Multiplex Ligation-Dependent Probe Amplification (MLPA) Compared to Immunohistochemistry (IHC) and Fluorescence In Situ Hybridization (FISH) for Assessing HER2 Amplification in Invasive Breast Carcinoma
Leela Elavathil, Asmaa Manan, Juan Moreno, Ronald Carter, Lisle Connie, Suvi Savola, Alice Lytwyn. Juravinski Hospital, Hamilton, Canada; McMaster University, Hamilton, Canada; Bay Area Genetics Laboratory, Hamilton, Canada; MRC Holland, Amsterdam, Netherlands
Background: HER2 amplification status determines optimal treatment regimens for patients with invasive breast carcinoma. FISH is currently the definitive test for HER2 status, however it is costly, and in many centers is performed only if IHC testing shows indeterminate results. MLPA is a PCR-based technique which uses 4 probes for HER2 gene and can detect sequence dosage differences in a semi-quantitative manner. MLPA costs less than FISH. We compared MLPA testing to FISH and IHC in invasive breast cancer and report the concordance among these tests.
Design: Thirty (30) breast carcinoma from excisional biopsy specimens with known HER2 status by IHC (A0485) were studied. There were 13 negative (score 0 or 1), 10 indeterminate (score 2+) and 7 positive cases (score 3+ in >30% of cells). HER2 gene amplification status was determined by FISH (Vysis Pathvision) and by MLPA (MRC-Holland). FISH scores were defined as: negative (<1.8), indeterminate (1.8-2.2) or positive (>2.2). MLPA ratio of 0.7-1.3 was considered negative, while a ratio >1.3 was positive for increased copy number. A ratio from 1.3 to 2 indicates low positive, while >2 indicates high positive results. There is no indeterminate scoring category in MLPA. Readers of one test were masked to the results of the other two tests, for example, those scoring MLPA did not know the results IHC or FISH.
Results: MLPA and FISH were concordant in 28 (93%) cases. Seven cases were positive for HER2 amplification by all 3 methods. Two cases scored as negative by both IHC and FISH were scored as low positive (1.44 and 1.35) by MLPA.
|Negative (n)||Positive (n)||Negative (n)||Positive (n)|