Prospective Validation of a 21-Gene Expression Assay in Breast Cancer

Princess Igwe, Mercer University College of Pharmacy

In 2014, more than 100,000 women in the United States were diagnosed with estrogen-receptor–positive breast cancer associated with negative axillary lymph nodes.1

It is proposed that tumor biomarker assays, used to identify tumor biomarkers, may be utilized to guide patient management by identifying patients who do not require any, or any additional, treatment, or by identifying patients whose tumors are unlikely to respond to treatment.2

Title: Prospective Validation of a 21-Gene Expression Assay in Breast Cancer3
Design Prospective trial; N = 10,253
Objective To further validate and refine the clinical usefulness of the 21-gene assay in a specified low-risk cohort of women with hormone-receptor-positive, human epidermal growth factor receptor 2 (HER2)-negative, axillary node-negative invasive breast cancer
Study Groups Endocrine therapy alone (breast cancer recurrence score of zero to 10, low risk of recurrence)
  • The study included women 18 to 75 years of age with axillary node–negative invasive breast cancer that was estrogen-receptor–positive or progesterone-receptor–positive (or both) and that did not overexpress HER2.
  • Patients met National Comprehensive Cancer Network guidelines for the recommendation of adjuvant chemotherapy: a primary tumor size of 1.1 to 5.0 cm in the greatest dimension for a tumor of any grade, or a size of 0.6 to 1.0 cm in the greatest dimension for a tumor of intermediate or high histologic grade or nuclear grade (or both).
  • A reverse-transcriptase–polymerase-chain-reaction assay of 21 genes was performed on the paraffin-embedded tumor tissue, and the results were used to calculate a score indicating the risk of breast-cancer recurrence.
  • Patients were assigned to receive endocrine therapy without chemotherapy if they had a recurrence score of zero to 10, indicating a very low risk of recurrence (on a scale of zero to 100, with higher scores indicating a greater risk of recurrence).
Duration April 7, 2006 to October 6, 2010
Primary Outcome Measure
  • A time-to-event analysis of the rate of survival free from invasive cancer
  • An invasive cancer event is defined as the first event of recurrence of ipsilateral breast tumor, local recurrence, regional recurrence, distant recurrence, contralateral second primary invasive cancer, second primary non-breast invasive cancer (excluding non-melanoma skin cancer), or death without evidence of recurrence.
Baseline Characteristics Characteristics Recurrence Score, 0 – 10 (n = 1626) Recurrence Score, 11 – 25 (n = 6897) p-value
Percent of all enrolled patients 15.9 67.3  
Age, mean – yr 57 ± 9 55 ± 9 < 0.001
Tumor size in the greatest dimension, mean – cm 1.74 ± 0.77 1.71 ± 0.79 0.23
Histologic grade of tumor – no./total no. (%) Low: 530/1,578 (34)

Intermediate: 937/1,578 (59)

High: 111/1,578 (7)

Low: 1,941/6,665 (29)

Intermediate: 3,812/6,665 (57)

High: 912/6,665 (14)

< 0.001
Results Event Rates at 5 Years, According to Histologic Grade*
Tumor Grade Invasive Disease–free Survival (95% CI) Freedom from Distant Recurrence (95% CI) Freedom from Any Recurrence (95% CI) Overall Survival (95% CI)
All grades 93.8 (92.4 – 94.9) 99.3 (98.7 – 99.6) 98.7 (97.9 – 99.2) 98.0 (97.1 – 98.6)
Low grade 95.8 (93.5 – 97.3) 99.8 (98.3 – 100) 99.8 (98.3 – 100) 98.7 (97.0 – 99.4)
Intermediate grade 93.6 (91.7 – 95.1) 99.0 (98.0 – 99.5) 98.2 (97.0 – 99.0) 97.9 (96.8 – 98.7)
High grade 91.3 (83.9 – 95.4) 100 (NC – NC) 98.7 (91.1 – 99.8) 97.3 (91.9 – 99.1)
  *The analyses for all tumor grades included data from all 1,626 patients who had a recurrence score of zero to 10, including the 48 patients with the tumor grade not reported. A total of 530 patients were included in the analyses for low-grade tumor, 937 in the analyses for intermediate-grade tumor, and 111 in the analyses for high-grade tumor.; NC = not calculated
Adverse Events Common Adverse Events: Not reported
Serious Adverse Events: Not reported
Percentage that Discontinued due to Adverse Events: Not reported
Study Author Conclusions Among patients with hormone-receptor–positive, HER2-negative, axillary node–negative breast cancer who met established guidelines for the recommendation of adjuvant chemotherapy on the basis of clinicopathologic features, those with tumors that had a favorable gene-expression profile had very low rates of recurrence at five years with endocrine therapy alone.

This prospectively-conducted study supports the use of the 21-gene assay to help avoid the use of chemotherapy in patients who would otherwise be recommended to receive it on the basis of clinicopathologic features. However, it is evident that more breast cancer recurrences occurred with longer follow-up (i.e. at 10 years). Therefore, although benefit was seen at five years, it is uncertain whether recurrences at 10 years may have been avoided by early administration of adjuvant chemotherapy.


  1. Siegel R, Ma J, Zou Z, Jemal A. Cancer statistics, 2014. CA Cancer J Clin 2014;64:9-29. doi: 10.3322/caac.21208.
  2. Hayes DF. Biomarker validation and testing. Mol Oncol 2015;9:960-6. doi:10.1016/j.molonc.2014.10.004.
  3. Sparano JA, Gray RJ, Makower DF, et al. Prospective Validation of a 21-Gene Expression Assay in Breast Cancer. N Engl J Med. 2015;373(21):2005-14. doi: 10.1056/NEJMoa1510764.




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