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Test Code NGFFPE (FFPE), NGSCS (Cytology Smear) OncoScreen- Extended RAS Assay

Specimen Type

FFPE or Cytology Smear

Container

Blocks or slides

Preferred Volume

n/a

Offsite Collection Instructions

  • Formalin-fixed, paraffin embedded tissue: Total tumor area on slides = 0.1 cm2 total (include top and bottom H&E guide slides).
  • Paraffin block acceptable.

UCMC Collection Instructions

  • FFPE tissues: a pathologist will review the respective case upon receiving a requisition to select the appropriate block for testing. Slides will be recut and evaluated, tumor-rich areas are selected for testing.
  • Cytology smears: immediate on-site adequacy assessment is performed by a pathologist and tumor-rich areas are selected for testing.

Availability

Monday – Friday, 8:00am – 5:00pm

Turnaround Time

8-10 business days

STAT Availability

Not Available

Test Methodology

KRAS, HRAS and NRAS genes are part of the OncoScreen ST2.0 NGS panel. The OncoScreen procedure is performed and data for the 3 genes only is analyzed.
The OncoScreen test is based on the Ion AmpliSeq Cancer Hotspot Panel (IACHP) v2 (Life Technologies). After DNA is isolated and its quality and quantity determined, a single multiplexed PCR is performed using a single reaction containing the 207 Ion AmpliSeq Cancer Hotspot primer pairs. It is then cleaned up using Ampure XP beads and a library is constructed using KAPA HTP Library Preparation Kit and Illumina compatible adapters. Through the library construction, adapter oligonucleotides containing unique patient-specific “barcode” indexes and P5/P7 sequences are ligated onto the amplicon PCR products to make an Illumina compatible sequencing library.All DNA samples for a given run are processed in duplicate and in parallel, and then following a library quantitation, samples with different bar-code sequences are pooled and sequenced in a 2 × 152 bp MiSeq run.

Additional Information

  • Reporting: Mutations with known or possible pathogenic or clinical significance are reported. Variants of unknown significance are reported as such.

STAT Turnaround Time

n/a

Reference Range

Interpretive report will be provided.

Test Limitations

  • This test will not detect mutations outside the targeted genomic regions.
  • Copy number alterations and translocations will not be detected. This test is not intended to detect minimal residual disease.
  • Assay sensitivity is 5% mutant allele frequency (MAF), thus false-negative results may occur when there is a lower mutant allele burden.
  • Variants present at oligonucleotide binding sites may negatively interfere with hybridization (as with any PCR-based assay), potentially resulting in underestimation of a particular allele frequency.

Test Includes

This is a next-generation Sequencing panel that tests hot spot regions of 3 genes that are part of the Solid Tumor OncoScreen ST 2.0 panel. The tested genes are: KRAS (exons 2, 3, and 4), NRAS (Exons 2, 3, and 4) and HRAS (Exons 2 and 3).