-FU™ tests.

Analytic Validity (technical performance, ie, reproducibility)

Beumer et al (2009) compared OnDose® (now called My5-FU™) assay results with liquid chromatography-tandem mass spectrometry results; the slope of the correlation was 1.04 (ideal=1.00) and the r value was 0.99 (ideal=1.00).(29)

Büchel et al (2013) compared My5-FU™ assay performance on the Roche Cobas® Integra 800 analyzer with liquid chromatography-tandem mass spectrometry and 3 other analyzers (Olympus AU400®, Roche Cobas® c6000, and Thermo Fisher CDx90®).(30) Serum samples were collected from 32 patients with gastrointestinal cancers who were receiving 5-FU infusion therapy at a single center in Switzerland. My5-FU™ was validated for linearity (ie, correlated linearly within 10% or less of true 5-FU concentrations from 100 mg/mL to 1750 mg/mL), precision, accuracy, recovery, sample carryover, and dilution integrity. Of several plasma compounds tested for potential interference, only lipids were found to exceed manufacturer’s specification. This was attributed to a freezing effect, and the authors recommended storage of plasma samples at 39°F (4°C) until analysis, or frozen for longer periods. In comparison with other tests, My5-FU™ had a 7% proportional (ie, dose-dependent) bias toward higher values compared with chromatography-spectrometry, and a 1.6% or less proportional bias toward higher values compared with the other 3 analyzers.

Clinical Validity (association with outcomes)

Kline et al (2013) assessed OnDose® (now called My5-FU™) in a retrospective study of patients with stage II/III (n=35) or stage IV or recurrent (n=49) CRC who received 5-FU regimens at a single center in the U.S.(31) Patients who required radiation therapy were excluded. Thirty-eight patients chose pharmacokinetic monitoring with OnDose®, and 46 patients were dosed by body surface area (BSA). Median PFS did not differ by dosing strategy in stage IV or recurrent patients (14 months with AUC monitoring vs 10 months BSA dosing; log-rank test, p=0.16), but did differ in stage II/III patients (p=0.04). Thirty-seven percent of stage IV or recurrent patients in both dosing strategy groups experienced grade 3 toxicity. Among stage II/III patients, 32% of AUC-monitored patients and 69% of BSA-dosed patients experienced grade 3 toxicity (Fisher exact test, p=0.04). Onset of adverse events also was delayed in the AUC-monitored group (6 or 7 months vs 2 months in the BSA-dose group; log-rank test, p=0.01).

OnDose® (now called My5-FU™) was clinically validated for patients with CRC in an observational analysis reported as a commentary by Saam et al (2011).(1) Sequential patients (n=357) were treated with constant infusion 5-FU using current adjuvant or metastatic treatment protocols with or without bevacizumab. Samples were drawn at least 2 hours after the start of and before the end of each infusion and sent to Myriad Genetics Laboratories for analysis. Sixty-two patients (17%) were studied longitudinally across 4 sequential sample submissions (ie, four 5-FU treatment infusions), of which 5% were within the target AUC after the first infusion. By the fourth infusion, this number rose to 37% and outliers were reduced. The use of bevacizumab did not affect results. No information on response or toxicity was reported.

Clinical Utility (impact on patient outcomes)

No prospective trials comparing outcomes with AUC-adjusted 5-FU dosing with standard BSA-based dosing were identified.


A 2009 TEC Assessment reviewed the evidence for pharmacogenetic testing to predict 5-FU toxicity.(32) DPYD and TYMS mutation testing did not meet TEC criteria. The author noted that the tests had “poor ability to identify patients likely to experience severe 5-FU toxicity. Although genotyping may identify a small fraction of patients for whom serious toxicity is a moderate to strong risk factor, most patients who develop serious toxicity do not have mutations in DPD or TS genes.”(32)

Analytic Validity

The Myriad Genetics website reports technical specifications for TheraGuide®.(33) DPYD and TYMS mutation testing both are PCR tests. The entire coding sequence of DPYD, comprising 23 coding exons and 690 introns, is analyzed. TYMS is analyzed for the number of base pair tandem repeats in the 5’ untranslated region. Analytic specificity and sensitivity were assessed in 60 samples from unselected individuals. No false positives or false negatives were reported. The estimated incidence of errors that may be due to specimen handling, amplification reactions, or analysis is less than 1%. Testing results are reported as high, moderate, or low risk or “genetic variant of uncertain significance.”

  • High risk: One of 3 mutations (IVS14 +1 G>A [also known as c.1905+1 G>A and DPYD*2A], c.2846A>T [D949V], or c.1679T>G [I560S and DPYD*13]) or other “variants with significant evidence indicating that they adversely affect protein production or function” is present in DPYD, regardless of TYMS genotype.
  • Moderate risk: Two tandem repeats (2R/2R) are present in TYMS, and the DPYD result is low risk.
  • Low risk: Both DPYD and TYMS must have low risk genotypes. For DPYD, this includes variants not predicted to affect protein production or function. For TYMS, this includes 2R/3R and 3R/3R genotypes.
  • Genetic variants of uncertain significance: Missense and/or intronic variants with uncertain clinical relevance are detected.

Specific recommendations for treatment selection and/or 5-FU dose modification or discontinuation based on genetic testing results are not provided. Some authors have developed dosing paradigms based on DPYD results,(4,34) but these have not been prospectively correlated with outcomes such as reduced toxicity.

ARUP Laboratories uses PCR to assess 5 mutations in DPYD (the 3 identified mutations in TheraGuide® plus c. 85T>C and c.-1590T>C) and 2 mutations in TYMS (5’ promoter-enhancer region and 3’ untranslated region.(5) Results are reported as positive (mutation detected) or negative (no mutation detected). On its website, ARUP Laboratories reports analytical sensitivity and specificity of 99 percent; clinical sensitivity and specificity are unknown. The website also notes, “Only targeted mutations in the DPYD and TYMS genes will be detected by this panel. Diagnostic errors can occur due to rare sequence variations [not detected by the test]…Genotyping does not replace the need for therapeutic drug monitoring or clinical observation.”(5)

Clinical Validity: Toxicity

Schwab et al (2008) enrolled 683 patients who were receiving 5-FU for colon or other gastrointestinal cancers, cancers of unknown primary, or breast cancer in a genotype study.(35) Seven different 5-FU regimens (monotherapy or in combination with folate or levamisole [not FDA-approved]) administered by bolus or by infusion were included. Patients were genotyped for the DPYD splice site mutation DPYD*2A (IVS14+1G>A) which leads to a nonfunctional enzyme, and for TYMS tandem repeats. Sensitivity, specificity, and positive and negative predictive value for overall toxicity, diarrhea, mucositis, and leukopenia were calculated (Table 1). Although heterozygosity for DPYD*2A had 99% specificity for serious toxicity, sensitivity ranged from 6% to 13%. Tandem repeats in TYMS were neither sensitive nor specific indicators of serious toxicity. Clinical factors also were examined for association with toxicity. Overall and in the group of 13 patients who were heterozygous for DPYD*2A, women were more likely than men to develop severe toxicity (overall odds ratio [OR], 1.9; 95% confidence interval, 1.26 to 2.87; p=0.002), most commonly mucositis. Bolus administration of 5-FU was a significant, independent predictor of severe toxicity overall. In an accompanying editorial, Ezzedin and Diasio (2008) observed that “genetic tests proposed for the prediction of patients at risk of developing toxicity to FU remain underdeveloped, with a high percentage of false-negative predictions because of the absence of a comprehensive molecular approach that could account for all elements associated with FU toxicity (genetic, epigenetic, and nongenetic), including impairment of cell signaling pathways and/or DNA damage response, which may significantly influence the cellular response to FU.”(36) The editorialists also commented that “the recent use of multiple treatment modalities in cancer patients has further complicated the development of a straightforward predictive test.”(36)

Table 1. Grade 3/4 Adverse Events and DPYD/TYMS Genotype in Schwab et al (2008)(35)







No specific code

ICD-9-CM diagnosis


Investigational for all relevant diagnoses

HCPCS S3722 Dose optimization by area-under-the-curve (AUC) analysis for infusional 5-fluorouracil (5-FU) (new code 2011)
ICD-10-CM (effective 10/1/15)   Investigational for all relevant diagnoses
   C18.0-C18.9 Malignant neoplasm of colon code range
   C19 Malignant neoplasm of rectosigmoid junction
ICD-10-PCS (effective 10/1/15)   Not applicable. ICD-10-PCS codes are only used for inpatient services. There are no ICD procedure codes for laboratory tests.


Area Under the Curve (AUC) Testing, 5-Fluorouracil (5-FU) Dosing 

Policy History

Date Action Reason
2/10/11 Add to Medicine section, Pathology/Laboratory subsection New policy. Considered investigational
03/08/12 Replace policy Policy updated with literature search. References 20-22 added. No change to policy statement.
03/14/13 Replace policy Policy updated with literature search through February 2013. References 18 and 21 added. No change to policy statement.
3/13/14 Replace policy Policy updated with literature review through February 17, 2014; references 2, 4-7, 12, 15-16, and 30-44 added; references 8, 17, and 29 updated. Investigational OnDose® policy statement modified to reflect new test name, My5-FU™. Investigational policy statement for TheraGuide® testing for genetic mutations in DPYD or TYMS added. Title changed to reflect incorporation of new test.



DPYD wt/*2Aa (n=13)

TYMS VNTR 2/3 or 3/3b (n=521)

Overall toxicity
























































NR, not reported; VNTR, variable number of tandem repeats.

a Heterozygous DPYD*2A compared with wt/wt.

b Homozygous (3R/3R) or mixed heterozygous (2R/3R) triple repeats compared with homozygous double repeats (2/2).

In 2013, Loganayagam et al reported similar results from a study of 430 patients treated with 5-FU-based (43%) or capecitabine-based chemotherapy (57%) for colorectal or other gastrointestinal cancers or cancers of unknown primary.(37) Sensitivity and specificity of the 3 identified DPYD mutations of the TheraGuide® test (c.1905+1 G>A, c.2846A>T, and c.1679T>G) for grade 3/4 diarrhea, mucositis, or neutropenia were 1% to 3% and 100%, respectively. Positive and negative predictive values were greater than 99% and 76% to 77%, respectively.

A 2011 review of DPYD mutations associated with 5-FU toxicity noted a lack of consistent correspondence between deleterious variants and DPYD activity across studies.(38) The authors attributed this to variation in allele frequencies across geographic populations studied, nonstandard toxicity assessments, and differences in 5-FU chemotherapy regimens.

Clinical Validity: Efficacy

A 2013 meta-analysis from China included 11 studies that assessed TYMS mutations (5’ tandem repeats and a single nucleotide substitution [G>C] within triplet repeats) and survival outcomes.(39) Patients had gastric or colorectal cancer and received 5-FU with or without leucovorin with or without levamisole. Three studies (total N=311) were eligible for pooled analysis of OS. Statistical heterogeneity was not assessed. Patients who were homozygous for triplet repeats (3R/3R) had improved OS compared with patients who were homozygous for doublet repeats (2R/2R) or compound heterozygous (2R/3R), contrary to expectation.

Clinical Utility (impact on patient outcomes)

Magnani et al (2013) reported a study of 180 cancer patients receiving fluoropyrimidines (5-FU or capecitabine) who underwent DPYD analysis for the 1905+1 G>A mutation by high-pressure liquid chromatography. (40) Four patients were heterozygous carriers. Of these, 3 patients received dose reduction of 50% to 60% but still experienced severe toxicities requiring hospitalization. One patient did not receive chemotherapy based on DPYD genotype and the presence of other mutations found in mismatch repair genes.

No prospective trials comparing outcomes with or without pretreatment DPYD and/or TYMS testing were identified. Online site currently lists an active comparative trial of DPYD testing to predict toxicity to fluoropyrimidines (NCT01547923). However, the estimated completion date was December 2012.


Prior evidence supports the wide variability of 5-fluorouracil (5-FU) plasma levels when patients are placed on a fixed-dose regimen; high exposure is associated with toxicity, but higher exposure up to the limits of toxicity is also associated with better tumor response to treatment. Area under the curve (AUC) laboratory testing methods to better measure 5-FU exposure during treatment of cancer and validated algorithms to modify subsequent dosing may improve response and reduce toxicity. However, currently available evidence is limited and insufficient to draw conclusions about the impact of 5-FU exposure measurement and AUC-targeted dose adjustment on outcomes of patients administered contemporary chemotherapy regimens for colorectal or head and neck cancer. Given the lack of relevant studies, a similar conclusion is reached for use of 5-FU in other cancers.

Impaired function of enzymes in 5-FU metabolic pathways may contribute to toxicity and/or reduced efficacy. However, current evidence for pretreatment testing for genetic mutations in dihydropyrimidine dehydrogenase (DPYD) and/or thymidylate synthase (TYMS) comprises associational studies only. Impacts on treatment selection and 5-FU dosing have not been demonstrated. Evidence for improved outcomes in patients eligible for 5-FU chemotherapy is lacking.

Ongoing Clinical Trials

Three clinical studies of AUC-guided dosing of 5-FU were identified at

  • NCT00943137 (Singapore) will determine the proportion of Asian patients achieving a target AUC using a pharmacokinetically guided 5-fluorouracil dose; the trial also will determine the safety and tolerability of dose adjusted 5-FU.
  • NCT02055560, sponsored by Saladax Biomedical, is a retrospective study of patients with CRC who were treated with a 5-FU chemotherapy regimen. AUC-guided dosing will be compared with BSA-based dosing. Outcomes include tumor response and survival. Expected completion is October 2014.
  • NCT01641458 will administer 5-FU or capecitabine (5-FU prodrug) at 50% reduced dose to patients with CRC and DPYD risk alleles, and employ AUC-guided dose adjustments. The primary outcome is toxicity. Tumor response and survival also will be assessed.

Three studies of DPYD and/or TYMS testing before use of fluoropyrimidines were identified:

  • NCT00131599 (Canada) will assess TYMS polymorphisms in 104 patients with stage III colon cancer to determine who may be at risk for increased adverse effects. Expected completion was December 2013.
  • NCT00515216 (U.S.) will assess TYMS polymorphisms in 75 patients with gastric or gastroesophageal junction adenocarcinoma to determine who may be at risk for resistance to 5-FU. Expected completion was August 2013.
  • NCT01547923 (France) will compare toxicity and mortality in 2296 patients with colorectal cancer who do and who do not undergo DPYD mutation screening before 5-FU treatment. Estimated completion was December 2012. No publication is cited.

Practice Guidelines and Position Statements

National Comprehensive Cancer Network Guidelines

Although current NCCN guidelines acknowledge that the “selection, dosing, and administration of anticancer agents and the management of associated toxicities are complex,”(41,42) they do not recommend AUC-guided 5-FU dosing or genetic testing for DPYD and/or TYMS mutations in patients with colon,(8) rectal,(43) breast,(41) gastric,(42) or pancreatic cancer.(44)

Clinical Pharmacogenetics Implementation Consortium

The CPIC was formed in 2009 as a shared project between PharmGKB, an internet research tool developed by Stanford University, and the Pharmacogenomics Research Network of the National Institutes of Health. In 2013, CPIC published an evidence-based guideline for DPYD genotype and fluoropyrimidine dosing.(4) The guideline does not address the issue of testing.

Medicare National Coverage

There is no national coverage determination (NCD). In the absence of an NCD, coverage decisions are left to the discretion of local Medicare carriers.


  1. Saam J, Critchfield GC, Hamilton SA et al. Body surface area-based dosing of 5-fluoruracil results in extensive interindividual variability in 5-fluorouracil exposure in colorectal cancer patients on FOLFOX regimens. Clin Colorectal Cancer 2011; 10(3):203-6.
  2. PRNewswire. Saladax Biomedical Laboratories to Offer the Full Portfolio of MyCare™ Therapeutic Dose Management Assays in the United States. February 11, 2013. Available online at: Last accessed February, 2014.
  3. Grem JL. 5-Fluorouracil and its biomodulation in the management of colorectal cancer. In: Saltz LB, ed. Colorectal Cancer: Multimodality Management. Totowa, NJ: Humana Press; 2002.
  4. Caudle KE, Thorn CF, Klein TE et al. Clinical Pharmacogenetics Implementation Consortium guidelines for dihydropyrimidine dehydrogenase genotype and fluoropyrimidine dosing. Clin Pharmacol Therap 2013; 94(6):640-5.
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