Current methods for the assessment of ER, PR, Her2, and Ki67 using FFPE tissues are hard to standardize and difficult to perform in Low and Middle Income Countries (LMIC). The GeneXpert® breast cancer stratifier assay (RUO) (BC Strat) is a cartridge-based, RT-qPCR assay of ESR1, PGR, ERBB2, and MKi67 mRNAs using FFPE specimens. The assay is fast (<2 hours, including <10 minutes of hands-on time) and easy to perform.

The aims of this study were: 1) to evaluate the concordance of BC Strat using different IHC antibodies and scoring methods in a preliminary dataset (Part I); and 2) to assess concordance between BC Strat and high quality standard methods in an expanded dataset (Part II).

Methods

Part I: IHC Antibody Variability

To assess BC Strat concordance with various IHC antibodies, 155 invasive ductal carcinoma blocks were sourced from 3 sites. Twenty-four adjacent slide sections from each block were prepared and shipped to different labs for BC Strat analysis(Cepheid) or IHC and FISH testing. Table 1 summarizes the IHC antibodies and scoring methods used in each reference lab.

Table 1:IHC antibodies and scoring methods used in reference labs

  Antibody for IHC lab IHC scoring 
ER SP1 MPLN Automated (Aperio) 
ER 6F11 Path Inc Automated (Aperio) 
ER 6F11 USC Manual 
PR IE2 MPLN Automated (Aperio) 
PR 16 Path Inc Automated (Aperio) 
PR PGR636 USC Manual 
Her2* 4B5 MPLN Automated (Aperio) 
Her2* HercepTest USC Manual 
Ki67 30-9 MPLN Automated (Aperio) 
Ki67 MIB1 Path Inc Automated (Aperio) 
Ki67 MIB1 USC Manual 
  Antibody for IHC lab IHC scoring 
ER SP1 MPLN Automated (Aperio) 
ER 6F11 Path Inc Automated (Aperio) 
ER 6F11 USC Manual 
PR IE2 MPLN Automated (Aperio) 
PR 16 Path Inc Automated (Aperio) 
PR PGR636 USC Manual 
Her2* 4B5 MPLN Automated (Aperio) 
Her2* HercepTest USC Manual 
Ki67 30-9 MPLN Automated (Aperio) 
Ki67 MIB1 Path Inc Automated (Aperio) 
Ki67 MIB1 USC Manual 

*HER2 FISH (all with PathVysion kit) was performed at USC

Part II: Concordance Study

522 invasive ductal carcinoma FFPE samples were sourced from 5 sites. All BC Strat analysis was performed at Cepheid and all IHC and FISH was performed in the Press laboratory at USC. Overall percent agreement (OPA), positive percent agreement (PPA), and negative percent agreement (NPA) between BC Strat and IHC were determined.

Results

Part I: IHC Antibody Variability

Table 2 summarizes the OPA for BC Strat analysis and IHC performed with different IHC antibodies and scoring methods. Slightly better concordance for ER and PR was observed between the BC Strat and the IHC methods performed at USC. Discordant IHC results were also observed among the reference labs' standard methodologies.

Table 2: Overall Percent Agreement between IHC and BC Strat

  Reference Lab OPA with BC Strat 
ER MPLN 92% 
ER Path Inc 96% 
ER USC 98% 
PR MPLN 84% 
PR Path Inc 83% 
PR USC 87% 
Her2* MPLN* 93% 
Her2* USC* 91% 
Ki67 MPLN 75% 
Ki67 Path Inc 67% 
Ki67 USC 81% 
  Reference Lab OPA with BC Strat 
ER MPLN 92% 
ER Path Inc 96% 
ER USC 98% 
PR MPLN 84% 
PR Path Inc 83% 
PR USC 87% 
Her2* MPLN* 93% 
Her2* USC* 91% 
Ki67 MPLN 75% 
Ki67 Path Inc 67% 
Ki67 USC 81% 

*for IHC 2+(equivocal), FISH HER2/CEP17 ratio was examined

Part II: Concordance Study

Of the 522 samples tested, 499 (96%) yielded valid results for both BC Strat and IHC (IHC and FISH for Her2). OPA between BC Strat and IHC was 98% for ESR1, 91% for PGR, 93% for ERBB2 (IHC and FISH, 97% for Her2 IHC excluding IHC2+), and 81% for MKi67.

Conclusion

BC Strat assay measurements for ESR1, PGR, ERBB2 and MKi67 mRNA expression in FFPE breast tumor tissues are highly concordant with IHC and FISH performed by high quality reference labs. Further investigations using clinical outcomes from independent studies including prospective-retrospective clinical trials are in progress.

Citation Format: Wu NC, Wong W, Ho KE, Chu VC, Rizo A, Davonport S, Kelly D, Makar R, Jassem J, Duchnowska R, Biernat W, Radecka B, Fujita T, Klein JL, Stonecypher M, Ohta S, Juhl H, Weidler JM, Bates M, Press MF. High concordance of ER, PR, HER2 and Ki67 by central IHC and FISH with mRNA measurements by GeneXpert® breast cancer stratifier assay [abstract]. In: Proceedings of the 2016 San Antonio Breast Cancer Symposium; 2016 Dec 6-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2017;77(4 Suppl):Abstract nr P1-03-03.