Mutational Analysis of EXON 9 of the CALR Gene (Reference )

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1 Mutational Analysis of EXON 9 of the CALR Gene (Reference ) Notice of Assessment June 2014 DISCLAIMER: This document was originally drafted in French by the Institut national d'excellence en santé et en services sociaux (INESSS), and that version can be consulted at biologie_medicale_juin_2014_4.pdf. It was translated into English by the Canadian Agency for Drugs and Technologies in Health (CADTH) with INESSS s permission. INESSS assumes no responsibility with regard to the quality or accuracy of the translation. While CADTH has taken care in the translation of the document to ensure it accurately represents the content of the original document, CADTH does not make any guarantee to that effect. CADTH is not responsible for any errors or omissions or injury, loss, or damage arising from or relating to the use (or misuse) of any information, statements, or conclusions contained in or implied by the information in this document, the original document, or in any of the source documentation.

2 1 GENERAL INFORMATION 1.1 Requester: Hôpital Maisonneuve-Rosemont 1.2 Application for Review Submitted to MSSS: January 15, Application Received by INESSS: March 1, Notice Issued: June 30, 2014 Note: This notice is based on the scientific and commercial information submitted by the requester and on a complementary review of the literature according to the data available at the time that this test was assessed by INESSS. 2 TECHNOLOGY, COMPANY, AND LICENCE(S) 2.1 Name of the Technology Analysis of the mutational status of exon 9 of the CALR 1 gene using fragment length analysis following a nucleic acid amplification test (NAAT) with fluorescent primers. 2.2 Brief Description of the Technology, and Clinical and Technical Specifications This test is conducted to confirm a diagnosis of myeloproliferative neoplasm, specifically essential thrombocythemia (ET) or primary myelofibrosis (PMF), in Philadelphia chromosome (Ph)-negative patients who tested negative for the JAK2 2 V617F 3 mutation. The requester uses a genetic analyzer (ABI 3130, Life Technologies) to accurately determine the size of insertions and deletions detected in patients who tested positive for the mutation. According to the information provided by the requester, the technique used is a NAAT with a fluorescent primer and a conventional primer. The coding sequence of exon 9 of the CALR (calreticulin 4 ) gene is fully amplified. Following amplification with a conventional thermal cycler, polymerase chain reaction (PCR) products are separated according to their size by capillary electrophoresis with a genetic analyzer. This device has single base-pair resolution, and the sensitivity is greater than 2%. This semi-quantitative analysis may be used to calculate the allelic ratio between the wild-type allele and the mutant allele. In fact, all of the mutations reported in the literature are somatic mutations, which accounts for the variability in the detected allelic ratios. The response time is 30 days and the test will be performed once a week. 2.3 Company or Developer: In-house test 2.4 Licence: Not applicable 2.5 Patent, If Any: Not applicable 1 The calreticulin gene, CALR, is located in chromosome 19p13.2 and contains 9 exons [Tefferi, 2014]. 2 JAK2: janus kinase 2, protein member of the family initially named Just Another Kinase [Olney, 2014]. 3 V617F: valine-to-phenylalanine alteration. 4 Calreticulin: endoplasmic reticulum protein that binds calcium ions. (TERMIUM Plus. Calreticulin [website] Available at: 1

3 2.6 Approval Status (Health Canada, FDA): Not applicable 2.7 Weighted Value: CLINICAL INDICATIONS, PRACTICE SETTINGS, AND TESTING PROCEDURES 3.1 Targeted Patient Group The test targets patients in whom essential thrombocythemia (ET) or myelofibrosis (MF) is suspected and who have tested negative for the mutation in JAK2 V617F. 3.2 Targeted Disease(s) According to the World Health Organization (WHO) classification of tumours of hematopoietic and lymphoid tissues, Ph-negative myeloproliferative neoplasms include polycythemia vera (PV), essential thrombocythemia (ET), and primary myelofibrosis (PMF) [Casini et al., 2013; Klampfl et al., 2013]. Polycythemia vera (PV) is a primary form of polyglobulia (hemoglobin > 185 g/l in men and > 165 g/l in women) and has an annual incidence of 2.8/100,000. It is more common in individuals of Ashkenazi Jewish origin. It can be distinguished from other polyglobulias by the increased risk of thrombotic complications associated with it. The mutation in JAK2 V617F is present in 98% of patients with the disease [Olney, 2014]. Thrombocytosis is defined as a platelet count greater than 450,000/µL 5 [Tefferi, 2014]. Essential thrombocythemia (ET) is characterized by a persistently high platelet count with a risk of thrombosis and hemorrhage. It can be asymptomatic or manifest as vasomotor (migraines, visual impairment, paresthesia, etc.), thrombotic or bleeding disturbances with severity and sequelae, depending on the location. Transformation into acute leukemia or myelodysplasia is uncommon (1.2/1,000 patient-years of follow up) [Olney, 2014] and generally of late-onset. It may occur in all age groups, but it is rare in children (median age at diagnosis 60 years to 65 years). Its prevalence in the general population is approximately 1/3,300 [Brière, 2007]. The annual incidence of ET is unknown, but it is estimated at 1.5/100,000 to 2.4/100,000 individuals. The mutation in JAK2 V617F is present in 50% to 60% of patients with the disease [Olney, 2014]. 5 Also expressed as > 450 x 10 9 /L. 2

4 One of the main diagnostic criteria for primary myelofibrosis (PMF) (WHO 2008, see criteria in the appendix) is the presence of bone marrow fibrosis with proliferation of atypical megakaryocytes (small-sized, with an abnormal nucleocytoplasmic ratio and a hyperchromatic, irregular nucleus) [Tefferi et al., 2009]. The annual incidence of PMF is estimated at 0.5 to 1.5 cases/100,000 individuals [Orphanet, 2007]. The mutation in JAK2 V617F is present in 53% of patients with the disease [Robert et al., 2008]. More than 80% of patients with Ph-negative myeloproliferative neoplasms experience fatigue, and more than half report having pruritus, bone pain and night sweats. More rarely, patients have fever and experience weight loss [Tefferi, 2014]. Approximately 40% of patients with ET or PMF do not have mutations in the JAK2 or MPL genes. However, between 60% and 88% of these patients are carriers of a mutation in the CALR gene. In 80% to 90% of cases, a 52-bp deletion or a 5-bp insertion is detected in exon 9 [Guglielmelli et al., 2014]. Tefferi [2014] reports that more than 80% of patients with myeloproliferative neoplasms have a mutually exclusive mutation in one of the three genes, JAK2, MPL or CALR. Mutations in the CALR gene do not occur in cases of PV or in the presence of JAK2 or MPL mutations. 3.3 Number of Patients Targeted Between 200 and 300 patients are targeted per year, representing approximately 10% to 20% of tests currently conducted to detect mutations in JAK2 V617F. 3.4 Medical Specialties and Other Professions Involved Molecular biology, oncology and hematology-oncology. 3.5 Testing Procedure Hematologists will prescribe the test if there is a suspicion of essential thrombocythemia or JAK2 V617F-negative myelofibrosis. The test is performed using DNA from bone marrowderived mononuclear cells or nucleated cells from total peripheral blood. 4 TECHNOLOGY BACKGROUND 4.1 Nature of the Diagnostic Technology The test is performed in conjunction with the mutational analysis of JAK2 V617F (Index code: 20967). It will be carried out if the mutation in JAK2 V617F is negative. 4.2 Brief Description of the Current Technological Context The current diagnostic algorithm provides screening for mutations in JAK2 V617F as a first step. This test allows a mutation to be detected in approximately 50% of patients with essential thrombocythemia or myelofibrosis. In the absence of this mutation, sequencing of exon 10 of the MPL gene is carried out. A mutation in this gene is detected in approximately 3% of patients [Tefferi et al., 2014c; 2014d]. 3

5 4.3 Brief Description of the Advantages Cited for the New Technology Approximately 50% of patients with ET who undergo a diagnostic test for the disease test negative for the mutation, as is also the case with 45% of patients with PMF. In 67% to 88% of cases, these patients test positive for the mutation, which will allow a definitive diagnosis [Klampfl et al., 2013]. According to the requester, the test results will not influence the choice of treatment or follow-up, but they will help establish a definitive diagnosis of myeloproliferative neoplasm and determine the proper course of treatment. Some patients currently have a diagnosis of exclusion that requires expensive screening. For some of these patients, this diagnostic uncertainty hinders therapeutic decisions (e.g., whether or not to proceed to chemotherapy). Moreover, tests for the CALR gene will reduce the number of tests for the mutation in MPL515 (uncommon) with the use of a more expensive method (sequencing) for patients who tested negative for the mutation in JAK2 V617F. Tests for mutations in the MPL515 gene will be conducted in a small number of patients, since most patients who tested negative for the JAK2 V617F gene will test positive for the CALR gene. The requester added that a local preliminary study indicated that it would be very easy to implement the diagnostic algorithm in its laboratory. 4.4 Cost of Technology and Options: Not assessed. 5 EVIDENCE 5.1 Clinical Relevance Other Tests Replaced: None Diagnostic or Prognostic Value As a result of the link between CALR gene mutations on the one hand, and ET and PMF in the absence JAK2 and MPL mutations on the other, CALR genotyping is a test indicated in patients in whom a myeloproliferative neoplasm is suspected and who tested negative for mutations in JAK2 and MPL [Guglielmelli et al., 2014]. Patients with a mutation in CALR or who are triple-negative obtained a significantly lower IPSET 6 score (p = 0.04) than those with ET who carry a mutation in JAK2 [Tefferi et al., 2014c]. Mortality Two studies assessed the risk of death associated with the presence of mutations in CALR, JAK2, and MPL in patients with PMF [Tefferi et al., 2014a] or ET and PMF [Klampfl et al., 2013]. The risk is 1.7 to 3.6 times higher in patients with a mutation in JAK2 or MPL or who are triple negative (JAK2, MPL, and CALR) than it is in patients with a mutation in the CALR gene (Table 1). 6 IPSET score: International Prognostic Score for Essential Thrombocytopenia. 4

6 Table 1: Mortality risk associated with the presence of various mutations in patients with ET or PMF STUDY NUMBER OF PATIENTS AND DIAGNOSIS RISK OF DEATH Comparison HR (95% CI) p Tefferi et al., 2014a 254 PMF Triple negatives* vs. CALR ( ) < JAK2 + vs. CALR ( ) < Klampfl et al., , ET and 321 PMF MPL + vs. CALR ( ) n. s. JAK2 + vs. CALR ( ) < MPL + vs. CALR ( ) < Abbreviations: CI = confidence interval; ET = essential thrombocythemia; HR = hazard ratio or instantaneous risk ratio; MPN = myeloproliferative neoplasm; n. s. = not a significant difference; p = statistical significance; PMF = primary myelofibrosis; PV = polycythemia vera; vs. = versus. * No mutations in JAK2, CALR, and MPL genes. When adjusted for age, the values of p are , 0.01 and 0.22, respectively. Tefferi et al. recently published a retrospective study of 299 patients who were diagnosed with ET between 1956 and 2005 at the Mayo Clinic, in the United States [Tefferi et al., 2014c]. The median follow-up was 12.7 years, and 47% of patients were monitored until death. Mutation rates were 53% (n = 159) for the JAK2 gene mutation, 32% (n = 95) for CALR mutation and 3% (n = 8) for MPL mutation. Thirty-seven patients (12%) were triple negative. Among carriers of the mutations in JAK2 and CALR, 42 (44%) and 82 (52%), respectively, died. Survival The median survival appears to be increased in patients with a mutation in the CALR gene than in those with other mutations or in those who are triple negative (Table 2). Table 2: Median and 10-year survival by type of mutation STUDY NUMBER OF PATIENTS AND DIAGNOSIS MUTATION MEDIAN SURVIVAL TIME (95% CI) OVERALL SURVIVAL AT 10 YEARS % (95% CI) Rumi et al., 2014a 127 MPN CALR + n. a. ET: 100 PMF: 100 JAK2 + n. a. ET: 96.8 ( ) PMF: 91.7 ( ) None n. a. ET: 100 PMF: 0 Tefferi et al., 2014c 299 ET CALR + 20 years* n. a. JAK years* n. a. MPL + 9 years n. a. Triple negatives n. a. n. a. Tefferi et al., 254 PMF CALR years n. a. 5

7 STUDY NUMBER OF PATIENTS AND DIAGNOSIS MUTATION MEDIAN SURVIVAL TIME (95% CI) OVERALL SURVIVAL AT 10 YEARS % (95% CI) 2014a JAK years n. a. MPL years n. a. Triple negatives 2.5 years n. a. Klampfl et al., , ET and 321 PMF CALR years ( ) JAK2 V617F years ( ) ET: 96.9 ( ) ET: 91.1 ( ) MLP years (2.0 - n.a.)* Abbreviations: DNA= deoxyribonucleic acid; ET = essential thrombocythemia; HR = hazard ratio or instantaneous risk ratio; MPN = myeloproliferative neoplasm; n.a. = not available; PMF = primary myelofibrosis. * Median follow-up: 12.7 years. Level of significance of the difference between the groups who tested positive for the mutations in genes CALR and JAK2: p = The difference also was not significant in the comparison with the group of individuals aged 65 years and under. Level of significance of the difference with the group having a mutation in the CALR gene: p < Level of significance of the difference with the group having a mutation in the CALR gene: p = However, a recent study of patients with ET showed that there is no difference in median survival between patients with ET who are carriers of a CALR mutation and those who are carriers of a JAK2 mutation. The mean duration of follow-up in this study was 12.7 years. The longest median survival time was 20 years in patients with a CALR gene mutation and was 19 years in patients with a JAK2 gene mutation. Patients with a mutation in the MPL gene have the shortest survival time [Tefferi et al., 2014c]. Survival time is also shorter in individuals with PMF. Rumi et al. [2014b] observed the overall survival after 15 years in 745 patients with ET: it was 95.3% in patients who tested positive for the CALR mutation and 90.4% in those with a JAK2 V617F gene mutation. The difference was not significant (p = 0.085) even after correcting for age. Morbidity Four studies assessed the cumulative incidence of thrombosis at 5 years, 10 years, and 15 years in patients with ET (Table 3). It was observed that the incidence of thrombosis is lower in the presence of a CALR gene mutation than it is in the presence of a JAK2 mutation. Tefferi et al. [2014c] also noted a lower rate of recurrent thrombosis in patients with ET who have CALR gene mutations (p = 0.04) and who are triple negative (p = 0.02) than in carriers of a JAK2 gene mutation. 6

8 Table 3: Cumulative incidence of thrombosis in patients with ET STUDY NUMBER OF PATIENTS MUTATION CUMULATIVE INCIDENCE OF THROMBOSIS % (95% CI) AT 5 YEARS AT 10 YEARS AT 15 YEARS Rumi et al., 2014a 127 MPN CALR + n.a. 0 n.a. JAK2 + n.a (4-27) n.a. None n.a. 0 n.a. Rumi et al., 2014b Rotunno et al., CALR + n.a. n.a JAK2 + n.a. n.a CALR + n.a ( ) n.a. JAK2 + n.a ( ) n.a. MPL + n.a ( ) n.a. Triple negatives n.a. 8.2 ( )* n.a. Klampfl et al., CALR ( ) 11 ( ) 12.8 (7.3-20) JAK2 V617F + 13 ( ) 21 ( ) 27.1 ( ) MLP ( ) 9.3 ( ) 17.6 ( ) Abbreviations: CI = confidence interval; ET = essential thrombocythemia; HR = hazard ratio or instantaneous risk ratio; MPN = myeloproliferative neoplasm; n.a. = not available. * If this group of patients (no mutations) were used as a reference group, the risk of thrombosis would be: HR: 0.74 (95% CI, 0.33 to 1.00) in the presence of a CALR mutation; 1.78 (95% CI: 1.06 to 3.18) in the presence of a JAK2 mutation, and 1.65 (1.7 to 3.92) in the presence of a MPL mutation. Significant difference: p = Significant difference: p = 0.001; the difference remains significant after age adjustment. Progression of the disease The progression of ET at 10 years or 15 years to leukemia or polycythemia is more common in patients who have a mutation in the JAK2 V617F gene. Myelofibrotic transformation is more common in patients with a CALR gene mutation, but the difference is not significant (Table 4). 7

9 Table 4: Cumulative incidence of disease progression in patients with ET, by type of mutation STUDY FOLLOW- UP TYPE OF PROGRESSION CALR + JAK2 + P % (95% CI) Rumi et al., 2014a 10 years Unspecified ( ) Rumi et al., 2014b 15 years Myelofibrotic 13.4 ( ) 8.4 ( ) n. s. Leukemic 2.5 ( ) 4.3 ( ) 0.026* Polycythemic ( ) n.a. Tefferi et al., 2014c 12.7 years Myelofibrotic Leukemic Polycythemic 0 5 Abbreviations: CI = confidence interval; n.a. = not available; n.s. = not significant; p = level of statistical significance. * No significant difference after age adjustment Therapeutic Value This test is not associated with a targeted treatment. 5.2 Clinical Validity Sensitivity Specificity Positive predictive value (PPV) Negative predictive value (NPV) Likelihood ratio (LR) ROC curve Accuracy Prevalence COMPONENT PRESENCE ABSENCE NOT APPLICABLE The presence of mutations in genes JAK2, MPL and CALR is very common in patients with myelodysplastic neoplasm (ET or PMF). Overall, fewer than 10% of these patients do not have a mutation in any of these three genes [Klampfl et al., 2013]. Moreover, exome sequencing in 151 patients with MPN found that 146 (97%) had mutually exclusive (q < 0,01 7 ) mutations in the JAK2, MPL or CALR genes [Nangalia et al., 2013]. Klampfl et al. [2013] found a mutation in the CALR gene in 73% of patients who do not have a mutation in JAK2 or MPL. Nangalia et al. [2013] showed that the JAK2 V617F mutation X X 7 The q value is the result of a p value (level of significance) adjustment and represents the proportion of false-positives (error or false discovery rate) [Storey, 2003]. 8

10 occurred in all 48 patients with PV (100%), 35 of the 62 patients with ET (56%), and 27 of the 39 patients with MF (69%). Table 5: Rate of CALR gene mutations in patients with or without JAK2 and MPL gene mutations STUDY DIAGNOSTIC TECHNOLOGY DIAGNOSIS JAK2 AND MPL MUTATIONS CALR GENE MUTATIONS Rotunno et al., 2014 Real-time PCR (for JAK2 and MPL) and high-resolution melting analysis followed by bidirectional Sanger sequencing for MPL. Bidirectional sequencing for CALR mutations in exon % ET 394 0/ / Tefferi et al., 2014a PCR PMF 147 1/ /85 74 Klampfl et al., 2013 High resolution sizing of fluorescent dye-labelled PCR products Confirmation using the conventional Sanger method PV None ET / PMF / Nangalia et al., 2013 Whole exome sequencing PV 0/48 ET 40 0/ /22 82 PMF 24 0/24 9 8/ Sanger method* PV 0/217 ET 138 0/ / PMF 115 0/ /46 65 Abbreviations: AML = acute myeloid leukemia; CML = chronic myeloid leukemia; CMML = chronic myelomonocytic leukemia; ET = essential thrombocythemia; MPN = myeloproliferative neoplasm; PCR = polymerase chain reaction; PMF = primary myelofibrosis; PV = polycythemia vera. * No CALR gene mutations were identified in 389 patients with other myeloid neoplasms, 502 patients with solid tumours, 287 patients with lymphoid neoplasms, or 550 healthy controls. The results of these four studies confirm the absence of mutations in the CALR gene in patients with PV and in those who do not have mutations in JAK2 and MPL. Only one study identified a case that tested positive for CALR in a patient with PMF who also had a mutation in JAK2 [Tefferi et al., 2014b]. Tests for CALR gene mutations were positive in 50% to 89% of patients with no JAK2 and MPL gene mutations. 9

11 5.3 Analytical (or Technical) Validity Repeatability Reproducibility Analytical sensitivity Analytical specificity Matrix effect Concordance COMPONENT PRESENCE ABSENCE NOT APPLICABLE Correlation between test and comparator The requester provided the results of a technical validation study to ensure result reliability and safety. For each test, internal quality controls (negative and positive) were included, and the results were to be validated twice. Lastly, the requester intends to implement external quality controls. Each of the parameters for the validation of the technology is described in the following paragraphs. 1) Validation study for the analytical technique using fragment length A validation study was conducted first to ensure the reliability of the technique. During the study, 124 patient samples previously tested and characterized as negative for the JAK2 V617F mutation were tested according to fragment length to detect the presence or absence of mutations. In total, 18 patients tested positive. Of the 18 patients who tested positive, 11 were analyzed using Sanger sequencing. Sequencing for each of these patients confirmed the presence of mutations (7 deletions and 4 insertions) in 100% of cases. Dilution tests were carried out to assess the sensitivity of the technique. In order to do this, the DNA of two patients who tested positive was compared with the DNA of a healthy patient. Results show a very high sensitivity of up to 1% when the mutation is detected beforehand. Consequently, the sensitivity threshold was raised slightly to a conservative level of 2%. The validation study allowed the results to be confirmed using fragment length analysis. As well, the requester was able to establish the technique's sensitivity threshold at 2%, identical to that established in the same laboratory for the JAK2 V617F gene test. X X 2) Validation of the results Each result is validated by two people; that is, there is a technical validation and a clinical or medical validation. 10

12 3) Implementing external quality controls The requester's laboratory is the first in Canada to offer this test. However, in the meantime, quality control will be carried out with Dr. Ross L. Levine s group from Memorial Sloan Kettering Cancer Center (MSKCC) in New York. They will exchange samples every six months. 5.4 Recommendations from Other Organizations Tefferi et al. [2014b] published a literature review focusing on CALR and CSF3R gene mutations and their potential role in improving the classification and diagnostic criteria for ET, PMF, and chronic myeloid leukemia (CML). Based on the literature reviewed, they propose a modification of the World Health Organization's 2008 diagnostic criteria for myeloproliferative neoplasm. These modifications would include the presence of mutations in the CALR and MPL genes as major criteria for the diagnosis of ET and PMF, on the same basis as the presence of JAK2 gene mutations in the current criteria [Tefferi et al., 2014b]. 6 ANTICIPATED OUTCOMES OF INTRODUCING THE TEST 6.1 Impact on Material and Human Resources: Not assessed. 6.2 Economic Consequences of Introducing Test Into Quebec's Health Care and Social Services System: Not assessed. 6.3 Main Organizational, Ethical, and Other (Social, Legal, Political) Issues: Not assessed. 7 IN BRIEF 7.1 Clinical Relevance This new test is useful to confirm the diagnosis and establish a prognosis for survival, the risk of thrombosis, and progression of the disease, especially in patients with ET and in conjunction with testing for JAK2 V617F gene mutations. 7.2 Clinical Validity The link between the frequency of CALR gene mutations in patients who tested negative for JAK2 and the various diagnoses has been shown in a number of studies. 7.3 Analytical Validity The clinical relevance of CALR gene mutation tests is recent. No technical validation studies have been published to date. However, the requester conducted a local study that confirmed the performance of the technique. External quality control is anticipated as well. 7.4 Recommendations from Other Organizations Recommendations from official organizations are not yet available, but a group of experts has proposed modifying current recommendations to include testing for CALR gene mutations in the diagnostic algorithm for ET and PMF, on the same basis as JAK2 gene mutation testing. 11

13 8 INESSS NOTICE IN BRIEF Mutational Analysis of Exon 9 of the CALR Gene Status of the Diagnostic Technology Established Innovative Experimental (for research purposes only) Replacement for technology which becomes obsolete INESSS Recommendation Inclusion of test in the Index conditional upon completion of the method's validation Do not include test in the Index Reassess test Additional Recommendation Draw connection with listing of drugs, if companion test Produce an optimal use manual Identify indicators, when monitoring is required 12

14 REFERENCES Brière J. Essential thrombocythemia [site Web]. Orphanet; Available at: (consulté le 16 avril 2014). Casini A, Boehlen F, Lecompte T, de Moerloose P. Risque thrombotique en cas de néoplasies myéloprolifératives : ce que devrait savoir le médecin praticien. Rev Med Suisse 2013;9(372):315-8, 320. Guglielmelli P, Nangalia J, Green AR, Vannucchi AM. CALR mutations in myeloproliferative neoplasms: Hidden behind the reticulum. Am J Hematol 2014; 89(5): 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(25): 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(25): Olney HJ. Les néoplasies myéloprolifératives. De la thrombocytose essentielle à la polycythémie vraie. Le Médecin du Québec 2014;49(3):31-6. Orphanet. Splénomégalie myéloïde [site Web] Available at: (consulté le 16 avril 2014). Robert DE, Stalder M, Lovey PY, Hutter P. Un test décisif pour le diagnostic des syndromes myéloprolifératifs : la recherche de la mutation V617F de la JAK2. Caduceus Express 2008;10(1). Rotunno G, Mannarelli C, Guglielmelli P, Pacilli A, Pancrazzi A, Pieri L, et al. Impact of calreticulin mutations on clinical and hematological phenotype and outcome in essential thrombocythemia. Blood 2014;123(10): Rumi E, Harutyunyan AS, Pietra D, Milosevic JD, Casetti IC, Bellini M, et al. CALR exon 9 mutations are somatically acquired events in familial cases of essential thrombocythemia or primary myelofibrosis. Blood 2014a;123(15): 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 2014b;123(10): Storey JD. The positive false discovery rate: A Bayesian interpretation and the q-value. Ann Statist 2003;31(6): Tefferi A. Overview of the myeloproliferative neoplasms. Waltham, MA : Wolters Kluwer Health; Available at: Tefferi A, Lasho TL, Finke CM, Knudson RA, Ketterling R, Hanson CH, et al. CALR vs JAK2 vs MPLmutated or triple-negative myelofibrosis: Clinical, cytogenetic and molecular comparisons. Leukemia 2014a;28(7):

15 Tefferi A, Thiele J, Vannucchi AM, Barbui T. An overview on CALR and CSF3R mutations and a proposal for revision of WHO diagnostic criteria for myeloproliferative neoplasms. Leukemia 2014b;28(7): Tefferi A, Wassie EA, Lasho TL, Finke C, Belachew AA, Ketterling RP, et al. Calreticulin mutations and long-term survival in essential thrombocythemia. Leukemia 2014c; [Epub ahead of print]. Tefferi A, Wassie EA, Guglielmelli P, Gangat N, Belachew AA, Lasho TL, et al. Type 1 vs Type 2 calreticulin mutations in essential thrombocythemia: A collaborative study of 1027 patients. Am J Hematol 2014d;89(8):E Tefferi A, Thiele J, Vardiman JW. The 2008 World Health Organization classification system for myeloproliferative neoplasms: Order out of chaos. Cancer 2009;115(17):

16 APPENDIX 2008 WHO Diagnostic Criteria for Myeloproliferative Neoplasms (from Tefferi et al., 2009) MAJOR DIAGNOSTIC CRITERIA 1 MINOR DIAGNOSTIC CRITERIA 1 PV* ET PMF Hb > 185 g/l (men) Hb > 165 g/l (women) 2 Presence of a JAK2 V617F gene mutation, or a similar mutation Medullary myeloproliferation for the three cell lines 2 Serum erythropoietin levels below normal values 3 Endogenous erythroid colony growth 1 Platelet count 450 x 10 9 /L 2 Megakaryocyte proliferation with large and mature morphology. Little or no proliferation of granulocytes or erythrocytes. 3 Lack of WHO diagnostic criteria for CML, PV, PMF, MDS or other MN. 4 Presence of the JAK2 V617F gene mutation or other clonal marker or lack of arguments to support a diagnosis of reactive thrombocytosis. 1 Proliferation of atypical megakaryocytes ǁ with reticulin or collagen fibrosis. In the absence of reticulin fibrosis, proliferation must be accompanied by medullary hypercellularity, a proliferation of granulocytes and, often, reduced erythropoiesis. 2 Lack of WHO diagnostic criteria for CML, PV, MDS or other MN 3 Presence of the JAK2 V617F gene mutation or other clonal marker or lack of arguments to support a diagnosis of reactive myelofibrosis 1 Leukoerythroblastosis 2 Increase in serum LDH 3 Anemia 4 Palpable splenomegaly Abbreviations: CML = chronic myeloid leukemia; ET = essential thrombocythemia; Hb = hemoglobin; Hct = hematocrit; LDH = lactate dehydrogenase; MDS = myelodysplastic syndrome; MN = myeloid neoplasm; PMF = primary myelofibrosis; PV = polycythemia vera; WHO = World Health Organization. * The diagnosis of PV requires two major criteria and one minor criterion or the first major criterion together with two minor criteria. Or Hb > 170 g/l (men) or Hb > 150 g/l (women) if associated with a sustained increase of 20 g/l from the baseline that cannot be attributed to the correction of an iron deficiency; or Hb or Hct above the 99th percentile of the reference range for age, sex, or altitude of residence; or erythrocyte volume > 25% above the mean normal predicted value. The diagnosis of ET requires the four major criteria. The diagnosis of PMF requires three major criteria and two minor criteria. ǁ Small megakaryocytes with an abnormal nucleocytoplasmic ratio, an irregular, hyperchromatic nucleus and dense clustering. 15