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 |
REPOSITORIES: biostudies-literature
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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.
| S-EPMC6485473 | biostudies-literature
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.
| S-EPMC3570596 | biostudies-literature
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.
| S-EPMC3873285 | biostudies-literature
Project description:A chromosomal region that includes the gene encoding HER2, a receptor tyrosine kinase (RTK), is amplified in 20% of breast cancers. Although these tumors tend to respond to drugs directed against HER2, they frequently become resistant and resume their malignant progression. Gene amplification in double minutes (DMs), which are extrachromosomal entities whose number can be dynamically regulated, has been suggested to facilitate the acquisition of resistance to therapies targeting RTKs. Here we show that ~30% of HER2-positive tumors show amplification in DMs. However, these tumors respond to trastuzumab in a similar fashion than those with amplification of the HER2 gene within chromosomes. Furthermore, in different models of resistance to anti-HER2 therapies, the number of DMs containing HER2 is maintained, even when the acquisition of resistance is concomitant with loss of HER2 protein expression. Thus, both clinical and preclinical data show that, despite expectations, loss of HER2 protein expression due to loss of DMs containing HER2 is not a likely mechanism of resistance to anti-HER2 therapies.
| S-EPMC4467984 | biostudies-literature
Project description:BackgroundIntra-tumor heterogeneity is a potential cause for failure of targeted therapy in gastric cancer, but the extent of heterogeneity of established (HER2) or potential (EGFR, CCND1) target genes and prognostic gene alterations (MYC) had not been systematically studied.MethodsTo study heterogeneity of these genes in a large patient cohort, a heterogeneity tissue microarray was constructed containing 0.6 mm tissue cores from 9 different areas of the primary gastric cancers of 113 patients and matched lymph node metastases from 61 of these patients. Dual color fluorescence in-situ hybridization was performed to assess amplification of HER2, EGFR, CCND1 and MYC using established thresholds (ratio ≥ 2.0). Her2 immunohistochemistry (IHC) was performed in addition.ResultsAmplification was found in 17.4% of 109 interpretable cases for HER2, 6.4% for EGFR, 17.4% for CCND1, and 24.8% for MYC. HER2 amplification was strongly linked to protein overexpression by IHC in a spot-by-spot analysis (p < 0.0001). Intra-tumor heterogeneity was found in the primary tumors of 9 of 19 (47.3%) cancers with HER2, 8 of 17 (47.0%) cancers with CCND1, 5 of 7 (71.4%) cancers with EGFR, and 23 of 27 (85.2%) cancers with MYC amplification. Amplification heterogeneity was particularly frequent in case of low-level amplification (<10 gene copies). While the amplification status was often different between metastases, unequivocal intra-tumor heterogeneity was not found in individual metastases.ConclusionThe data of our study demonstrate that heterogeneity is common for biomarkers in gastric cancer. Given that both TMA tissue cores and clinical tumor biopsies analyze only a small fraction of the tumor bulk, it can be concluded that such heterogeneity may potentially limit treatment decisions based on the analysis of a single clinical cancer biopsy.
| S-EPMC4324419 | biostudies-literature
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.
| S-EPMC5535706 | biostudies-other
Project description:Gastric carcinomas are among the most common cancers in Germany, with approximately 18,000 new cases per year. About 10 years ago, based on results of the Trastuzumab for gastric cancer (ToGA) trial, the addition of the monoclonal antibody trastuzumab to a platinum-fluoropyrimidine chemotherapy backbone became the standard-of-care 1st-line therapy for human epidermal growth factor receptor 2 (HER2)-positive gastric cancers. Only patients with primary HER2 gene amplification benefit from this therapy. Thus, accurate HER2 gene amplification detection is predictive and critical for therapy selection. As a gold standard the HER2 status is currently determined in tumor tissue specimens using immune histochemistry and fluorescent in situ hybridisation. However, HER2 amplification is detectable in only about 20 % of gastric carcinomas. The recent approval of an antibody-drug conjugate Trastuzumab deruxtecan (T-DXd) and the establishment of a new subgroup of HER2-low tumors due to the bystander effect associated with T-DXd increases the relevance of precise HER2 diagnostics. Aim of this analysis was to determine the HER2 amplification status from circulating DNA fragments in blood using a HER2 Copy Number Variation assay to establish a minimal invasive approach. For the present study, a digital droplet PCR-based method was validated relative to established tissue-based methods. Furthermore and most importantly, the changes of HER2 status during therapy were investigated in seven patients indicating that the changes of HER2 status and number of HER2 copies detected in blood can reflect on therapy efficiency and uncover treatment resistance.
| S-EPMC10665680 | biostudies-literature
Project description:BackgroundPCR is a molecular technique for herpes simplex virus (HSV) detection that can cause life-threatening infections such as encephalitis and keratitis. However, the main issues, false-negative results causing by PCR inhibitors, of this technique that reduce PCR efficiency. To overcome this problem, a competitive internal amplification control (IAC) was constructed for conventional PCR using the PCR-cloning technique.ObjectivesThe purpose of this study is the design of competitive IAC for PCR diagnosis of HSV, which in fact is the main cause of keratitis and viral encephalitis in developed countries.Materials and methodsComposite primers for PCR amplification of Leishmania major kDNA (kinetoplast DNA) were designed and optimized to use as IAC-HSV. IAC-HSV amplified in a non-stringent condition, ligated into pTZ57R plasmid vector, and transformed into Escherichia coli JM207 and then cloned. Resulting IAC was used for 105 CSF and 78 keratitis specimens.ResultsPCR amplicons for HSV and IAC-HSV were 454-bp and 662-bp, respectively. Detection limit of IAC was determined as 1000 plasmids per PCR reaction. IAC sensitivity for HSV detection was determined as 1000 plasmids per PCR reaction. IAC sensitivity for HSV detection was 500 copies/mL of HSV DNA. Among all specimens, 7 inhibited specimens were detected.ConclusionsIndeed, using other DNA as an IAC is expected to detect false-negative results and amplification of the DNA is the key tool to examine the accuracy of amplification and detection steps. This internal amplification control is applicable for early reliable diagnosis of HSV in different loads of virus in different specimens.
| S-EPMC4353061 | biostudies-literature
Project description:Moringa oleifera is a promising plant species for oil and forage, but its genetic improvement is limited. Our current breeding program in this species focuses on exploiting the functional genes associated with important agronomical traits. Here, we screened reliable reference genes for accurately quantifying the expression of target genes using the technique of real-time quantitative polymerase chain reaction (RT-qPCR) in M. oleifera. Eighteen candidate reference genes were selected from a transcriptome database, and their expression stabilities were examined in 90 samples collected from the pods in different developmental stages, various tissues, and the roots and leaves under different conditions (low or high temperature, sodium chloride (NaCl)- or polyethyleneglycol (PEG)- simulated water stress). Analyses with geNorm, NormFinder and BestKeeper algorithms revealed that the reliable reference genes differed across sample designs and that ribosomal protein L1 (RPL1) and acyl carrier protein 2 (ACP2) were the most suitable reference genes in all tested samples. The experiment results demonstrated the significance of using the properly validated reference genes and suggested the use of more than one reference gene to achieve reliable expression profiles. In addition, we applied three isotypes of the superoxide dismutase (SOD) gene that are associated with plant adaptation to abiotic stress to confirm the efficacy of the validated reference genes under NaCl and PEG water stresses. Our results provide a valuable reference for future studies on identifying important functional genes from their transcriptional expressions via RT-qPCR technique in M. oleifera.
| S-EPMC4991797 | biostudies-literature
Project description:Evaluating HER2 gene amplification is an essential component of therapeutic decision-making for advanced or metastatic gastric cancer. A simple method that is applicable to small, formalin-fixed, paraffin-embedded biopsy specimens is desirable as an adjunct to or as a substitute for currently used HER2 immunohistochemistry and in situ hybridization protocols. In this study, we developed a microfluidics-based digital PCR method for determining HER2 and chromosome 17 centromere (CEP17) copy numbers and estimating tumor content ratio (TCR). The HER2/CEP17 ratio is determined by three variables-TCR and absolute copy numbers of HER2 and CEP17-by examining tumor cells; only the ratio of the latter two can be obtained by digital PCR using the whole specimen without purifying tumor cells. TCR was determined by semi-automatic image analysis. We developed a Tumor Content chart, which is a plane of rectangular coordinates consisting of HER2/CEP17 digital PCR data and TCR that delineates amplified, non-amplified, and equivocal areas. By applying this method, 44 clinical gastric cancer biopsy samples were classified as amplified (n = 13), non-amplified (n = 25), or equivocal (n = 6). By comparison, 11 samples were positive, 11 were negative, and 22 were equivocally immunohistochemistry. Thus, our novel method reduced the number of equivocal samples from 22 to 6, thereby obviating the need for confirmation by fluorescence or dual-probe in situ hybridization to < 30% of cases. Tumor content chart-assisted digital PCR analysis is also applicable to multiple sites in surgically resected tissues. These results indicate that this analysis is a useful alternative to HER2 immunohistochemistry in gastric cancers that can serve as a basis for the automated evaluation of HER2 status.
| S-EPMC4847903 | biostudies-literature