The combinatorial approach of laser-captured microdissection and reverse transcription quantitative polymerase chain reaction accurately determines HER2 status in breast cancer

Abstract

BackgroundHER2 expression in breast cancer correlates with increased metastatic potential, higher tumor recurrence rates and improved response to targeted therapies. Fluorescence in situ hybridization (FISH) and immunohistochemistry (IHC) are two methods commonly used for the analysis of HER2 in the clinic. However, lack of standardization, technical variability in laboratory protocols and subjective interpretation are major problems associated with these testing procedures.MethodsHere we evaluated the applicability of reverse-transcription quantitative polymerase chain reaction (RT-qPCR) for HER2 testing in breast cancer. We tested thirty formaldehyde-fixed and paraffin-embedded tumor samples by RT-qPCR, FISH and IHC and analysed and compared the data from the three methods.ResultsWe found that laser-captured microdissection is essential for the accurate determination of HER2 expression by RT-qPCR. When isolating RNA from total tumor tissue we obtained a significant number of false negative results. However, when using RNA from purified cancer cells the RT-qPCR data were fully consistent with FISH and IHC. In addition we provide evidence that ductal carcinomas might be further classified by the differential expression of HER3 and HER4.ConclusionsLaser-captured microdissection in combination with RT-qPCR is a precise and cost-effective diagnostic approach for HER2 testing in cancer. The PCR assay is simple, accurate and robust and can easily be implemented and standardized in clinical laboratories.

Establishment of Rapid and Accurate Screening System for Molecular Target Therapy of Osteosarcoma

“…Elevated HER2 protein levels are shown to be tightly associated with gene amplification; thus, HER2 protein levels can be semi-quantitatively assessed by immunohistochemistry (IHC), and the rates of concordance between IHC and FISH range from 80% to 90% in this setting. [12][13][14][15][16][17][18] However, these assays have not yet been established to estimate the gene amplification status of these actionable/potential loci in OS.…”

“…Another method to evaluate the gene amplification status is fluorescence in situ hybridization (FISH), for example, HER2 FISH, which is commonly used to evaluate therapeutic applications such as trastuzumab in breast cancer. [12][13][14][15][16] Although its TAT is short, FISH targets only one gene status, and we cannot obtain further information even if the result is negative. Immunohistochemistry is an easy and cost-effective method with a short TAT that can be used to examine protein expression status and to test the application of molecular targeted therapies such as trastuzumab in breast cancer.…”

ErbB Family Signalling: A Paradigm for Oncogene Addiction and Personalized Oncology

“…In the last years, ErbB3 signalling has gained considerable attention in cancer research. ErbB3 predominantly forms a heterodimer with ErbB2 and was found to be critically involved in tumour initiation and progression and is now considered one of the most active signalling dimers of the ErbB family in cancer [ 98 , 99 , 100 ]. Consistent with the findings of other groups, we could recently identify two distinct breast cancer populations that expressed either high (~60% of tumours) or low (~40% of tumours) levels of ErbB3.…”

“…Interestingly, the highest ErbB3 expression was detected in ErbB2-positive specimen. Increased co-expression of ErbB2 and ErbB3 might critically influence oncogenic signalling, oncogene addiction and thereby responsiveness to TKIs [ 99 ]. Somatic mutations in the ErbB3 gene occur in approximately 10% of colon and gastric cancers and increased activity may also be involved in melanoma formation [ 17 , 101 ].…”