Background: Breast cancer is the most common type of malignancy and the leading cause of cancer-related death among women.Among its risk factors, excess body fat is one of the most remarkable.Body Mass Index (BMI) is the most frequently used indice to determine body fat percentage.However, other estimators also exist, such as Clinica Universidad de Navarra e Body Adiposity Estimator (CUN-BAE).Our aim is to compare the attributable fraction of body fat among postmenopausal women with breast cancer by comparing BMI versus CUN-BAE.Methods: We performed a case-control study by using the MCC-Spain database.It is a population multi-case control study that includes high incidence tumours in Spain.We calculated the BMI and CUN-BAE after dividing the total number of cases into four respective categories.Lastly, we compared the population attributable fraction of body fat with both indices.Results: We included a total of 2176 women, 1143 (52.52%) in the control group and 1033 (47,47%) cases of women with breast cancer.The body fat distribution data for the different BMI groups in cases and controls were the following: 36,5% vs 45,6%, 38,8% vs 34,6%, 18,5 vs 14,7%,6 % vs 5%, respectively.The data for CUN-BAE in cases and control were: 14% vs 20%, 31,7% vs 33%, 33,5% vs 29%, 2 % vs 17%, respectively.(Table ).As a result, the population attributable fraction was 28,6% by using the BMI and 46,2% in CUN-BAE. Conclusions:The increase in body fat determined by CUN-BAE, after adjusting it based on the menopausal status and hormonal factors, has shown to directly correlate with an increased risk of breast cancer.We conclude that CUN-BAE is a more precise measure than BMI.Table .

Background: Due to the compelling predictive value of companion diagnostic (CDx) biomarkers tied to targeted and immune-based therapies, well-characterized robust analytic and clinical validation of genomic assays has become mandatory. An NGS-based CGP (comprehensive genomic profiling) platform was developed in compliance with FDA guidelines for CDx indications. Methods: DNA extracted from FFPE tumor tissue underwent whole-genome shotgun library construction and hybridization-based capture, followed by sequencing using Illumina HiSeq 4000. Sequence data were processed using a proprietary analysis pipeline designed to identify sub substitutions, indels, copy number alterations, genomic rearrangements, microsatellite instability (MSI), and tumor mutational burden (TMB) in 324 genes. Results: Clinical validity was demonstrated by establishing statistical non-inferiority between CGP and the respective approved CDx, e.g. cobas EGFR and BRAF mutational testing, ALK rearrangements with FISH and IHC, ERBB2 amplification with FISH, and others. For analytical validity, concordance with an orthogonal NGS platform was 94.6% for substitutions and indels, and within-assay reproducibility had positive percent agreement (PPA) of 99.4%. TMB was analytically validated via concordance with whole-exome sequencing. For the first 616 patients (25% non-small cell lung cancer) assayed in clinical care, 6.8% of cases had TMB exceeding 20 mut/Mb, with 25% of these also harboring microsatellite instability. For 143 NSCLC cases, >50% harbored 10 mut/Mb. Of 354 cases with CDx findings possible, 25.6% had such findings, which were split nearly evenly between indications to benefit from and contraindications to targeted therapies. Conclusions: We developed a CGP assay and demonstrated clinical and analytical validity for CDx biomarkers for targeted therapy, with clinical validation for TMB in progress via correlation with prospective immunotherapy trials. Initial oncologist feedback indicates impact of assay results on course of treatment decisions in patient care. Legal entity responsible for the study: Foundation Medicine, Inc. Funding: Foundation Medicine, Inc. Disclosure: Y. Li: Employee of and stockholder: Foundation Medicine Inc. J.X. Sun: Stockholder: Foundation Medicine Inc. J. Skoletsky, C. Milbury, C. Burns, W-K. Yip, N. Dewal, J. He, J. Tuesdell, J.A. Elvin, G. Otto, D. Lipson, J.S. Ross, V.A. Miller, M. Doherty, C. Vietz: Employee and stockholder: Foundation Medicine Inc. E. Peters, E. Schleifman, J. Noe: Employee and stockholder: Genentech Inc. S. Jenkins: Employee and stockholder: AstraZeneca.

Advanced NSCLC patients may benefit from treatment with ALK inhibitors if they harbor ALK rearrangements such as an EML4-ALK fusion. While the FDA has approved companion diagnostics (CDx) using IHC and FISH-based assays for ALK, molecular diagnostic testing in NSCLC is rapidly evolving towards comprehensive genomic profiling (CGP) to test for a growing number of established predictive biomarkers. Clinical validity of ALK testing using CGP however, has not yet been demonstrated in an FDA approved manner. We present here the first follow-on CDx ALK test using CGP as a part of our universal CDx platform.

The increase in targeted therapies and associated companion diagnostics (CDx) has led to the need for efficient determination of therapeutic eligibility from a single assay. Comprehensive genomic profiling (CGP) provides a solution, but due to the complexity and number of assays available today, standardization of validation has become critically important. We present here the first NGS-based universal CDx platform developed and performed in compliance with FDA 21 CFR part 820. The assay interrogates 324 genes, and is anticipated initially to have eight CDx indications (Table 1). The versatile assay design will facilitate streamlined development of future CDx indications. DNA extracted from FFPE tumor tissue underwent whole-genome shotgun library construction and hybridization-based capture, followed by sequencing using Illumina HiSeq 4000. Sequence data were processed using a proprietary analysis pipeline designed to detect base substitutions, indels, copy number alterations, genomic rearrangements, microsatellite instability (MSI), and tumor mutational burden (TMB). Concordance with FDA-approved CDx are shown in Table 1. Clinical validity was established such that the concordance between CGP and approved CDx were statistically non-inferior to that of two runs of approved CDx. For analytical validity, limit of detection (LoD) was at allele frequency 4% for known substitutions and indels. LoD was 16% tumor content for copy number amplifications, 30% for homozygous deletions, 11% for genomic rearrangements, 12% for MSI, and estimated 20% for TMB. Positive percent agreement (PPA) with an orthogonal NGS platform was 95.8% in substitutions and indels. PPA with FoundationOne was 98.3% across all variant types. Within-assay reproducibility was measured with PPA 99.4%. Rapid expansion of targeted therapies and CDx has necessitated a new approach and urgency to defining performance standards. We developed a universal CDx assay and established a robust approach for demonstrating clinical and analytical validity to support and accelerate the use of CGP for routine clinical care.

Late-stage NSCLC patients may benefit from treatment with EGFR tyrosine kinase inhibitors if they harbor certain activating mutations in EGFR. While the FDA has approved companion diagnostics (CDx) using PCR to detect EGFR mutations, molecular diagnostic testing is evolving towards comprehensive genomic profiling (CGP). However, clinical validity of EGFR testing using CGP has not yet been demonstrated in an FDA-approved manner. We present here the first follow-on CDx EGFR test using CGP as part of our universal CDx platform.

BACKGROUND Increased DNA methylation is an epigenetic alteration that is common in human cancers and is often associated with transcriptional silencing. Aberrantly methylated DNA has also been proposed as a potential tumor marker. However, genes such as vimentin, which are transcriptionally silent in normal epithelium, have not until now been considered as targets for cancer-associated aberrant methylation and for use as cancer markers. METHODS We applied methylation-specific polymerase chain reaction to the vimentin gene, which is transcriptionally silent in normal colonocytes, and compared methylation of vimentin exon 1 in cancer tissues and in fecal DNA from colon cancer patients versus control samples from healthy subjects. RESULTS Vimentin exon-1 sequences were unmethylated in 45 of 46 normal colon tissues. In contrast, vimentin exon-1 sequences were methylated in 83% (38 of 46) and 53% (57 of 107) of tumors from two independently collected groups of colon cancer patients. When evaluated as a marker for colon cancer detection in fecal DNA from another set of colon cancer patients, aberrant vimentin methylation was detected in fecal DNA from 43 of 94 patients, for a sensitivity of 46% (95% confidence interval [CI] = 35% to 56%). The sensitivity for detecting stage I and II cancers was 43% (26 of 60 case patients) (95% CI = 31% to 57%). Only 10% (20 of 198 case patients) of control fecal DNA samples from cancer-free individuals tested positive for vimentin methylation, for a specificity of 90% (95% CI = 85% to 94%). CONCLUSIONS Aberrant methylation of exon-1 sequences within the nontranscribed vimentin gene is a novel molecular biomarker of colon cancer and can be successfully detected in fecal DNA to identify nearly half of individuals with colon cancer.

Colorectal cancer accounts for more than 10% of all cancer deaths but is curable, if detected early. We reported previously on a stool-based screening test in which DNA from stool samples is subjected to genome analysis; sensitivity of the test has been limited in part by inefficiency of retrieving DNA from stool. Our aim was to test the impact of a new purification method that would increase the yield of human DNA from stool. DNA from 86 cancer and 100 non-cancer subjects (diagnosed by colonoscopy) were purified from stool with a new method for DNA recovery based on sequence-specific capture with acrylamide gel immobilized capture probes as well as with a previously developed magnetic bead-capture procedure. The new purification method gives an average 5.4-fold increase in the quantity of human DNA that can routinely be retrieved from fecal samples. The increased recovery of DNA corresponds with an increase in assay sensitivity from 53% (CI: 42 to 64%) to 70% (CI: 59 to 79%); P = 0.0005 (by McNemar's test), with no change in specificity. The newly developed sample preparation method mitigates a major problem in detecting rare cancer-associated genetic changes in heterogeneous clinical samples such as stool.

As more mutations are identified in genes of known sequence, there is a crucial need in the areas of medical genetics and genome analysis for rapid, accurate and cost-effective methods of mutation detection. We have developed a multiplex allele-specific diagnostic assay (MASDA) for analysis of large numbers of samples (> 500) simultaneously for a large number of known mutations (> 100) in a single assay. MASDA utilizes oligonucleotide hybridization to interrogate DNA sequences. Multiplex DNA samples are immobilized on a solid support and a single hybridization is performed with a pool of allele-specific oligonucleotide (ASO) probes. Any probes complementary to specific mutations present in a given sample are in effect affinity purified from the pool by the target DNA. Sequence-specific band patterns (fingerprints), generated by chemical or enzymatic sequencing of the bound ASO(s), easily identify the specific mutation(s). Using this design, in a single diagnostic assay, we tested samples for 66 cystic fibrosis (CF) mutations, 14 beta-thalassemia mutations, two sickle cell anemia (SCA) mutations, three Tay-Sachs mutations, eight Gaucher mutations, four mutations in Canavan disease, four mutations in Fanconi anemia, and five mutations in BRCA1. Each mutation was correctly identified. Finally, in a blinded study of 106 of these mutations in > 500 patients, all mutations were properly identified. There were no false positives or false negatives. The MASDA assay is capable of detecting point mutations as well as small insertion or deletion mutations. This technology is amenable to automation and is suitable for immediate utilization for high-throughput genetic diagnostics in clinical and research laboratories.

Three major methods have been described for the isolation of fetal cells from maternal blood: fluorescence-activated cell sorting (FACS), immunomagnetic beads, and magnetic-activated cell sorting (MACS). To date, no study has directly compared fetal cell recovery using each of these methods. Here we describe our system using a "model' male fetal cell mixed into female peripheral blood mononuclear cells. Fetal cell yields and purities were assayed by a quantitative polymerase chain reaction (qPCR) using chromosomes Y- and 7-specific sequences. Fetal cell recovery was investigated by selection of CD71+ cells or depletion of CD45+ cells. Our data demonstrated variation in fetal cell recovery for all methods tested, although CD71+ selection by FACS gave the best and most consistent results.

The identification of the cystic fibrosis transmembrane conductance regulator (CFTR) gene has led to the identification of more than 225 presumed disease-causing mutations at the locus. The diagnosis of cystic fibrosis or the carrier state by direct DNA analysis is hindered by this large number. A practical assay must be able to detect enough mutations to achieve clinically significant sensitivity. The use of allele-specific oligonucleotide probes is the most promising of the available methods. However, to date this has generally involved tedious probe-by-probe hybridizations, due to variations in the oligonucleotides' denaturation temperatures caused by differences in their G-C base-pair content. We have developed a rapid, cost-effective assay that simultaneously detects 12 CFTR mutations after multiplex polymerase-chain-reaction amplification of genomic DNA. The test may be readily extended to detect additional mutations at minimal increase in the cost per test or the turnaround time. We improve specificity and avoid the need for individual hybridizations by the use of tetramethylammonium chloride to virtually eliminate the effects of G-C differences. Coupled with non-invasive sample-collection methods, this is an immediately practical assay for cystic fibrosis. More generally, it will serve as a model for the development of diagnostic tests in other genetic disorders involving complex mutation analysis.

Traditionally, DNA used for PCR-based diagnostic analysis has originated from white cells fractionated from whole blood. Although this method yields substantial quantities of DNA, there are some drawbacks to the procedure, including the inconvenience of drawing blood, risk of exposure to blood-borne pathogens, liquid sample handling, and the somewhat involved extraction procedure. Alternatively, DNA for genetic diagnosis has been derived from finger stick blood samples, hair roots, cheek scrapings, and urine samples. Oral saline rinses have also been used extensively as a means of collecting buccal epithelial cells as a DNA source. However, this method still requires liquid sample handling. Herein, we present our results involving the rapid extraction of DNA from buccal cells collected on cytology brushes and swabs for use in PCR reactions, specifically the multiplex amplification of 5 exons within the CFTR gene. The quality of DNA isolated from buccal cells, collected in this manner, has been sufficient to reproducibly support multiplex amplification. Cheek cell samples and the DNA prepared from them as described here are highly stable. The success rate of PCR amplification on DNA prepared from buccal cells is 99%. In a blind study comparing the analysis of 12 mutations responsible for cystic fibrosis in multiplex products amplified with DNA from both blood and buccal cell samples from 464 individuals, there was 100% correlation of results for blood and cheek cell DNA, validating the use of DNA extracted from cheek cells collected on cytology brushes for use in genetic testing.