PCMP - Personalized Cancer Mutation Panel

Test Details

Targeted Mutation Detection by Next Generation Sequencing in:

  • Breast Cancer
  • Colorectal Cancer
  • Lung Cancer
  • Brain Cancer
  • Ovarian Cancer
  • Renal Cancer
  • Malignant Melanoma

Most cancers result from accumulation of multiple genetic alterations that lead to dysfunction of cancer-related genes. These genetic alterations are important diagnostic, prognostic, and predictive biologic markers. Next generation sequencing technologies permit deep sequencing of hundreds of cancer genes concurrently. This targeted next generation sequencing test utilizes multiplex PCR with ion semiconductor sequencing (AmpliSeq, Ion Torrent/Life Technologies) to sequence a panel of key cancer gene mutations including many clinically actionable mutations. This test can provide important individual information regarding tumor development and progression, and a more reliable prediction of personalized cancer therapies.

This Personalized Cancer Mutation Panel is designed to target 2,800 mutations in the following 50 key cancer genes: ABL1, AKT1, ALK, APC, ATM, BRAF, CDH1, CDKN2A, CSF1R, CTNNB1, EGFR, ERBB2, ERBB4, EZH2, FBXW7, FGFR1, FGFR2, FGFR3, FLT3, GNAQ, GNAS, GNA11, HNF1A, HRAS, IDH1, IDH2, JAK2, JAK3, KDR, KIT, KRAS, MET, MLH1, MPL, NOTCH1, NPM1, NRAS, PDGFRA, PIK3CA, PTEN, PTPN11, RB1, RET, SMAD4, SMARCB1, SMO, SRC, STK11, TP53, and VHL. The full list of mutations can be found at http://path.upmc.edu/divisions/map/. Mutation details can be obtained from the Catalogue of Somatic Mutations in Cancer (COSMIC) database with the corresponding COSMIC ID at http://cancer.sanger.ac.uk/cancergenome/projects/cosmic/. DNA sequences used as references for this panel of genes can be found at http://www.ncbi.nlm.nih.gov/refseq/rsg/. The mutation nomenclature is based on the convention recommended by the Human Genome Variation Society at http://www.hgvs.org/mutnomen/.

This mutation panel is designed to detect targeted mutations only. The 50 genes are not sequenced in their entirety. Mutations outside the targeted regions will not be detected. The limit of detection is 5% at 500X coverage and 10% at 300X coverage. This technology cannot reliably detect mutations at coverage below 300X. Confirmation of mutation is performed by Sanger Sequencing, real-time PCR/FMCA, or SNAPshot assay.

Methodology

For enrichment of tumor cell population, all surgically removed tumor specimens are microdissected from unstained slides under the microscope with H&E guidance. Specimens with a minimum of 30-50% of tumor cells or at least 300 tumor cells in a microdissection target are accepted for analysis.

Genomic DNA was used for multiplex PCR amplification of 207 amplicons, which target 2,800 mutations in 50 key cancer genes, with the Ion AmpliSeq™ Kit. Amplicons were barcoded, purified and ligated with specific adapters. A final check of library preparation was performed using the Agilent 2100 Bioanalyzer or Tapestation. The Ion One Touch and One Touch ES were used to prepare and enrich templates and enable testing via Ion Sphere Particles on a semi conductor chip. Next generation bidirectional sequencing was performed on the Ion Torrent Personal Genome Machine and analyzed with the Torrent Suite Software. Variant annotation and reporting was performed with SeqRepoter software (UPMC).

Specimen Requirements and Shipping Instructions

Paraffin embedded tissue sections

  • Tissue should be fixed in formalin and not exposed to decalcification solution. The paraffin block should contain no less than 3 mm area of tumor.
  • Slides should prepared by histology using a specific protocol for cutting molecular sections to avoid contamination of the tissue sections (available upon request).
  • One H&E and 6 unstained sections are required for most of the tests. Ten unstained sections or more are required for some tests (indicated by * in test menu) or if the tissue is small. Please call the lab if you have questions.
  • Inclusion of normal patient tissue (either adjacent to tumor in the same block or separate block) is optimal for LOH and MSI analyses.
  • Slides should be properly labeled with a block label that matches the surgical pathology specimen number on the surgical pathology report.
  • Slides should be sent ambient temperature in proper storage containers (plastic slide boxes) to protect them during shipment.
  • A surgical pathology and/or cytology report and completed requisition for must accompany all specimens.

Frozen tissue

  • A minimum of 2 x 2 x 2 mm of frozen tissue is required; however, 5 x 5 x 5 mm is optimal.
  • Collection date and time should be stated.
  • Tissue specimen containing at least 50% of tumor cells can be either placed into cryogenic tube and snap frozen in liquid nitrogen, or placed into a tube with preservative solution provided by the Molecular & Genomic Pathology laboratory (request solution from the lab) and frozen at -20° C.
  • Ship overnight on dry ice. A surgical pathology and/or cytology report and completed requisition for must accompany all specimens.

Fresh Fine Needle Aspiration (FNA) samples

  • Fresh specimens should be collected into preservative solution provided by the Molecular & Genomic Pathology laboratory (request solution from the lab). Collection instruction will be provided with the solution.
  • Collection date and time should be stated.
  • Specimen can be refrigerated at 4° C for 12 hours or stored at -20° C prior to shipment.
  • Ship at room temperature when using "next business morning" delivery or with ice packs by overnight delivery. A surgical pathology and/or cytology report and completed requisition for must accompany all specimens.

Fixed Fine Needle Aspiration (FNA) samples

  • One H&E and 4-6 unstained sections from cell block are required. A minimum of 300 tumor cells should be present on a slide. Please call the lab if you have any questions.
  • Slides should be properly labeled with a number that matches the specimen number on the cytology report.
  • Slides should be sent in proper storage containers (plastic slide boxes) to protect them during shipment. A surgical pathology and/or cytology report and completed requisition for must accompany all specimens.

Peripheral blood

  • 2-5 ml of fresh peripheral blood collected in EDTA (purple top) tube or ACD (yellow top) tube.
  • Blood should be refrigerated until shipment at 4°C.
  • Shipment is at ambient temperature by overnight delivery in a properly labeled shipping container for biohazard substances. A surgical pathology and/or cytology report and completed requisition for must accompany all specimens.

Buccal brush

  • Collection should be performed using the CytoSoft Cytology Brush or similar collection device following the manufacturer's instructions.
  • Collection date and time should be stated.
  • Shipping Conditions: Sample should be refrigerated until shipment at 4° C.
  • Shipment is at ambient temperature by overnight delivery in a properly labeled shipping container for biohazard substances. A surgical pathology and/or cytology report and completed requisition for must accompany all specimens.

Turnaround Time

7 – 14 days

Billing Information

*For insurance or Institutional Prices, please call.