The clinical utility of a custom-developed targeted next-generation sequencing assay for detection of mutations associated with Philadelphia-negative chronic myeloproliferative neoplasms: Two case examples with CALR exon 9 mutations

Vijay Ramanan, Ketki Kelkar, Shatakshi Ranade, Priyanka Gangodkar, Nikhita Gogate, Kunal Patil, Trupti Ragte-Wathare, Meenal Agarwal, Nikhil A. Phadke


We have developed a highly targeted custom next generation sequencing-based test targeting JAK2 exons 12 and 14, CALR exon 9, and MPL exon 10, which are implicated in Philadelphia-negative chronic myeloproliferative neoplasms. The assay is capable of ultra-high sequencing depth and producing mutational detection sensitivities of 0.5% and below. We have validated the performance of the test through orthogonal testing and demonstrated a high degree of multiplexing with up to 50 samples in a single run. We show the clinical utility of this test through the description of a couple of cases with myeloproliferative disorders with Type-II and Type-II-like mutations in exon 9 of the CALR gene.


NGS, multiplexing, CALR, JAK2, Hemato-oncology, Philadelphia-negative chronic myeloproliferative neoplasms

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Naidoo N, Pawitan Y, Soong R, Cooper DN, Ku CS. Human genetics and genomics a decade after the release of the draft sequence of the human genome. Hum Genomics 2011;5:577-622.

Meldrum C, Doyle MA, Tothill RW. Next-generation sequencing for cancer diagnostics: A practical perspective. Clin Biochem Rev 2011;32:177-95.

Gilissen C, Hoischen A, Brunner HG, Veltman JA. Unlocking mendelian disease using exome sequencing. Genome Biol 2011;12:228.

Goldman JM. Chronic myeloid leukemia: A historical perspective. Semin Hematol 2010;47:302-11.

Mesa RA, Passamonti F. Individualizing care for patients with myeloproliferative neoplasms: Integrating genetics, evolving therapies, and patient-specific disease burden. Am Soc Clin Oncol Educ Book 2016;35:e324-35.

Rumi E, Pietra D, Ferretti V, Klampfl T, Harutyunyan AS, Milosevic JD, et al. JAK2 or CALR mutation status defines subtypes of essential thrombocythemia with substantially different clinical course and outcomes. Blood 2014;123:1544-51.

Pikman Y, Lee BH, Mercher T, McDowell E, Ebert BL, Gozo M, et al. MPLW515L is a novel somatic activating mutation in myelofibrosis with myeloid metaplasia. PLoS Med 2006;3:e270.

Nangalia J, Massie CE, Baxter EJ, Nice FL, Gundem G, Wedge DC, et al. Somatic CALR mutations in myeloproliferative neoplasms with nonmutated JAK2. N Engl J Med 2013;369:2391-405.

Klampfl T, Gisslinger H, Harutyunyan AS, Nivarthi H, Rumi E, Milosevic JD, et al. Somatic mutations of calreticulin in myeloproliferative neoplasms. N Engl J Med 2013;369:2379-90.

Jones AV, Ward D, Lyon M, Leung W, Callaway A, Chase A, et al. Evaluation of methods to detect CALR mutations in myeloproliferative neoplasms. Leuk Res 2015;39:82-7.

Tefferi A, Lasho TL, Tischer A, Wassie EA, Finke CM, Belachew AA, et al. The prognostic advantage of calreticulin mutations in myelofibrosis might be confined to type 1 or type 1-like CALR variants. Blood 2014;124:2465-6.

Pietra D, Rumi E, Ferretti VV, Buduo CA, Milanesi C, Cavalloni C, Sant'Antonio E, Abbonante V, Moccia F, Casetti IC, Bellini M, Renna MC, Roncoroni E, Fugazza E, Astori C, Boveri E, Rosti V, Barosi G, Balduini A, Cazzola M. Differential clinical effects of different mutation subtypes in CALR-mutant myeloproliferative neoplasms. Leukemia 2016;30:431-8.

Rampal R, Al-Shahrour F, Abdel-Wahab O, Patel JP, Brunel JP, Mermel CH, et al. Integrated genomic analysis illustrates the central role of JAK-STAT pathway activation in myeloproliferative neoplasm pathogenesis. Blood 2014;123:e123-33.

Cazzola M, Kralovics R. JAK inhibitor in CALR-mutant myelofibrosis. N Engl J Med 2014;370:1169.

Fric J, Lim CX, Koh EG, Hofmann B, Chen J, Tay HS, et al. Calcineurin/NFAT signalling inhibits myeloid haematopoiesis. EMBO Mol Med 2012;4:269-82.

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