Alberta Precision Laboratories

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Organization:

Alberta Precision Laboratories

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Test Name/Synonym:

Lung Carcinoma Mutation Analysis (Synonyms: lung adenocarcinoma, lung, oncomine focus, EGFR, ALK, ROS1, RET, MET and hot spot mutations)

Clinical Indications:

Predictive biomarker testing in advanced stage lung adenocarcinoma.

Test Includes:

Molecular Pathology North
Evaluated for single nucleotide variants (SNVs) and small insertions/deletions (indels) are the hotspot regions of AKT1, ALK, AR, BRAF, CDK4, CTNNB1, DDR2, EGFR, ERBB2, ERBB3, ERBB4, ESR1, FGFR2, FGFR3, GNA11, GNAQ, HRAS, IDH1, IDH2, JAK1, JAK2, JAK3, KIT, KRAS, MAP2K1, MAP2K2, MET, MTOR, NRAS, PDGFRA, PIK3CA, RAF1, RET, ROS1 and SMO.
Evaluated for copy number alterations (CNA) are ALK, AR, BRAF, CCND1, CDK4, CDK6, EGFR, ERBB2, FGFR1, FGFR2, FGFR3, FGFR4, KIT, KRAS, MET, MYC, MYCN, PDGFRA, and PIK3CA.
Evaluated for fusions are ABL1, AKT3, ALK, AXL, BRAF, EGFR, ERBB2, ERG, ETV1, ETV4, ETV5, FGFR1, FGFR2, FGFR3, MET, NTRK1, NTRK2, NTRK3, PDGFRA, PPARG, RAF1, RET and ROS1.

Molecular Pathology South:
The Agena iPLEX HS Lung Panel (DNA) is validated to detect selected single nucleotides (SNVs) and insertion/deletion mutations (indels) in 5 genes: EGFR, KRAS, ERBB2, BRAF, PIK3CA. The Lung Carcinoma Fusion RNA Panel is validated to detect gene fusions, oncogenic isoforms and/or selected single nucleotide variants (SNVs) and insertions/deletions (indels) involving the following 17 genes: ALK, BRAF, EGFR, ERBB2, FGFR1, FGFR2, FGFR3, KRAS, MET, NRG1, NTRK1, NTRK2, NTRK3, NUTM1, PIK3CA, RET, ROS1.

LABID (Connect Care):

LAB4850

Specimen Type:

Paraffin-Embedded Tissue

Specimen Source:

Indicate specimen source.

Primary Container:

See Specimen Collection Requirements

Specimen Collection Requirements:

For submissions to Molecular Pathology North

A formalin-fixed, paraffin-embedded tissue block is preferred.
Alternatively, one slide stained with Hematoxylin & Eosin and five 10-micron non-baked, unstained slides can be submitted.
Please provide a copy of the corresponding pathology report.

For submissions to Molecular Pathology South

A formalin-fixed, paraffin-embedded tissue block (resection specimen, biopsy specimen, cytology cell block) accompanied by a representative H&E (ideally the deepest level) is preferred. The assay requires a minimum of 5% tumor in the selected area (5 tumor nuclei per 100 total nuclei). When possible, non-Cytolyt®-exposed FFPE tissue is preferred over Cytolyt®-exposed FFPE tissue (cytology cell blocks).
Alternatively, five 10-micron non-baked unstained slides accompanied by a representative H&E (ideally the deepest level) can be submitted. If the sample is relatively small (less than 10mm squared, for example a core biopsy 1mm in diameter and less than 1cm in length), particularly if the tissue has a low cell density, more than 5 (up to 10) 10-micron non-baked unstained slides may be sent to increase nucleic acid yield for testing.
Alternatively, precut scrolls can be submitted, accompanied by a representative H&E (ideally cut after the scrolls).
Note: The tissue should not be exposed to decalcification solution.

Test Resources:

Submit request using the applicable requisition or Connect Care online order entry:

Molecular Pathology North		Molecular Pathology South
	Molecular Pathology Requisition		Molecular Pathology Requisition

Please ensure all required information, including patient's clinical history/indication, is provided on the requisition.

Stability and Storage:

Room temperature: paraffin-embedded tissue blocks and slides

Transportation:

Molecular Pathology North	Molecular Pathology South
Transport at room temperature to Molecular Pathology at University of Alberta Hospital.	Transport at room temperature to Pathology Department at Foothills Medical Center.

Method:

Molecular Pathology North
Next-Generation Sequencing (NGS)

Molecular Pathology South
DNA testing: PCR followed by single base extension and mass spectrometry.
Reflexive RNA testing (when indicated): Next Generation Sequencing (NGS).

Method and Interpretation of Results:

Molecular Pathology North
DNA and RNA extraction from formalin-fixed paraffin-embedded (FFPE) tissue was performed using the Thermo Fisher MagMAX FFPE DNA/RNA Ultra Kit on the KingFisher Apex purification system.

The Oncomine™ Focus Assay from Thermo Fisher (Waltham, MA) comprises DNA and RNA panels. Ion AmpliSeq-based library preparation is performed by the Ion Chef System and sequencing is performed by the Ion GeneStudio S5 System. Base calling is generated by the Torrent Suite Software (version 5.12.1). Variant calling and annotations are generated using the Ion Reporter software (version 5.20) with alignment to reference human genome GRCh37/hg19. Copy number evaluation is performed using OncoCNV software (version 6.9).
Tier I, II, and III variants (per AMP/ASCO/CAP guidelines [1]) present at >5% variant allele fraction (VAF) are reported. As per these guidelines, Tier I variants have strong clinical significance with level A and B evidence. Tier II variants have potential clinical significance (level C and D evidence). Tier III variants are variants of unknown clinical significance. All genomic coordinates are given with respect to GRCh37 (hg19). For cDNA and protein coordinates, reference transcripts are listed in the report.
PERFORMANCE METRICS: For SNVs and small indels, the limit of detection of this assay is 5% variant allele fraction (VAF) with 100% sensitivity. For gene fusion, the limit of detection is 1% of fusion transcript present in the total RNA and an estimated clinical sensitivity of 95% for the specific fusion genes included in the panel design. For CNA, the limit of detection of this assay is 4 copies. The sensitivity of CNA detection is affected by the tumor cellularity (fraction of tumor cells) and the copy number in the tumor cells. The minimum depth of coverage is 300X.
TEST LIMITATIONS: This assay requires a minimum 10% tumor cellularity for SNV, small indels and gene fusions. For CNA, this assay requires a variable range of tumor cellularity (from 100% tumor cellularity with 4 copies per tumor cell to 10% tumor cellularity with 22 copies per tumor cell).

This assay does not distinguish between somatic (tumor acquired) and germline (inherited) mutations.

Molecular Pathology South
OVERVIEW: This test algorithm is intended for predictive biomarker assessment in advanced stage lung adenocarcinoma. DNA and RNA is extracted from formalin-fixed paraffin-embedded (FFPE) using the Promega Maxwell® RSC DNA FFPE Kit and Promega Maxwell® RSC RNA FFPE Kit. DNA testing is performed using the Agena iPLEX HS Lung panel and analyzed using an Agena MassArray matrix assisted laser desorption-ionization time-of-flight mass spectrophotometer (MALDI-TOF MS). Cases negative for a mutation in EGFR, KRAS, ERBB2, and BRAF on the Agena iPLEX HS Lung Panel are reflexively tested on the Lung Carcinoma Fusion RNA Panel. RNA is converted to cDNA and library preparation is performed using ArcherDx FusionPlex reagents (Invitae) for Ion Torrent™, with a custom primer design. Next generation sequencing (NGS) is performed using an Ion Torrent Genexus System (ThermoFisher Scientific), software version 6.6. Sequence output files are analyzed on Archer Analysis v6.2.7 (ArcherDX, Inc.). File transfers and additional data processing are performed using custom in-house Python™ scripts. All results are reviewed by a molecular pathologist.
PANEL CONTENT: The assay utilizes anchored multiplex PCR (AMP), which enables partner-agnostic fusion detection. Assessment for SNVs/indels on the RNA panel is limited to specific targeted mutations, and is not performed across all genes / gene regions on the panel; the list of assessed SNVs/indels is available upon request (also see below Test Limitations for SNVs/indels in the RNA panel). Further details on the RNA panel content including the breakdown of specific molecular alterations covered per gene, and specific breakpoints covered, are available on request.
ANALYTIC INTERPRETATION: Agena iPLEX HS Lung Panel (DNA) testing, and reflex Lung Carcinoma Fusion RNA Panel testing (when indicated/performed), are interpreted and integrated in one report. For the Agena iPLEX HS Lung Panel, Tier I, II, and III single nucleotide variants (SNVs) and insertion/deletion variants (indels) with sufficient signal on MALDI-TOF MS are reported. For the Lung Carcinoma Fusion RNA Panel, Tier I, II, and III single nucleotide variants (SNVs) and insertion/deletion variants (indels) with sufficient unique supporting reads and expressed variant allele fraction (VAF) > 5% are reported. Tier I, II, and III fusions and oncogenic isoforms with sufficient supporting reads and without evidence of mispriming, paralog fusion partners, low confidence alignment, and/or transcriptional read-through are reported. Fusions often demonstrate multiple splicing isoforms within one sample; in such cases, the isoform with the greatest unique read support is reported. The list of all detected fusion isoforms in a sample is available on request. All genomic coordinates are given with respect to GRCh37 (hg19). For cDNA and protein coordinates, reference transcripts are listed in the report.
TEST LIMITATIONS: Both the Agena iPLEX HS Lung Panel and Lung Carcinoma Fusion RNA Panel require a minimum of 5% tumor (5 tumor nuclei / 100 total nuclei) and are not validated for sample types other than FFPE. The Lung Carcinoma Fusion RNA Panel requires a minimum of 50ng RNA input (250ng preferred), and sufficient sample RNA quality, as evaluated by Archer PreSeq qPCR +/- sequencing metrics. The Lung Carcinoma Fusion RNA Panel cannot exclude SNVs/indels when not detected. Sensitivity for SNVs/indels depends on sequencing depth, which varies significantly across targets on RNA NGS panels due to variable expression profiles. For similar reasons, expressed VAF in RNA may not correspond to VAF in DNA. Indels near exon-exon junctions may not be called due to alignment challenges. RNA is not a suitable analyte for detecting loss-of-function mutations in tumor suppressors. RNA NGS cannot detect copy number variants (CNVs) including deletions and amplifications. On rare occasions, gene fusions may not be detected despite adequate tumor percentage and sample quality due to low fusion expression level, rare breakpoints not targeted by primers, or bioinformatics challenges (paralogs, alignment issues, etc).

This assay does not distinguish between somatic (tumor acquired) and germline (inherited) mutations.

Routine Turn Around Time:

3 weeks

Testing Schedule:

Weekly

Testing Area:

Molecular Pathology

Performing Site:

University of Alberta Hospital or Foothills Medical Center

Contact Comments:

Molecular Pathology North	Molecular Pathology South
780-407-6648	403-220-4240

Last Updated On:

Thursday, February 8, 2024

Date of Last Review:

Feb 7 2024 12:00AM

Registered Trademark Comments:

CYTOLYT® is a trademark of Hologic, Inc.
FusionPlex® is a registered trademark of ARCHERDX, LLC
Genexus™ is a trademark of Life Technologies Corporation