The relationship between quantitative human epidermal growth factor receptor 2 gene expression by the 21-gene reverse transcriptase polymerase chain reaction assay and adjuvant trastuzumab benefit in Alliance N9831

Abstract

Introduction: The N9831 trial demonstrated the efficacy of adjuvant trastuzumab for patients with human epidermal growth factor receptor 2 (HER2) locally positive tumors by protein or gene analysis. We used the 21-gene assay to examine the association of quantitative HER2 messenger RNA (mRNA) gene expression and benefit from trastuzumab.

Methods: N9831 tested the addition of trastuzumab to chemotherapy in stage I-III HER2-positive breast cancer. For two of the arms of the trial, doxorubicin and cyclophosphamide followed by paclitaxel (AC-T) and doxorubicin and cyclophosphamide followed by paclitaxel and trastuzumab concurrent chemotherapy-trastuzumab (AC-TH), recurrence score (RS) and HER2 mRNA expression were determined by the 21-gene assay (Oncotype DX®) (negative <10.7, equivocal 10.7 to <11.5, and positive ≥11.5 log2 expression units). Cox regression was used to assess the association of HER2 expression with trastuzumab benefit in preventing distant recurrence.

Results: Median follow-up was 7.4 years. Of 1,940 total patients, 901 had consent and sufficient tissue. HER2 by reverse transcriptase polymerase chain reaction (RT-PCR) was negative in 130 (14 %), equivocal in 85 (9 %), and positive in 686 (76 %) patients. Concordance between HER2 assessments was 95 % for RT-PCR versus central immunohistochemistry (IHC) (>10 % positive cells = positive), 91 % for RT-PCR versus central fluorescence in situ hybridization (FISH) (≥2.0 = positive) and 94 % for central IHC versus central FISH. In the primary analysis, the association of HER2 expression by 21-gene assay with trastuzumab benefit was marginally nonsignificant (nonlinear p = 0.057). In hormone receptor-positive patients (local IHC) the association was significant (p = 0.002). The association was nonlinear with the greatest estimated benefit at lower and higher HER2 expression levels.

Conclusions: Concordance among HER2 assessments by central IHC, FISH, and RT-PCR were similar and high. Association of HER2 mRNA expression with trastuzumab benefit as measured by time to distant recurrence was nonsignificant. A consistent benefit of trastuzumab irrespective of mHER2 levels was observed in patients with either IHC-positive or FISH-positive tumors. Trend for benefit was observed also for the small groups of patients with negative results by any or all of the central assays.

Trial registration: Clinicaltrials.gov NCT00005970 . Registered 5 July 2000.

Fig. 1

CONSORT diagram. *Reasons for clinical ineligibility: failed central and reference laboratory review for HER2 positivity (193), patient cancelled (22), patient lost to follow-up (96), and other reasons for clinical ineligibility (42), where the numbers are combined for Arms A and C. Note that Arm A includes 148 otherwise eligible patients who were enrolled during the Arm C closure

Fig. 2

Distribution of HER2 by RT-PCR according to central HER2 by IHC. HER2 human epidermal growth factor receptor 2, IHC immunohistochemistry, RT-PCR reverse transcriptase polymerase chain reaction

Fig. 3

Distribution of HER2 by RT-PCR according to central FISH ratio. FISH fluorescence in situ hybridization, HER2 human epidermal growth factor receptor 2, RT-PCR reverse transcriptase polymerase chain reaction

Fig. 4

Hazard ratio for trastuzumab benefit as a continuous function of HER2 expression by RT-PCR. Estimate and 95 % confidence limits obtained from a Cox PH model for DRFI with a main effect for treatment arm (C versus A), a natural cubic spline for the main effect of HER2 by RT-PCR, a natural cubic spline for the interaction of HER2 by RT-PCR with treatment arm, and three indicator variables to adjust for nodal status (0, 1–3, 4–9 and 10+ positive nodes). Solid line = estimate of hazard ratio; dashed lines = lower and upper 95 % confidence limits. DRFI distant recurrence-free interval, HER2 human epidermal growth factor receptor 2, RT-PCR reverse transcriptase polymerase chain reaction

Fig. 5

Trastuzumab benefit by HER2 status by RT-PCR, FISH and IHC. FISH fluorescence in situ hybridization, HER2 human epidermal growth factor receptor 2, IHC immunohistochemistry, RT-PCR reverse transcriptase polymerase chain reaction