Quantitative Assessment of Her-2 mRNA by PCR in Breast Carcinomas Failing To Hybridize with HER-2 Fish Probes
KJ Bloom, P Choppa, TA Bloom, A Kyshtoobayeva, M Hibbard. Clarient, Aliso Viejo, CA
Background: The determination of HER-2 status is essential to aid the oncologist in the selection of an appropriate chemotherapeutic regimen. Although the failure rate of FISH is relatively low, 2-4% in most reported series, failure to hybridize still occurs several times per day in a high volume laboratory. In addition, specimens exposed to acids or bases or inappropriate fixatives also render them unable to hybridize. We hypothesized that the quantitative assessment of HER-2 mRNA in this setting may be possible. After establishing that HER-2 mRNA levels correlate tightly with HER-2 protein expression and gene copy number, we assessed 198 consecutive breast carcinomas that failed to hybridize with the PathVysion FISH assay by quantitative PCR.
Design: One hundred ninety eight consecutive breast carcinomas which failed to hybridize with the PathVysion HER-2 FISH assay (Abbott) were submitted for PCR testing. Tissue sections were cut at 7 microns and mounted on uncharged slides. The samples were stained with H&E using a Leica Autostainer (Meyer Instruments, Houston, TX). Cells of interest were identified and removed from the sections using a PALM laser capture microdissection system (Zeiss, Jena, Germany). RNA was isolated from the captured cells using RNeasy mini kit (Qiagen, Valencia, CA). Gene expression analysis was performed using one step real-time RT-PCR chemistry on an Applied Biosystems 7900HT (ABI, Foster City, CA). Standard curves were generated from universal RNA to determine the relative expression of HER-2 and GUSB. The HER-2 quantitative values are expressed as a ratio to GUSB.
Results: One hundred fifty four of the 198 tumors analyzed (78%) yielded a quantitative PCR result. One hundred twenty three (80%) revealed low expression of HER-2 mRNA (less than 30% relative to the reference gene), 10 (6.5%) revealed borderline expression of HER-2 mRNA (30-40% relative to the reference gene) and 21 (13.5%) revealed high expression of HER-2 mRNA ranging from (42% to 491% relative to the reference gene).
Conclusions: Assessment of HER-2 mRNA has been shown to tightly correlate with HER-2 protein expression and HER-2 gene copy number. The expression of HER-2 mRNA could be reliably determined in 78% of samples which failed to hybridize with the PathVysion HER-2 FISH assay with approximately 80% of samples lacking significant over-expression . HER-2 mRNA determination may be a viable alternative when HER-2 FISH probes fail to hybridize.
Tuesday, March 10, 2009 9:30 AM
Poster Session III # 60, Tuesday Morning