[COMPREHENSIVE GENETIC ASSAY PANEL ON

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1 2014 SN GENELAB AND RESEARCH CENTER DR. SALIL VANIAWALA, PH.D [COMPREHENSIVE GENETIC ASSAY PANEL ON MYELOPROLIFERATIVE NEOPLASMS] SN Genelab presents one of the most comprehensive genetic assay panel for myeloproliferative neoplasms ranging from BCR-ABL to CALR mutation analysis. `1

2 The myeloproliferative neoplasms (MPNs), previously myeloproliferative diseases (MPDs), are a group of diseases of the bone marrow in which excess cells are produced. There are four main myeloproliferative diseases, which can be further categorized by the presence of the chromosome. Leukemia Myeloproliferative neoplasms chromosome-based clasification chromosome positive (Chronic myeloid leukemia or CML) chromosome negative chromosome negative blood disorders Essential thrombocytosis or ET Polycythemia vera or PV Myelofibrosis or MF Chronic myeloid leukemia- The science This myeloproliferative disorder is characterized by parts of two chromosomes (the 9th and 22nd) switch places. As a result, part of the BCR ("breakpoint cluster region") gene from chromosome 22 is fused with the ABL gene on chromosome 9. This abnormal "fusion" gene generates a protein of p210 or sometimes p185 weight. Because abl carries a domain that can add phosphate groups to tyrosine residues (a tyrosine kinase), the bcr-abl fusion gene product is also a tyrosine kinase. This tyrosine kinase is continuously active and does not require activation by other cellular messaging proteins. This results in activation of a cascade of proteins that control the cell cycle, speeding up cell division and therefore, oncologic events. 2

3 With defining translocation t(9;22) BCR-ABL translocation which has three breakpoints: 1. µ-bcr-abl (p230) that leads to CML with usual neutrophilia and basophilia. 2. Minor-BCR-ABL (p190) that leads to CML which has a tendency to become acute lymphoblastic leukemia (ALL) 3. Major-BCR-ABL (p210) which is the normal and usual breakpoint. Drug resistance Imatinib is an effective drug to treat CML patients. The major mechanism of imatinib resistance for patients with chronic myeloid leukemia (CML) is clonal expansion of leukemic cells with mutations in the Bcr-Abl fusion tyrosine kinase that reduce the capacity of imatinib to inhibit kinase activity. The early detection of such mutations may allow timely treatment intervention to prevent or overcome resistance. Direct sequencing of the BCR-ABL kinase domain is relatively rapid and allows detection of emerging mutations at a sensitivity of approx 20%. The genetic tests available at SN GeneLab FISH Drug resistance BCR ABL Real Time PCR to detect breakpoints Minimal residual disease 3

4 JAK 2 AND MPL gene mutations- The science Jak2 gene mutations Diagnostic criteria for ET, MF, and PV adopted by the World Health Organization (WHO) include identification of a clonal marker, with a specific recommendation to test for the JAK2 V617F mutation in exon 14. JAK2V617F is a gain-of-function mutation that leads to clonal proliferation; it is present in about 95% of PV cases and about half of ET and MF cases. The JAK2 allele burden decreases with successful therapy, disappears in some patients, and reappears during relapse. Thus, quantitative JAK2 analysis may be useful for diagnosis and patient management. In JAK2 V617F-negative patients, the presence of a JAK2 exon 12 mutation also meets the WHO criterion for establishing clonality. 4

5 MPL gene mutations MPL gene mutation Because a significant proportion of patients with ET or MF are negative for JAK2 mutations, other somatic mutations were sought. Hence mutations in the myeloproliferative leukemia gene (MPL) were identified. MPL gene mutation MPL, found at chromosome 1p34, encodes the thrombopoietin receptor that works in concert with thrombopoietin for platelet production. Acquired MPL mutations (eg, W515L and W515K) are associated with severe anemia and have been detected in patients with ET or MF but not in patients with PV. Prevalence of Somatic Mutations in PV, ET, and MF JAK2 V617F Mutation,% JAK2 Exon 12 Mutation,% MPL W515 Mutation,% Polycythemia vera (PV) Essential thrombocythemia (ET) Myelofibrosis (MF) The genetic tests available at SN GeneLab: Jak2 PCR and gene sequencing analysis negative myeloproliferative disorders MPL PCR and gene sequencing analysis 5

6 CALR Mutation Analysis New Calreticulin molecular testing aids diagnosis and classification of MPNs by revealing mutations in the majority of JAK2 and MPL mutation-negative patients. THE SCIENCE: Somatic mutations in the calreticulin gene CALR are detected in peripheral blood in the ~65-85% of essential thrombocythemia (ET) and primary myelofibrosis (PMF) patients that are JAK2- and MPL-mutation negative. Molecular analysis of these three genes now allows these disease markers to be identified in >90% of MPN patients, helping to classify the disease and differentiate it from a reactive process. CALR mutation testing also has prognostic value as CALR mutations are associated with longer survival and fewer thrombotic events compared to JAK2-mutations. The quantitative reporting of CALR mutation levels in positive patients allows repeat testing to serve as a monitor of therapy efficacy and predictor of progression. 6

7 CALR in Primary Myelofibrosis (PMF) CALR mutations were detected in 88% PMF patients without JAK2 and MPL mutations. PMF patients with CALR mutations are reported to have longer overall survival than those with JAK2 and MPL mutations. CALR in Polycythemia Vera (PV) and other hematologic disorders CALR mutations are not reported in PV patients. In two studies with 430 total PV patients, no mutations were found.testing can therefore be used to distinguish PV patients from those with ET or PMF. Mutations are reported in 8% of patients with MDS, rarely found in CMML or atypical CML, and not reported in lymphoid leukemia or solid tumors. Clinical significance: CALR mutation analysis aids diagnostic confirmation of -chromosome negative and JAK2/MPLmutation negative MPN. CALR mutations are mutually exclusive with JAK2 and MPL mutations, and are detected in peripheral blood in the majority (~70-85%) of essential thrombocythemia (ET) and primary myelofibrosis (PMF) cases that are JAK2- and MPL-mutation negative. CALR mutations are not reported in polycythemia vera (PV) and can distinguish ET and PMF from PV. Presence of CALR mutations is also associated with a better clinical course than JAK2 mutations. The genetic tests available at SN GeneLab CALR gene analysis Bi-directional sequencing of exon 9 of the CALR (calreticulin) gene 7

8 TESTING CASCADE: SNGENE TEST AVAILABLE AT ALL POINTS BCR ABL SN GeneLab - FISH Real time PCR Imatinib drug resistance by PCR sequencing Positive Negative No further testing Jak2 V617F mutation SN Genelab - PCR-Sequencing assay Positive Negative No further testing Jak2 exon 12 & 13 mutation SN Genelab - PCR-Sequencing assay Positive Negative No further testing required Positive MPL mutation Negative SN Genelab - PCR- Sequencing assay No further testing CALR mutation NEW SN Genelab - PCR-Sequencing 8

9 For further information, contact us at: SN GeneLab (P) Limited 2nd Floor, President Plaza-A, Near RTO Circle, Nanpura, Ring Road Surat, GJ , INDIA Phone: