Alberta Precision Laboratories

--- Query Results ---

Organization:

Alberta Precision Laboratories

Message Alert:

PLEASE NOTE: This Test Directory is in an interim state and due to changes pertaining to the DynaLIFE transition, performing site for routing has not been updated. Please refer to Laboratory Information System for appropriate routing.

Test Name/Synonym:

Myeloid Panel (Synonyms: Myeloid Molecular Analysis by NGS, CALR, MPL, JAK2 exon 12, CEBPA, NPM1 and MYD88)

Clinical Indications:

For the investigation of acute myeloid leukemia (AML), myeloid dysplastic syndrome (MDS), myeloproliferative neoplasms (MPN), chronic myeloid leukemia (CML), chronic myelomonocytic leukemia (CMML), and juvenile myelomonocytic leukemia (JMML).

Test Includes:

Molecular Pathology North
Hot spot regions of ABL1, BRAF, CBL, CSF3R, DNMT3A, FLT3, GATA2, HRAS, IDH1, IDH2, JAK2, KIT, KRAS, MPL, MYD88, NPM1, NRAS, PTPN11, SETBP1, SF3B1, SRSF2, U2AF1, and WT1 and full coding sequences of ASXL1, BCOR, CALR, CEBPA, ETV6, EZH2, IKZF1, NF1, PHF6, PRPF8, RB1, RUNX1, SH2B3, STAG2, TET2, TP53 and ZRSR2 are evaluated for single nucleotide variants (SNVs) and insertions/deletions.

ABL1, ALK, BCL2, BRAF, CCND1, CREBBP, EGFR, ETV6, FGFR1, FGFR2, FUS, HMGA2, JAK2, KMT2A (MLL), MECOM, MET, MLLT10, MLLT3, MYBL1, MYH11, NTRK3, NUP214, PDGFRA, PDGFRB, RARA, RBM15, RUNX1, TCF3, and TFE3 are evaluated for translocations.

Molecular Pathology South
Hot spot regions of ABL1, ANKRD26, BRAF, CBL, CSF3R, DDX41, DNMT3A, FLT3, GATA2, HRAS, IDH1, IDH2, JAK2, KIT, KRAS, MPL, MYD88, NPM1, NRAS, PPM1D, PTPN11, SETBP1, SF3B1, SMC1A, SMC3, SRSF2, U2AF1, and WT1 and full coding sequences of ASXL1, BCOR, CALR, CEBPA, ETV6, EZH2, IKZF1, NF1, PHF6, PRPF8, RB1, RUNX1, SH2B3, STAG2, TET2, TP53 and ZRSR2 are evaluated for single nucleotide variants (SNVs) and insertions/deletions.

ABL1, ALK, BCL2, BRAF, CCND1, CREBBP, EGFR, ETV6, FGFR1, FGFR2, FUS, HMGA2, JAK2, KMT2A (MLL), MECOM, MET, MLLT10, MLLT3, MYBL1, MYH11, NTRK3, NUP214, NUP98, PDGFRA, PDGFRB, RARA, RBM15, RUNX1, TCF3, and TFE3 are evaluated for gene fusions.

Ordering Alert:

Test ordering restricted to hematologists, hematopathologists and molecular pathologists.

Collection Alert:

Unopened dedicated tubes are required for Molecular testing.

LABID (Connect Care):

LAB2770

Specimen Type:

Bone marrow, Peripheral blood

Primary Container:

See Specimen Collection Requirements

Specimen Collection Requirements:

Molecular Pathology North

Molecular Pathology South

Bone Marrow:

Collect one EDTA (lavender top) tube containing 1.0 mL of bone marrow and
Collect one PAXgene^® Bone Marrow RNA tube containing a maximum of 2.0 mL of bone marrow.

Bone Marrow:

Collect one EDTA (lavender top) containing 1.0 mL of bone marrow.
Pediatric collections may be in Sodium citrate (light blue top) tube.
Bone marrow in bone marrow flow media is also acceptable.

Peripheral Blood:

Collect one EDTA tube containing 1.0 mL of blood and
Collect one PAXgene^® Blood RNA tube containing 2.5 mL of blood.
An additional EDTA tube will be required for CBC testing if no CBC has been done in the last month (reflex action in Epic Beaker).

Ensure to follow the specific specimen collection requirements.

Using a butterfly needle and holding the tube below the arm, first collect the EDTA tube(s), then the PAXgene^® Blood RNA tube.
Note: Collection must be in the correct order of draw to ensure accurate white count determination

Peripheral Blood:

Collect one EDTA containing 4.0 mL of blood.

Test Resources:

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

Molecular Pathology North		Molecular Pathology South
	Molecular Pathology Requisition
			Calgary Bone Marrow Collections: Bone Marrow Pathology Requisition
			Calgary Peripheral Blood Collections: Molecular Hematology Requisition

Calgary Hematopathologists complete and fax the BM Pathology or Molecular Hematology requisition to Molecular Pathology, South at 587-231-4586.

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

Stability and Storage:

Keep specimens refrigerated until ready for transport.

Processing:

Molecular Pathology North	Molecular Pathology South
Immediately following collection, place all tubes ON ICE. Do not aliquot or centrifuge. For Peripheral Blood: Submit one EDTA tube and the PAXgene^® Blood RNA tube to Molecular Pathology. Send EDTA tube for a CBC to be processed as per local handling practice (only if no CBC has been done in the last month). This is a reflex action in Epic Beaker.	Immediately following collection, place all tubes ON ICE. Do not aliquot or centrifuge.

Transportation:

Molecular Pathology North	Molecular Pathology South
Transport specimens as soon as possible, on ice or cold pack, to Molecular Pathology via UAH Specimen Control WMC 4B2.10. Specimens must be received within 24 hours of collection. If transport is anticipated to be greater than 24 hours, contact Molecular Pathology at 780-407-6648.	Transport on ice or cold pack to Arthur J.E. Child Comprehensive Cancer Centre. Protect from freezing.

Method:

Next-generation sequencing

Method and Interpretation of Results:

Molecular Pathology North
DNA was extracted using the QIAsymphony DSP DNA MiniKit from Qiagen (Valencia, CA). RNA was extracted using the QIAsymphony.

The Oncomine^™ Myeloid Research Assay from ThermoFisher (Waltham, MA) contains targeted, multi-biomarker panels that enable simultaneous detection of variants across 40 key genes and a fusion panel of 29 driver genes relevant to major myeloid disorders associated with acute myeloid leukemia (AML), myeloid dysplastic syndrome (MDS), myeloproliferative neoplasms (MPN), chronic myeloid leukemia (CML), chronic myelomonocytic leukemia (CMML), and juvenile myelomonocytic leukemia (JMML). Pooled libraries, generated on the Ion Chef (ThermoFisher) are loaded into an Ion Torrent S5 sequencing system (Thermo Fisher) for sequencing and data analysis.

Hotspot regions of ABL1, BRAF, CBL, CSF3R, DNMT3A, FLT3, GATA2, HRAS, IDH1, IDH2, JAK2, KIT, KRAS, MPL, MYD88, NPM1, NRAS, PTPN11, SETBP1, SF3B1, SRSF2, U2AF1, and WT1 and full coding sequences of ASXL1, BCOR, CALR, CEBPA, ETV6, EZH2, IKZF1, NF1, PHF6, PRPF8, RB1, RUNX1, SH2B3, STAG2, TET2, TP53, and ZRSR2 are evaluated for single nucleotide variants (SNVs) and insertions/deletions. ABL1, ALK, BCL2, BRAF, CCND1, CREBBP, EGFR, ETV6, FGFR1, FGFR2, FUS, HMGA2, JAK2, KMT2A (MLL), MECOM, MET, MLLT10, MLLT3, MYBL1, MYH11, NTRK3, NUP214, PDGFRA, PDGFRB, RARA, RBM15, RUNX1, TCF3, and TFE3 are evaluated for translocations. Tier I, II, and III variants (per AMP guidelines) present at >5% allele fraction 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.

Reference: Li et al. Standards and Guidelines for the Interpretation and Reporting of Sequence Variants in Cancer. Journal of Molecular Diagnostics. 2017; 19(1): 4-23.

Genes with areas of low coverage (<500x): ***

The variants listed below are known to be non-reportable using the current methodology: • ASXL1c.1934_1935insG, p.G646fs*12 (homopolymer insertion-difficult variant) • CEBPA Variants: c.1021_1022 ins882_898 inv920_1021, p.lle341Argfs*17 (Variant occurring at edge of the amplicon) • CEBPA c.dup914_1047 (134 nt duplication), p.Leu350Serfs*13 (Resulting amplicon at edge of sequencing length: ~400) • CEBPA c.68_69insC, p.H24fs*84 (Homopolymer insertion – difficult variant) • FLT3-ITD mutations with lengths > 176bp

After DNA extraction, a fluorescent multiplex polymerase chain reaction (PCR) was performed for ASXL1. The amplified DNA was then subjected to fragment analysis by capillary electrophoresis using the ABI 3500xL genetic analyzer. Results were analyzed by Genemapper software. This assay detects indels in the region of ASXL1 exon 13 that contains the G homopolymer in which the NM_015338:c.1934dupG (p.G646fs) mutation occurs. The sensitivity of this assay is 10%.

Disclaimer: This test does not discriminate between germline and somatic variants. Although ThermoFisher markets this kit for research use only, the assay was validated and its performance characteristics determined by the Molecular Pathology Laboratory, University of Alberta Hospital. At the University of Alberta Hospital this test is used for clinical purposes. It should not be regarded as investigational or for research.

Molecular Pathology South
After isolation of DNA and/or RNA from patient bone marrow or peripheral blood, the sample is run on the Genexus^TM Intergrated Sequencer using Ion Torrent^TM Oncomine^TM Myeloid Assay GX v.2 chemistry. This chemistry is a comprehensive targeted next generation sequencing (NGS) assay designed for detection of DNA variants and fusion transcripts associated with myeloid disorders. The assay kit includes 3 pools of Ion AmpliSeq^TM oligonucleotide primers (a 2 pool DNA panel and 1 pool RNA panel), and Genexus^TM library reagents.

Multiplex primer design and sample barcoding leverage Ion AmpliSeq^TM technology to generate comprehensive results from multiple samples in a single run. Sequencing results are analyzed in the Genexus^TM Software using the optimized assay-specific analysis workflow.

Germline variants are not reported by this test. In the absence of confirmed somatic status in current databases, this assay cannot distinguish somatic heterozygous from germline variants. A follow-up germline testing may therefore be indicated.

Unless otherwise stated, the karyotype is assumed to be normal. Interpretation of pathogenicity of detected variants is as of current reports and databases. Sensitivity of the test is 5% for somatic variants detection. Details on low coverage regions can be provided upon request.

Routine Turn Around Time:

Molecular Pathology North	Molecular Pathology South
AML: 2 weeks MDS/MPN: 4 weeks	AML: 2 weeks MDS/MPN: 4 weeks

Testing Schedule:

Weekly

Testing Area:

Molecular Pathology

Performing Site:

University of Alberta Hospital, Arthur J.E. Child Comprehensive Cancer Centre

Additional Test Information:

PAXgene^® collection tubes are available upon request.
Submit an order as per online Client Order Form Submission process.
Details in following document: RR01-005 Ordering Supplies from Reagent Room

Contact Comments:

Molecular Pathology North	Molecular Pathology South
780-407-6648	587-231-3199

Last Updated On:

Tuesday, December 17, 2024

Date of Last Review:

Feb 7 2023 12:00AM

Registered Trademark Comments:

PAXgene^® is a registered trademark of PreAnalytiX GmbH.
Genexus^TM, Ion Torrent^TM, Oncomine^TM and Ion AmpliSeq^TM are trademarks of Life Technologies.