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

Specimen and Container/Tube

FFPE or
Cytology Smear

Turnaround Time

n/a

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).

Specimen Type

FFPE or
Cytology Smear

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.

STAT Turnaround Time

n/a

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.

Additional Information

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

Container/Tube

Blocks or slides

Day(s) Performed

Monday – Friday, 8:00am – 5:00pm

STAT DAY(S) AND TIME(S) PERFORMED

Not Available

Method Name

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.

Reference Values

Interpretive report will be provided.

Transport 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.

Test Components

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).

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.