Myeloproliferative neoplasms (MPNs) are a group of rare blood cancers derived from myeloid stem cells. The most well-known MPN is chronic myeloid leukemia, (CML), characterized by the classic BCR-ABL1 rearrangement. There are BCR-ABL1-negative MPNs as well (polycythemia vera (PV), essential thrombocythemia (ET), primary myelofibrosis (PMF)), which are also characterized by their unique somatic mutations, or lack thereof. In >95% of cases, one of three genes drives the MPN phenotype in a mutually exclusive manner: JAK2, CALR, or MPL1, 4.
Typical diagnostic tests for MPNs target JAK2 V617F (exon12) and JAK2 exon 14 mutations, exon 9 of CALR and exon 10 of MPL. Currently, recommendations are to screen for these mutations in a sequential manner using clinical/laboratory findings and bone marrow morphology to guide the diagnostic testing. Typically, the algorithm begins with JAK2 V617F mutation testing (usually by allele-specific oligonucleotide PCR). If negative, testing reflexes to CALR exon 9 insertion/deletion analysis by PCR (some laboratories use high resolution melting curve analysis). If that is negative, testing reflexes to MPL codon W515 mutation testing by pyrosequencing. Alternatively, some institutions may use next-generation sequencing myeloid malignancy panels to identify single gene mutations or “hot spots” (substitutions, insertions, deletions) all at once. Each method has its pros and cons, and largely depends on laboratory workflow, clinical need, and financial resources2, 3, 10.
Molecular diagnostics have certainly broadened our ability to diagnose and identify prognostic markers in cases of MPNs. However, challenges remain. So-called “triple-negative” MPNs are negative for mutations in all three of the aforementioned genes (about 12% of ET and 5% of PMF fall into this category), and also alert the diagnostician to the possibility of a reactive or hereditary process rather than a neoplasm. Triple-negative status carries prognostic significance in MPNs: In PMF, triple-negative status is associated with poorer survival, while triple-negative ETs have favorable prognosis3, 8.
Recent studies have turned their attention to triple-negative MPNs and the search for other possible driver mutations using whole exome sequencing (WES)5, 6, 7. Both somatic and germline gain-of-function mutations in non-traditional exons of MPL and JAK2 were identified, as well as cases with no mutation that showed polyclonal hematopoiesis. About 10% of cases of triple-negative ET and PMF showed mutations in exons 3, 4, 5, 6, 12 of MPL and 10% showed germline mutations in exons 8, 13, and 15 of JAK2. WES methodology is limited, however, in that it can only detect mutation(s) in the coding exons of genes, and cannot detect mutations in coding sequences at low variant allele frequency6, 7.
RNA sequencing for detection of fusion oncogenes or whole genome sequencing of regulatory regions could be another approach. Next-generation sequencing has identified very low allele frequency mutations in cases of MPN with the driver mutation JAK2 V617F, suggesting a lack of sensitivity of other methods5. These findings emphasize the heterogeneity of triple-negative MPNs, as well as the possibility of hereditary MPN-like disorders in those with polyclonal hematopoiesis.
It is important for physicians to note that MPN-associated mutations are not entirely represented in currently available commercial panels used by routine diagnostic pathology laboratories. One research group sought to develop a comprehensive custom assay to capture the heterogeneous mutations described in triple-negative MPNs using targeted exon sequencing11. They covered 86 genes extending beyond JAK2, CALR, and MPL to rare mutations in ASXL1, TET2, SH2B3, and others and showed that although next-generation sequencing is useful for exploring well-established mutations, their custom panel has a distinct advantage by providing a full snapshot of an individual patient’s MPN which can be used to follow molecular progression, clonal heterogeneity, and drug resistance.
Not only are molecular advances occurring to identify futher mutations, but there are also new technologies bringing testing closer to the patient9. A new point-of-care platform for the simultaneous analysis of JAK2 V617F and MPL W515K/L mutations (developed in 2017) aimed to improve early diagnosis of MPNs and decrease potential associated arterial thrombosis. It is a portable microfluidic platform with a glass capillary containing polypropylene matrix that extracts genomic DNA from a drop of whole blood, a microchip for simultaneous multi-gene mutation screening, and a handheld battery-powered heating device.
These recent studies indicate that a heterogeneous landscape of mutations is co-occurring, rather than single mutations, and that there is a need to explore a broader approach to disease pathogenesis in cases of triple-negative MPNs. The presence of non-traditional mutations in triple-negative cases signals a need for further work-up to cover the entire coding region of JAK2 and MPL and suggests that identifying the key driver mutations will become only a portion of the diagnostic work-up to identify the landscape of genetic mutations in these cases.
- Swerdlow et al. The 2016 revision of the World Health organization classification of lymphoid neoplasms. Blood 2016; 127-2375.
- Tefferi et al. Polycythemia vera and essential thrombocythemia: algorithmic approach. Curr Opin Hematol. 2018 Mar;25(2):112-119. doi: 10.1097/MOH.0000000000000402.
- Tefferi et al. Polycythemia vera and essential thrombocythemia: 2017 update on diagnosis, risk-stratification, and management. Am J Hematol. 2017 Jan;92(1):94-108. doi: 10.1002/ajh.24607.
- Schischlik et al. Mutations in myeloproliferative neoplasms- their significance and clinical use. Expert Rev Hematol. 2017 Nov;10(11):961-973. doi: 10.1080/17474086.2017.1380515.
- Chang et al. Targeted next-generation sequencing identified novel mutations in triple-negative myeloproliferative neoplasms. Med Oncol. 2017 May;34(5):83. doi: 10.1007/s12032-017-0944-z.
- Feenstra et al. Whole-exome sequencing identifies novel MPL and JAK2 mutations in triple-negative myeloproliferative neoplasms. Blood. 2016 Jan 21; 127(3): 325–332.
- Cabagnols et al. Presence of atypical thrombopoietin receptor (MPL) mutations in triple-negative essential thrombocythemia patients. Blood. 2016 Jan 21;127(3):333-42.
- Rumi et al. Diagnosis, risk stratification, and response evaluation in classical myeloproliferative neoplasms. Blood. 2017;129(6):680-692. doi:10.1182/blood-2016-10-695957.
- Wang et al. A portable microfluidic platform for rapid molecular diagnostic testing of patients with myeloproliferative neoplasms. Nature. Scientific Reports. 7: 8596. 2017. DOI:10.1038/s41598-017-08674-8
- Langabeer SE. Chasing down the triple-negative myeloproliferative neoplasms: Implications for molecular diagnostics. JAK-STAT. 2016;5(2-4):e1248011. doi:10.1080/21623996.2016.1248011.
- Magor et al. Rapid Molecular Profiling of Myeloproliferative Neoplasms Using Targeted Exon Resequencing of 86 Genes Involved in JAK-STAT Signaling and Epigenetic Regulation. J Mol Diagn. 2016 Sep;18(5):707-718. doi: 10.1016/j.jmoldx.2016.05.006.