A Reliable and Standardizable Differential PCR and qPCR Methodology Assesses HER2 Gene Amplification in Gastric Cancer.
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ABSTRACT:
SUBMITTER: Juarez I
PROVIDER: S-EPMC8230392 | biostudies-literature | 2021 Jun
REPOSITORIES: biostudies-literature
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A Reliable and Standardizable Differential PCR and qPCR Methodology Assesses HER2 Gene Amplification in Gastric Cancer.
Juarez Ignacio I Toro-Fernandez Juan Francisco JF Vaquero-Yuste Christian C Molina-Alejandre Marta M Lasa Inmaculada I Gomez Remedios R Lopez Adela A Martin-Villa Jose Manuel JM Gutierrez Alberto A
Biology 20210610 6
We have applied two PCR techniques, differential PCR (diffPCR) and qPCR for the identification of HER2 gene amplifications in genomic DNA of tumor and distal gastric samples from patients with gastric cancer. The diffPCR technique consists of the simultaneous amplification of the HER2 gene and a housekeeping gene by conventional PCR and the densitometric analysis of the bands obtained. We established a cut-off point based on the mean and standard deviation analyzing the DNA of 30 gastric tissues ...[more]
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Project description:PURPOSE:Digital PCR is a highly accurate method of determining DNA concentration. We adapted digital PCR to determine the presence of oncogenic amplification through noninvasive analysis of circulating free plasma DNA and exemplify this approach by developing a plasma DNA digital PCR assay for HER2 copy number. EXPERIMENTAL DESIGN:The reference gene for copy number assessment was assessed experimentally and bioinformatically. Chromosome 17 pericentromeric probes were shown to be suboptimal, and EFTUD2 at chromosome position 17q21.31 was selected for analysis. Digital PCR assay parameters were determined on plasma samples from a development cohort of 65 patients and assessed in an independent validation cohort of plasma samples from 58 patients with metastatic breast cancer. The sequential probability ratio test was used to assign the plasma DNA digital PCR test as being HER2-positive or -negative in the validation cohort. RESULTS:In the development cohort, the HER2:EFTUD2 plasma DNA copy number ratio had a receiver operator area under the curve (AUC) = 0.92 [95% confidence interval (CI), 0.86-0.99, P = 0.0003]. In the independent validation cohort, 64% (7 of 11) of patients with HER2-amplified cancers were classified as plasma digital PCR HER2-positive and 94% (44 of 47) of patients with HER2-nonamplified cancers were classified as digital PCR HER2-negative, with a positive and negative predictive value of 70% and 92%, respectively. CONCLUSION:Analysis of plasma DNA with digital PCR has the potential to screen for the acquisition of HER2 amplification in metastatic breast cancer. This approach could potentially be adapted to the analysis of any locus amplified in cancer.
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Project description:Data from 8 breast cancer genome-sequencing projects identified 25 patients with HER2 somatic mutations in cancers lacking HER2 gene amplification. To determine the phenotype of these mutations, we functionally characterized 13 HER2 mutations using in vitro kinase assays, protein structure analysis, cell culture, and xenograft experiments. Seven of these mutations are activating mutations, including G309A, D769H, D769Y, V777L, P780ins, V842I, and R896C. HER2 in-frame deletion 755-759, which is homologous to EGF receptor (EGFR) exon 19 in-frame deletions, had a neomorphic phenotype with increased phosphorylation of EGFR or HER3. L755S produced lapatinib resistance, but was not an activating mutation in our experimental systems. All of these mutations were sensitive to the irreversible kinase inhibitor, neratinib. These findings show that HER2 somatic mutation is an alternative mechanism to activate HER2 in breast cancer and they validate HER2 somatic mutations as drug targets for breast cancer treatment.We show that the majority of HER2 somatic mutations in breast cancer patients are activating mutations that likely drive tumorigenesis. Several patients had mutations that are resistant to the reversible HER2 inhibitor lapatinib, but are sensitive to the irreversible HER2 inhibitor, neratinib. Our results suggest that patients with HER2 mutation–positive breast cancers could benefit from existing HER2-targeted drugs.
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Project description:Determination of the presence of HER2 amplification by quantitative PCR has been challenging, in part due to chromosomal instability and identification of a robust a reference region. We assessed the potential of digital PCR for highly accurate assessment of DNA concentration with EFTUD2 as chromosome 17 reference probe. We assessed a HER2:EFTDU2 ratio by digital PCR assay in the microdissected DNA from 18 HER2 amplified and 58 HER2 non-amplified cancers. The HER2:EFTUD2 ratio had high concordance with conventionally defined HER2 status with a sensitivity of 100% (18/18) and a specificity of 98% (57/58). The HER2:EFTUD2 digital PCR assay has potential to accurately assess HER2 amplification status.
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Project description:Background: HER2 serves as an important therapeutic target in gastroesophageal cancer. Differences in HER2 gene signal distribution patterns can be observed at the tissue level, but how it influences the HER2 amplification status has not been studied so far. Here, we investigated the link between HER2 amplification and the different types of gene signal distribution. Methods: Tumor samples from 140 patients with gastroesophageal adenocarcinoma where analyzed using the HER2 IQFISH pharmDx™ assay. Specimens covered non-amplified and amplified cases with a preselected high proportion of HER2 amplified cases. Based on the HER2/CEN-17 ratio, specimens were categorized into amplified or non-amplified. The signal distribution patterns were divided into homogeneous, heterogeneous focal or heterogeneous mosaic. The study was conducted based on anonymized specimens with limited access to clinicopathological data. Results: Among the 140 analyzed specimens 83 had a heterogeneous HER2 signal distribution, with 62 being focal and 21 of the mosaic type. The remaining 57 specimens had a homogeneous signal distribution. HER2 amplification was observed in 63 of the 140 specimens, and nearly all (93.7%) were found among specimens with a heterogeneous focal signal distribution (p<0.0001). The mean HER2/CEN-17 ratio for the focal heterogeneous group was 8.75 (CI95%: 6.87 - 10.63), compared to 1.53 (CI95%: 1.45 - 1.61) and 1.70 (CI95%: 1.22 - 2.18) for the heterogeneous mosaic and homogeneous groups, respectively, (p<0.0001). Conclusions: A clear relationship between HER2 amplification and the focal heterogeneous signal distribution was demonstrated in tumor specimens from patients with gastroesophageal cancer. Furthermore, we raise the hypothesis that the signal distribution patterns observed with FISH might be related to different subpopulations of HER2 positive tumor cells.
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