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DNA from breast cancer tumors could lead to genotype misclassification
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Loss of heterozygosity at the CYP2D6 locus frequently occurs in breast cancer, leading to the potential misclassification of germline CYP2D6 genotypes, according to study results.
“Tumor DNA should not be used to determine germline CYP2D6 genotype without sensitive techniques to detect low frequency alleles and quality control procedures appropriate for somatic DNA,” Matthew P. Goetz, MD, professor of oncology at Mayo Clinic in Rochester, Minn., and colleagues concluded.
The impact of CYP2D6 genotype on patients’ response to tamoxifen has been the subject of debate. Goetz and colleagues assessed whether loss of heterozygosity at the CYP2D6 locus affected genotyping error when tumor tissue is used as a DNA source.
Researchers obtained genomic tumor data from The Cancer Genome Atlas and Foundation Medicine. They used the data, from both the adjuvant and metastatic settings, to evaluate the impact of CYP2D6 copy number alterations and loss of heterozygosity on Hardy Weinberg equilibrium.
They also investigated CYP2D6*4 genotype from formalin-fixed paraffin-embedded tumor blocks containing non-malignant tissue and germline samples. Those samples were obtained from patients in the North Central Cancer Treatment Group tamoxifen trial.
In 627 samples obtained from The Cancer Genome Atlas, the loss of heterozygosity in CYP2D6 was similar in ER-positive (41.2%) and ER-negative (35.2%) tumors, but it was considerably lower in HER-2–positive tumors (15.1%; P<.001).
In 290 ER-positive samples obtained from Foundation Medicine, the loss of heterozygosity rate (40.8%) was similar to that observed from The Cancer Genome Atlas samples.
In 190 samples from the North Central Cancer Treatment Group, the agreement between CYP2D6 genotypes derived from formalin-fixed paraffin-embedded tumors and tumors of the same type containing nonmalignant tissue was moderate, with a weighted Cohen’s Kappa coefficient of 0.74 (95% CI, 0.63-0.84).
When researchers compared CYP2D6 enzymes derived from germline samples to the formalin-fixed, paraffin-embedded tumor DNA, the CYP2D6*4 genotype was discordant in six of 31 cases (19.4%). By contrast, researchers observed no disagreement when they compared CYP2D6 genotypes derived from germline samples from the tumors that had nonmalignant tissue.
“We have provided definitive data from independent data sets that over 40% of primary and metastatic breast tumors exhibit tumor loss of heterozygosity at the CYP2D6 locus and that the use of standard polymerase chain reaction genotyping techniques applied to purified tumor DNA to detect germline CYP2D6 variation results in genotyping error because of an excess number of homozygotes and departure from Hardy-Weinberg equilibrium,” Goetz and colleagues concluded.
“Based on these results, we recommend that CYP2D6 genotyping be repeated in those studies in which the use of tumor DNA to derive germline CYP2D6 genotype resulted in substantial departure from Hardy-Weinberg equilibrium,” they wrote. “Furthermore, recommendations and/or guidelines for the use of CYP2D6 genotyping should not be derived from studies with evidence for genotyping error.”
Disclosure: The researchers report no relevant financial disclosures
Perspective
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Antoinette R. Tan, MD, MHSc, FACP
Tamoxifen is a major endocrine treatment for hormone receptor-positive breast cancer. Cytochrome P450 CYP2D6 is the primary enzyme responsible for converting tamoxifen to the active metabolite endoxifen. The rationale for CYP2D6 testing is based on the hypothesis that tamoxifen may have less benefit in CYP2D6 poor metabolizers who have low endoxifen concentrations than patients who are extensive metabolizers.Retrospective studies evaluating the impact of CYP2D6 genotype on clinical outcomes of patients with breast cancer treated with adjuvant tamoxifen have reported mixed results. Recently, retrospective pharmacogenetic analyses from several major adjuvant trials with prospectively collected DNA — which include BIG 1-98, ATAC and TEAM — demonstrated no significant association between CYP2D6 genotype and breast cancer recurrence, and utilized DNA from tumor sections for genotyping. The clinical utility of CYP2D6 testing has now come into question.
Subsequently, Goetz and colleagues analyzed genomic tumor data from The Cancer Genome Atlas and Foundation Medicine to conclude that CYP2D6 genotyping should not be done on tumor-derived DNA, as it is not representative of the germline genome due to potential loss of heterozygosity. Additionally, researchers found 100% concordance in CYP2D6 genotypes between formalin-fixed paraffin-embedded nonmalignant tissue and buccal cell (germline) samples from patients on the North Central Cancer Treatment Group trial.
Although these study results are scientifically interesting and add to the literature, they do not impact our daily clinical practice. ASCO and the National Comprehensive Cancer Network do not include CYP2D6 testing in their guidelines for use of tamoxifen. Prospective data are lacking to show that poor metabolizers experience higher recurrence rates.
Two ongoing prospective clinical trials, ECOG E3108 and the European CYPTAM-BRUT2, are evaluating how CYP2D6 metabolizer status influences the efficacy of tamoxifen in a metastatic breast cancer population. Ideally, the most definitive way to answer this question is a prospective study of adjuvant tamoxifen comparing clinical outcome in patients with breast cancer with and without CYP2D6 variants. Moving forward, it is important to better understand how genotype-guided dosing of tamoxifen can improve outcomes for hormone receptor-positive breast cancer to more clearly define the role of CYP2D6 gentoyping for clinicians.
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