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A subset of different variant types detected by this assay in 46 clinical samples have been confirmed using range of orthogonal methods like qPCR, FISH, Agena's MassArray, and pyrosequencing. Specificity was established across 14, characterized loci.
For CNV and SV, a total of 10 loci were used for sensitivity and specificity calculations as very few control samples cell lines with these rare events exist. A wide variety of validation samples including commercially available reference controls, pools of cell lines, clinical samples, and unaffected tissue samples were used for the analytical validation.
Obtaining fresh tissue samples is not practical in clinical settings. Improper fixation causes severe damage to the nucleic acids in these samples and results in DNA fragments of shorter length.
The variant data are then interpreted using indigenously developed software StrandOmics, v. The interpretation process involves integration of both the NGS variant information as well as evaluation of protein marker expression status to provide a report with recommendations of therapy options that might work effectively or not; in addition to providing prognosis level information that make certain tumors refractory to therapy.
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This comprehensive reporting and its clinical utility have not been previously evaluated extensively in literature. Louis, MO.
Cells were cultured as per the recommended protocol. FFPE normal tissues breast, colon, kidney, liver, lung, pancreas, prostate, bladder, lymph node, spleen were obtained from BioChain Institute, Inc. Newark, NJ.
The details of the samples appear in Table S1 A. For sensitivity analysis, we used 19 different cell lines pooled to create a range of variant frequencies expected in somatic cancer samples. Clinical samples for validation of the SA test Clinical samples were obtained from two sources.
Ethics committee approval was obtained from participating hospitals and laboratories as well as from Strand's internal ethics committee. Selection of genes for the SA test Genes involved in cancer therapy or other categories were selected using literature survey. A complete list of the genes assayed by NGS and their functional relevance with respect to therapy and prognosis is listed in Table S3. Genes were grouped into those associated with 1 approved targeted therapies Percentage of genes in each category is mentioned in parenthesis.
Definition of SOC test A survey of literature enabled the definition of a subpanel of relevant markers specific to each tumor type. Evidence from literature associating these markers with these therapies was ordered into the following categories: Substantial clinical evidence in the same tissue type. Substantial evidence in the same as well as a different tissue type.
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Moderate clinical evidence in the same tissue type supplemented with preclinical evidence in other tissue types. The appropriate testing method was chosen NGS for mutations and IHC for proteins depending on whether the evidence was related to gene mutation or protein expression, respectively.
For tumor suppressor genes all coding exons RefSeq hg19 were included. Exons were extended to include 10 intronic bases on either side to capture splice site mutations. The assay is designed such that it includes probes that cover all the known breakpoints in these three genes from COSMIC irrespective of the fusion partner, novel or known.
A schematic to illustrate this design strategy is shown in Figure S1. For genes with reported copy number variations, multiple regions of variable length interspersed across coding and noncoding regions of the gene were included.
The number of PCR cycles to obtain indexed precapture libraries was fixed at eight cycles.
The loading was optimized to get 10—15 million reads per sample. Different antigen retrieval methods were tested along with different antibody dilutions to arrive at a standardized protocol. Interpretation of the results being positive or negative for clinical samples, for each stain, was determined as described in Table S5.
Sources of the antibodies are listed in Table S6. FFPE tissues were sectioned at 4 microns and mounted on adhesive slides, along with sample being studied. After drying, the slides were deparaffinized in xylene and rehydrated in graded alcohols to distilled water.
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