The prospective-retrospective NSABP B-20 trial evaluating the Oncotype DX® 21-gene assay as a predictor of benefit from adjuvant chemotherapy in node-negative, estrogen-receptor (ER)-positive breast cancer demonstrated that a high recurrence score (RS), defined as 31 or higher, was predictive of chemotherapy benefit.1,2 In preparation for the TAILORx trial, the analysis of the B-20 trial was repeated with the cutoffs ultimately employed in TAILORx: <11, 11–25, >25, and demonstrated that the patients with RS > 25 also had a large benefit from the addition of chemotherapy.3 Supportive findings of chemotherapy benefit in patients with RS ≥ 31 were subsequently demonstrated in a similar prospective-retrospective analysis of SWOG-8814, conducted in patients with node-positive, ER-positive breast cancer.4

The 21-gene assay is based on RT-PCR analysis and integration of expression of 16 breast cancer-related genes and 5 reference genes.5 HER2 is one of the genes in the RS algorithm with a positive coefficient and contributes to a higher RS value. As a result, cancers with HER2 overexpression generally have higher RS values. In NSABP B-20, patients were accrued from October 1988 to March 1993, prior to establishment of routine clinical testing for HER2, so a portion of the patients accrued to B-20 were likely to have had HER2-positive disease.6

Formal tests for interaction between quantitative individual HER2 gene expression as well as expression of the HER2 gene group by RT-PCR and benefit from chemotherapy were conducted as part of the original analysis of the 21-gene assay and were negative, so positive HER2 status should not be a prerequisite for benefit from chemotherapy in patients with high RS.5 However, because the 21-gene assay is used clinically as a predictive biomarker of chemotherapy benefit for patients with hormone-receptor positive, HER2-negative disease, questions have persisted regarding the performance of the 21-gene assay in B-20 if patients with HER2-positive tumors were excluded. Insufficient tumor material remains in the blocks used in the prospective-retrospective B-20 study to allow sectioning for routine immunohistochemical (IHC) or in situ hybridization (ISH) testing for HER2 expression without risk of wastage of the remaining material. However, the quantitative HER2 individual gene score component of the RS assay was compared to assessment of HER2 status by FISH in a case–controlled study using specimens from patients identified in the Kaiser Permanente Northern California Cancer Registry. The study demonstrated concordance of 97% (95% CI: 96–99%) by central FISH and the RS assay with a definition of HER2 positive as quantitative RT-PCR ≥ 11.5 units.7 A second study8 using 901 specimens from Alliance N9831 compared HER2 status determined by RT-PCR using the same cutoff with results determined by IHC and FISH. Concordance for HER2 assessment by RT-PCR was 95% vs. IHC and 91 % vs. FISH. Although quantitative RT-PCR has not been validated as a companion diagnostic for identifying individual candidates for HER2-directed therapies, the concordance with HER2 determination by IHC and FISH documented in these two large studies justifies use of the single-gene expression by RT-PCR to identify the patients in B-20 who were likely to have been HER2-positive by routine testing methods.

We report here for the first time results of an exploratory reanalysis of chemotherapy benefit in B-20 when excluding the cohort of patients with quantitative RT-PCR ≥ 11.5 units, in order to demonstrate the performance of the assay in predicting chemotherapy benefit for patients with node-negative, ER-positive, HER2-negative disease. In addition, we assessed the performance of the 21-gene assay in predicting chemotherapy benefit in the B-20, HER2-negative population using the RS cutoffs employed in the TAILORx study.3