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Myeloproliferative Neoplasm Diagnosis: Molecular Evaluation

Test code(s) 92472, 92473, 92474, 92475, 92476, 92477, 91065, 90665

Tests used for diagnostic evaluation of MPNs vary based on the particular hematologic abnormalities observed, as shown below. If the specimen is negative for the BCR/ABL1 translocation, then additional testing, such as the JAK2 V617F Cascading Reflex, is a consideration.

The suggestions are based primarily on the 2017 World Health Organization classification of myeloid neoplasms and acute leukemia.1 Genes most closely associated with the various MPNs are shown below.

This cascading reflex includes multiplex PCR and sequencing analysis for JAK2 mutations at codon 617 (exon 14) and exon 12, CALR frame shift mutations in exon 9, MPL mutations at codons 505 and 515 (exon 10), and CSF3R mutations in exons 14 and 17. The analysis includes all the currently well-characterized MPN-associated mutations in these genes.

Each mutation is essentially mutually exclusive in any given MPN case.2 Therefore, results are reported in a cascading manner (ie, detection of any MPN-associated mutation terminates data analysis/reporting of subsequent genes in the cascading reflex). For example, detection of JAK2 V617F mutation will result in a “Test not indicated/not reported” result for the other 4 analytes. The order of the reflex cascade is based on mutation frequency in MPNs; data are analyzed for genes in the following order: JAK2 (V617F), CALR, JAK2 (exon 12), MPL, and CSF3R.

Yes, the following single-analyte test codes are available:

18950: JAK2 V617F Mutation, Quantitative
92473: JAK2 V617F Mutation Analysis
92474: JAK2 Exon 12 Mutation Analysis
92475: Calreticulin (CALR) Mutation Analysis
92476: MPL Mutation Analysis
92477: CSF3R Mutation Analysis

Peripheral blood and bone marrow aspirate are the acceptable specimen types. Specimens are stable for 7 days, but they should be sent immediately after collection whenever possible. See the online Test Directory for specimen volumes and acceptable collection tubes.

Mutations in JAK2, CALR, MPL, and CSF3R are all detected in DNA extracted from leukocytes using an advanced sequencing method. The sensitivity for mutation detection is set at 5% mutant alleles, which is lower than that of traditional Sanger DNA sequencing. Given the unclear significance of occult or low levels of these mutations,6 the 5% sensitivity is appropriate for a diagnostic MPN assay. The percentage of mutation reads is reported, so it can be compared with that in subsequent tests.

As of December 3, 2018, the Leumeta plasma-based versions (test codes 16175, 16536X, 16538X, 16539X, 16184X) are no longer available.

Owing to the improved precision and sensitivity of advanced sequencing methods, the Leumeta plasma RNA-based assays are no longer considered state-of-the-art for hematological neoplasms. DNA-based assays have longer specimen stability and provide a cell-equivalent level of mutations (ie, the percentage of mutation reads corresponds directly to the number of cells in the sample bearing that mutation).

The recommended alternative codes for the discontinued Leumeta-based tests are shown below.

Yes. This assay, which tests for the percentage of JAK2 V617F mutation in DNA extracted from leukocytes, will still be available. This assay can be performed using formalin-fixed, paraffin-embedded tissue samples, including bone marrow trephine core biopsies.

The BCR/ABL1 gene rearrangement test is not included in JAK2 V617F Cascading Reflex because it is an RNA-based test rather than a DNA-based test. RNA-based technology is better for detecting fusion transcripts such as BCR/ABL1. Additionally, BCR/ABL1 fusion transcript results must be normalized and reported according to the

International Scale (IS). This requires copy number of both BCR/ABL1 and ABL1 RNA transcripts. For additional information on the BCR/ABL1 test, including identification of the P190 vs P210 breakpoint, see the following:

If eosinophilia is a prominent feature in a patient with suspected MPN, the following tests should be considered.

  • BCR/ABL1 Gene Rearrangement, Quantitative, PCR (91065)
  • FISH, Myeloproliferative Neoplasms (Eosinophilia) (90665)

The FISH test includes probes for PDGFRA (4q12), PDGFRB (5q33.1), and FGFR1 (8p11-12). PDGFRA, PDGFRB, and FGFR1 rearrangements occur in MPN with eosinophilia (including hypereosinophilic syndrome) and result in activation of tyrosine kinases. 

Other causes of eosinophilia and myeloid proliferation, including coexistent lymphoma, should also be considered.

References

  1. Swerdlow SH, Campo E, Harris NL, et al (eds). WHO Classification of Tumours of Haematopoietic and Lymphoid Tissues. Revised 4th ed. Lyon, France: The International Agency for Research on Cancer; 2017.
  2. Tefferi A, Thiele J, Vannucchi AM, et al. An overview on CALR and CSF3R mutations and a proposal for revision of WHO diagnostic criteria for myeloproliferative neoplasms. Leukemia. 2014;28:1407-1413.
  3. Lundberg P, Karow A, Nienhold R, et al. Clonal evolution and clinical correlates of somatic mutations in myeloproliferative neoplasms. Blood. 2014;123:2220-2228.
  4. Schnittger S, Bacher U, Eder C, et al. Molecular analyses of 15,542 patients with suspected BCR/ABL1-negative myeloproliferative disorders allow to develop a stepwise diagnostic workflow. Haematologica. 2012;97:1582-1585.
  5. Cervera N, Itzykson R, Coppin E, et al. Gene mutations differently impact the prognosis of the myelodysplastic and myeloproliferative classes of chronic myelomonocytic leukemia. Am J Hematol. 2014;89:604-609.
  6. Pardanani A, Lasho TL, Hussein K, et al. JAK2V617F mutation screening as part of the hypercoagulable work-up in the absence of splanchnic venous thrombosis or overt myeloproliferative neoplasm: assessment of value in a series of 664 consecutive patients. Mayo Clin Proc. 2008;83:457-459.
  7. Gong, JZ, et al. Laboratory practice guidelines for detecting and reporting JAK2 and MPL mutations in myeloproliferative neoplasms: a report of the Association for Molecular Pathology. J Mol Diagn. 2013;15:733-744.
  8. Guglielmelli P, Lasho TL, Rotunno G, et al. The number of prognostically detrimental mutations and prognosis in primary myelofibrosis: an international study of 797 patients. Leukemia, 2014;28:1804-1810.
  9. Lundberg P, Karow A, Nienhold R, et al. Clonal evolution and clinical correlates of somatic mutations in myeloproliferative neoplasms. Blood. 2014;123:2220-2228.
  10. Ortmann, CA, Kent DG, Nangalia J, et al. Effect of mutation order on myeloproliferative neoplasms. N Engl J Med. 2015;372:601-612.

 

This FAQ is provided for informational purposes only and is not intended as medical advice. A clinician’s test selection and interpretation, diagnosis, and patient management decisions should be based on his/her education, clinical expertise, and assessment of the patient.

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