Validation of quantitative PCR-based assays for detection of gene copy number aberrations in formalin-fixed, paraffin embedded solid tumor samples.

Gene copy number changes are important somatic alterations in cancers. A number of high throughput methods, such as next generation sequencing, are capable of detecting copy number aberrations, but their use can be challenging and cost prohibitive for screening a small number of markers. Furthermore, detection of CNAs by high throughput platforms needs confirmation by an orthogonal technique, especially in cases with low level CNAs. Here, we have validated TaqMan based quantitative PCR (qPCR) assays to detect CNAs in genes of high clinical importance in formalin-fixed, paraffin-embedded (FFPE) samples. A cohort of 22 tumors of various types that harbor 67 CNAs in 13 genes was assessed. The abnormalities in these tumors were detected by using a NGS-based 50 gene hotspot panel on Ion Torrent PGM and molecular inversion probe (MIP) array. The CNAs included ERBB2 (n = 6), PDGFRA (n = 6), KIT (n = 7), NRAS (n = 3), PIK3CA (n = 6), MYC (n = 7), MET (n = 4), FLT3 (n = 6), FGFR3 (n = 3), FGFR2 (n = 3), EGFR (n = 7), KRAS (n = 6) and FGFR1 (n = 5). Different amounts of input DNA were tested and 5 ng FFPE DNA was found to be adequate without limiting detection sensitivity. All 22 (100%) positive tumor samples revealed by MIP array were confirmed by real time qPCR and 17 of 22 (77.2%) samples tested by NGS were confirmed. The limit of detection of the qPCR assay was determined by serial dilution of SKBR3 cell line DNA (with amplified ERBB2) and showed an ability to detect 3 copies consistently up to 0.75% dilution. The ability to use low input of FFPE DNA, high sensitivity, and short turnaround time makes qPCR a valuable and economically viable platform for detecting single gene CNAs as well as for confirmation of CNAs detected by high throughput screening assays.

Genome-wide copy number aberrations and HER2 and FGFR1 alterations in primary breast cancer by molecular inversion probe microarray.

Breast cancer remains the second leading cause of cancer-related death in women despite stratification based on standard hormonal receptor (HR) and HER2 testing. Additional prognostic markers are needed to improve breast cancer treatment. Chromothripsis, a catastrophic genome rearrangement, has been described recently in various cancer genomes and affects cancer progression and prognosis. However, little is known about chromothripsis in breast cancer. To identify novel prognostic biomarkers in breast cancer, we used molecular inversion probe (MIP) microarray to explore genome-wide copy number aberrations (CNA) and breast cancer-related gene alterations in DNA extracted from formalin-fixed paraffin-embedded tissue. We examined 42 primary breast cancers with known HR and HER2 status assessed via immunohistochemistry and FISH and analyzed MIP microarray results for correlation with standard tests and survival outcomes. Global genome-wide CNA ranged from 0.2% to 65.7%. Chromothripsis-like patterns were observed in 23/38 (61%) cases and were more prevalent in cases with ≥10% CNA (20/26, 77%) than in cases with <10% CNA (3/12, 25%; p<0.01). Most frequently involved chromosomal segment was 17q12-q21, the HER2 locus. Chromothripsis-like patterns involving 17q12 were observed in 8/19 (42%) of HER2-amplified tumors but not in any of the tumors without HER2 amplification (0/19; p<0.01). HER2 amplification detected by MIP microarray was 95% concordant with conventional testing (39/41). Interestingly, 21% of patients (9/42) had fibroblast growth factor receptor 1 (FGFR1)amplification and had a 460% higher risk for mortality than those without FGFR1 amplification (p<0.01). In summary, MIP microarray provided a robust assessment of genomic CNA of breast cancer.

Comprehensive Screening of Gene Copy Number Aberrations in Formalin-Fixed, Paraffin-Embedded Solid Tumors Using Molecular Inversion Probe-Based Single-Nucleotide Polymorphism Array.

Gene copy number aberrations (CNAs) represent a major class of cancer-related genomic alterations that drive solid tumors. Comprehensive and sensitive detection of CNAs is challenging because of often low quality and quantity of DNA isolated from the formalin-fixed, paraffin-embedded (FFPE) solid tumor samples. Here, in a clinical molecular diagnostic laboratory, we tested the utility and validated a molecular inversion probe-based (MIP) array to routinely screen for CNAs in solid tumors. Using low-input FFPE DNA, the array detects genome-wide CNAs with a special focus on 900 cancer-related genes. A cohort of 76 solid tumors of various types and tumor cellularity (20% to 100%), and four cancer cell lines were used. These harbored CNAs in clinically important genes (ERBB2, EGFR, FGFR1, KRAS, MYC) as detected by orthogonal techniques like next-generation sequencing or fluorescence in situ hybridization. Results of the MIP array were concordant with results from orthogonal techniques, and also provided additional information regarding the allelic nature of the CNAs. Limit-of-detection and assay reproducibility studies showed a high degree of sensitivity and reproducibility of detection, respectively. FFPE compatibility, ability to detect CNAs with high sensitivity, accuracy, and provide valuable information such as loss of heterozygosity along with relatively short turnaround times makes the MIP array a desirable clinical platform for routine screening of solid tumors in a clinical laboratory.

Identification of clinically important chromosomal aberrations in acute myeloid leukemia by array-based comparative genomic hybridization.

Array-based comparative genomic hybridization (aCGH) chromosomal analysis facilitates rapid detection of cytogenetic abnormalities previously undetectable by conventional cytogenetics. In this study, we analyzed 48 uniformly treated patients with acute myeloid leukemia (AML) by 44K aCGH and correlated the findings with clinical outcome. aCGH identified previously undetected aberrations, as small as 5 kb, of currently unknown significance. The 36.7 Mb minimally deleted region on chromosome 5 lies between 5q14.3 and 5q33.3 and contains 634 genes and 15 microRNAs, whereas loss of chromosome 17 spans 3194 kb and involves 342 genes and 12 microRNAs. Loss of a 155 kb region on 5q33.3 (p < 0.05) was associated with achievement of complete remission (CR). In contrast, loss of 17p11.2-q11.1 was associated with a lower CR rate and poorer overall survival (Kaplan-Meier analysis, p < 0.0096). aCGH detected loss of 17p in 12/48 patients as compared to 9/48 by conventional karyotyping. In conclusion, aCGH analysis adds to the prognostic stratification of patients with AML.

Cytotoxic effects of ultra-diluted remedies on breast cancer cells.

The use of ultra-diluted natural products in the management of disease and treatment of cancer has generated a lot of interest and controversy. We conducted an in vitro study to determine if products prescribed by a clinic in India have any effect on breast cancer cell lines. We studied four ultra-diluted remedies (Carcinosin, Phytolacca, Conium and Thuja) against two human breast adenocarcinoma cell lines (MCF-7 and MDA-MB-231) and a cell line derived from immortalized normal human mammary epithelial cells (HMLE). The remedies exerted preferential cytotoxic effects against the two breast cancer cell lines, causing cell cycle delay/arrest and apoptosis. These effects were accompanied by altered expression of the cell cycle regulatory proteins, including downregulation of phosphorylated Rb and upregulation of the CDK inhibitor p27, which were likely responsible for the cell cycle delay/arrest as well as induction of the apoptotic cascade that manifested in the activation of caspase 7 and cleavage of PARP in the treated cells. The findings demonstrate biological activity of these natural products when presented at ultra-diluted doses. Further in-depth studies with additional cell lines and animal models are warranted to explore the clinical applicability of these agents.