January 31, 2024

MYELOID MALIGNANCIES MUTATION PANEL by NEXT GEN. SEQUENCING – Warde Medical Laboratory (wardelab.com)

Effective January 31, 2024, Warde Medical Laboratory performs targeted next-generation sequencing (NGS) for patients suspected of having acute myeloid leukemia (AML), myeloproliferative neoplasms (MPN), myelodysplastic syndromes (MDS), MDS/MPN overlap syndromes, or related clonal expansions of the myeloid lineage. The new assay (MNGS) is a Lab Developed Test (LDT) test utilizing the Archer VariantPlex® Myeloid Panel kit and Illumina sequencing technology. Variant classification and interpretation will follow joint standards from ACMG¹, AMP^1,2, ASCO², and CAP².

Selected exon regions from the following genes are targeted in the MNGS panel:

ABL1	ANKRD26	ASXL1	BCOR
CALR	CBL	CEBPA	CSF3R
DDX41	DNMT3A	EZH2	FLT3
GATA2	IDH1	IDH2	JAK2
KIT	KMT2A	KRAS	MPL
NPM1	NRAS	PTPN11	RUNX1
SETBP1	SF3B1	SRSF2	STAG2
TET2	TP53	U2AF1	WT1
ZRSR2

Variant allele frequencies (VAF) are reported for mutations, insertions, deletions, and clinically impactful copy number variations. Assay sensitivity is 5% VAF, with lower VAF reported in selected cases based on case characteristics.

The expected turn-around time will be roughly one week, which includes DNA extraction, library preparation, massively parallel sequencing, data analysis, variant interpretation, and director signout. In instances requiring repeat DNA preparation after initial analysis, cases may take up to 14 days.

What to send:

MNGS uses genomic DNA extracted from whole blood or bone marrow collected in lavender top EDTA tubes. An aliquot specimen is acceptable; a minimum of 1 mL is required. In many cases it will be possible to add MNGS testing to an existing flow cytometry specimen (call to inquire). Specimens should be shipped refrigerated and are stable for up to 7 days at that temperature. DNA extracted at a site other than Warde Lab is not an acceptable specimen.

It is common for pathologists or hematologists to refine diagnostic classification in the days between when the specimen is ordered and when MNGS results become available for interpretation by Warde directors. We always welcome additional information about morphology, phenotype, genetic results, or clinical findings, as a greater level of specificity can be achieved in the report. Please call Warde Lab at (734)214-0300 and ask to speak to Dr. Sitwala or Dr. Sekedat to establish an email line of communication regarding pending MNGS cases.

Indications for use:

Generally, a patient with a known or suspected myeloid neoplasm could benefit from MNGS, depending on overall clinicopathologic context.

Per the 5^th edition of WHO Classification of Tumours online – Haematolymphoid Tumours, screening hematologically normal patients for clonal haematopoiesis of indeterminate potential (CHIP) is not recommended³. If somatic mutation testing is performed, and one or more somatic mutations are detected at variant allele frequency >2% in the absence of unexplained cytopenias or other diagnostic criteria for defined myeloid neoplasms, then a patient can be designated as CHIP. CHIP progresses to malignancy at a rate of about 1%/year, though specific NGS findings (including VAF, number, and identity of mutated genes) greatly affect risk of progression. Highest risk is associated with mutation of TP53, U2AF1, SRSF2, IDH2, IDH1, SF3B1, and ASXL1. Studies have also found associations between CHIP and cardiovascular disease.

MNGS can be appropriate in investigation of unexplained cytopenias; the timing of when to order the assay may depend on the pace of the clinical workup. Generally, cytopenias are not considered “unexplained” before bone marrow assessment has been performed, as biopsy may uncover a non-myeloid explanation that would not benefit from myeloid-targeted NGS (for example, lymphoma, myeloma, metastatic solid tumor involvement, infection, vitamin deficiency, or other causes). Given the week-long specimen stability for MNGS, it is reasonable for a hematologist or hematopathologist to wait until after initial histologic examination of bone marrow before ordering MNGS.

If there are contraindications to bone marrow examination, a patient with cytopenias, myeloproliferation, or other findings suspicious for a myeloid neoplasm can be evaluated with peripheral blood MNGS. Peripheral blood is also a reasonable sample in instances of dry tap bone marrow or short specimen.

When bone marrow examination does reveal evidence of a myeloid neoplasm, NGS testing is often advisable and sometimes necessary for precise disease subclassification. It is currently standard of care to perform NGS on AML cases, as choice of therapy can be affected by detected mutations. Prognostic stratification of myelodysplastic syndrome and related disorders can rely upon NGS results. In some myeloproliferative/myeloproliferative neoplasms, specific mutations (not detectable by conventional cytogenetics or FISH) are definitional to classification, and results have prognostic significance as well. A subset of myeloproliferative neoplasms can be diagnosed through more targeted testing, though MNGS does cover the JAK2/CALR/MPL triad useful in primary myelofibrosis, essential thrombocythemia, and polycythemia vera. Note that diagnosis of chronic myeloid leukemia (CML) is not possible with NGS.

References:

1. Richards et. al. Standards and Guidelines for the Interpretation of Sequence Variants: A Joint Consensus Recommendation of the American College of Medical Genetics and Genomics and the Association for Medical Pathology. Genetic Med 2015, 17(5):405-424
2. Li et. al. Standards and Guidelines for the Interpretation and Reporting of Sequence Variants in Cancer: A Joint Consensus Recommendation of the Association for Molecular Pathology, American Society of Clinical Oncology, and College of American Pathologists. J Mol Diagn 2017, 19:4-23
3. WHO Classification of Tumours online – Haematolymphoid Tumours (5^th ed.) website beta version (International Agency for Research on Cancer, World Health Organization).