TP53 Mutation Is Rare in Primary Myelofibrosis by High Resolution Melting Curve Analysis and Sanger Sequencing
Wesley O Greaves, Shalini Verma, Tigist Bisrat, Hamed Rahimi, Abhaya Paladugu, Hui Yao, Keyur P Patel, Rajyalakshmi Luthra, L Jeffrey Medeiros, Srdan Verstovsek, Carlos E Bueso-Ramos. The University of Texas MD Anderson Cancer Center, Houston, TX
Background: TP53 is the most frequently mutated gene in human cancers and is usually associated with an aggressive disease course. TP53 mutation has been described in a variety of hematopoietic neoplasms, and has been suggested to play a role in leukemic transformation of myeloproliferative neoplasms (MPNs). However, the frequency and clinicopathologic implications of TP53 mutation in primary myelofibrosis (PMF), a common type of MPN, have not been systematically assessed. In this study, we interrogated a large series of patients with PMF for TP53 mutations using two distinct molecular methods.
Design: We assessed archival bone marrow DNA samples from patients with PMF at our institution. Diagnosis was based on morphologic, immunophenotypic, cytogenetic and molecular evaluation of peripheral blood and bone marrow in conjunction with clinical data. DNA samples were assessed for sequence variation in exons 4 through 9 of TP53 by both high resolution melting curve (HRM) analysis using LightCycler® 480 System (Roche, Indiana IN) and bidirectional Sanger sequencing using 3730XL DNA Analyzer (Life Technologies, Carlsbad CA). All patients were previously assessed and negative for BCR-ABL1 translocation.
Results: We identified 50 patients with PMF and available DNA. Twenty-nine (58%) patients showed JAK2 p.V617F mutation. Four patients showed elevated blast counts ≥ 10% (10%-14% blasts). By Sanger sequencing, only 1 (2%) case showed an amino acid-altering mutation in TP53: c.707A>G (TAC to TGC) in codon 236 (p.Y236C) of exon 7. In addition, 8 cases showed silent mutations/single nucleotide polymorphisms of unknown significance - c.36G>A (CCG to CCA) in exon 4 (n=3) and c.213A>G (CGA to CGG) in exon 6 (n=6). The p.R72P polymorphism in exon 4 which has been described in other hematopoietic neoplasms was present in 1 patient. All cases with a mutant sequence by Sanger sequencing also showed a variant melting curve pattern by HRM analysis. The mean overall survival was 5.7 years. Five patients developed acute leukemia, all of whom died of disease. Wild type TP53 was identified by Sanger sequencing in 3 of 5 available acute leukemia specimens. The patient with TP53 mutation died 2 years after presentation from progressive PMF without developing acute leukemia.
Conclusions: TP53 mutation is rare in PMF and therefore gene mutation does not seem to play a significant role in the pathogenesis of PMF or transformation to AML. HRM is a convenient approach to screen for p53 mutations.
Wednesday, March 21, 2012 9:30 AM
Poster Session V # 224, Wednesday Morning