Karl Haslam

Molecular diagnostics of myeloproliferative neoplasms.

Since the discovery of the JAK2 V617F mutation in the majority of the myeloproliferative neoplasms (MPN) of polycythemia vera, essential thrombocythemia and primary myelofibrosis ten years ago, further MPN‐specific mutational events, notably in JAK2 exon 12, MPL exon 10 and CALR exon 9 have been identified. These discoveries have been rapidly incorporated into evolving molecular diagnostic algorithms. Whilst many of these mutations appear to have prognostic implications, establishing MPN diagnosis is of immediate clinical importance with selection, implementation and the continual evaluation of the appropriate laboratory methodology to achieve this diagnosis similarly vital. The advantages and limitations of these approaches in identifying and quantitating the common MPN‐associated mutations are considered herein with particular regard to their clinical utility. The evolution of molecular diagnostic applications and platforms has occurred in parallel with the discovery of MPN‐associated mutations, and it therefore appears likely that emerging technologies such as next‐generation sequencing and digital PCR will in the future play an increasing role in the molecular diagnosis of MPN.

Incidence of CALR mutations in patients with splanchnic vein thrombosis

kemia. The Journal of Experimental Medicine, 194, 1639–1647. Rossi, F.M., Del Principe, M.I., Rossi, D., Irno Consalvo, M., Luciano, F., Zucchetto, A., Bulian, P., Bomben, R., Dal Bo, M., Fangazio, M., Benedetti, D., Degan, M., Gaidano, G., Del Poeta, G. & Gattei, V. (2010) Prognostic impact of ZAP-70 expression in chronic lymphocytic leukemia: mean fluorescence intensity T/B ratio versus percentage of positive cells. Journal of Translational Medicine, 8, 8– 23. Sloan-Lancaster, J., Zhang, W., Presley, J., Williams, B.L., Abraham, R.T., Lippincott-Schwartz, J. & Samelson, L.E. (1997) Regulation of ZAP-70 Intracellular Localization: visualization with the Green Fluorescent Protein. The Journal of Experimental Medicine, 186, 1713– 1724. Tripepi, G., Heinze, G., Jager, K.J., Stel, V.S., Dekker, F.W. & Zoccali, C. (2013) Risk prediction models. Nephrology Dialysis Transplantation, 28, 1975–1980. Vroblova, V., Vrbacky, F., Hrudkova, M., Jankovicova, K., Schmitzova, D., Maly, J., Krejsek, J. & Smolej, L. (2010) Significant change in ZAP-70 expression during the course of chronic lymphocytic leukemia. European Journal of Haematology, 84, 513–517.

Assessment of CALR mutations in myelofibrosis patients, post-allogeneic stem cell transplantation

Study Coordinator, Principal Investigator; Baylor College of Medicine: Bogden Dino, Study Coordinator; Weill Medical College of Cornell University: Dorothy Kleinert, RN, Research Nurse, Patricia Giardina, MD; The Children’s Hospital of Philadelphia: Alan Cohen, MD, Janet Kwiatkowski, MD, Marie Martin, RN, Research Nurse, Principal Investigator, Sage Green, Study Coordinator; Children’s Memorial Hospital, Chicago, IL: Alexis Thompson, MD, Janice Beatty, RN, Research Nurse, Diane Calamaras, RN, CPNP, Research Nurse, Pauline Hess, Study Coordinator; Children’s Hospital & Research Center Oakland: Dru Haines, CPNP, Research Nurse, Principal Investigator, Olivia Oliveros, Study Coordinator, Elliott Vichinsky, MD; Children’s Hospital of Los Angeles: Thomas Coates, MD, Principal Investigator, Susan Carson, RN, Research Nurse, Principal Investigator, Ani Dongelyan, Study Coordinator, Tatiana Hernandez, Study Coordinator; Toronto General Hospital, Toronto, Ontario, Canada: Nancy Oliveri, MD, Cecilia Kim, BS, Study Coordinator; NHLBI oversight: Kathryn Hassell, MD; Data Coordinating Center: New England Research Institutes: Sonja McKinlay, PhD, Principal Investigator, Lisa Virzi, RN, MS, MBA, Project Director, Felicia Trachtenberg, PhD, Senior Statistician, Eric Gerstenberger, MS, Statistician.

CALR mutations are rare in childhood essential thrombocythemia

To the Editor: The incidence of childhood myeloproliferative neoplasms is significantly lower than that in adults. Also, the common JAK2 V617F mutation and other MPN-associated mutations are detected less frequently [1] inferring differences in both the etiology and molecular pathogenesis of these diseases in adults and children. Two recent reports have identified acquired insertion/deletion mutations of CALR, exclusively in exon 9, in approximately 70–80% of adult essential thrombocythemia (ET) and primary myelofibrosis (PMF) that do not possess JAK2 orMPL mutations by exome sequencing approaches. Furthermore they have demonstrated a previously unrecognized molecular complexity to these malignancies [2,3]. These two studies prompted the investigation of CALR mutations in pediatric MPN. The presence of CALR exon 9 mutations was retrospectively investigated using fluorescent PCR fragment analysis [2] in the blood or bone marrow DNA from a relatively small single-center cohort of unrelated, sporadic pediatric MPN classified according to established hematological, histo-morphological and molecular criteria [4]. This comprised of JAK2 V617F-negative ET (n1⁄4 4), JAK2 V617F-positive ET (n1⁄4 2) (Table I), JAK2 V617F-positive polycythemia vera (PV; n1⁄4 1), JAK2 V617F-negative polycythemia (n1⁄4 2), and reactive thrombocytosis (n1⁄4 8). All MPN patients had noMPLW515L or W515Kmutations. Two different mutations found in adult MPN acted as positive controls. CALR mutations were not detected in the four JAK2 V617F-negative ET patients. Also, no CALR mutations were detected in three JAK2 V617Fpositive MPN patients or in the eight children with reactive thrombocytosis (Table I). While this brief study demonstrates absence of CALR exon 9 mutations in JAK2V617F-positive childhood MPN, no CALR exon 9 mutations were found in the four JAK2 V617F-negative children with ET. If the molecular pathogenesis of pediatric and adult MPN was similar then one might expect to detect CALR mutations in a certain proportion of young JAK2and MPL-negative ET patients. Whether CALR mutations are present and will have a role in the diagnosis and classification of childhoodMPN requires verification in a larger cohort. This study serves to highlight another potential biological difference between childhood and adult MPN.

Allogeneic Hematopoietic Stem Cell Transplantation for a BCR-FGFR1 Myeloproliferative Neoplasm Presenting as Acute Lymphoblastic Leukemia

Hematopoietic myeloproliferative neoplasms (MPNS) with rearrangements of the receptor tyrosine kinase FGFR1 gene, located on chromosome 8p11, are uncommon and associated with diverse presentations such as atypical chronic myeloid leukemia, acute myeloid leukemia, or an acute T- or B-lymphoblastic leukemia, reflecting the hematopoietic stem cell origin of the disease. A review of MPN patients with the t(8;22) translocation that results in a chimeric BCR-FGFR1 fusion gene reveals that this disease either presents or rapidly transforms into an acute leukemia that is generally unresponsive to currently available chemotherapeutic regimens including tyrosine kinase inhibitors (TKIS). The first case of a rare BCR-FGFR1 MPN presenting in a B-acute lymphoblastic phase who underwent allogeneic hematopoietic stem cell transplantation (HSCT) with a subsequent sustained complete molecular remission is described. Allogeneic HSCT is currently the only available therapy capable of achieving long-term remission in BCR-FGFR1 MPN patients.

The CSF3R T618I mutation as a disease-specific marker of atypical CML post allo-SCT.

Atypical CML (aCML) is a rare myelodysplastic/myeloproliferative neoplasm (MDS/MPN) characterized by neutrophilia with left-shift maturation and dysgranulopoietic features. Diagnosis also requires the exclusion of BCR-ABL1, PDGFRA and PDFRB rearrangements. The prognosis of aCML is poor with survival times of generally o30 months: patients may undergo transformation to AML or succumb to the effects of BM failure. To date, allo-SCT in the chronic phase of the disease has been the only option to improve OS. No specific, recurrent cytogenetic or molecular abnormalities have been described in aCML until the recent characterization of mutations within the genes encoding the G-CSF receptor (CSF3R) and SET binding protein 1 (SETBP1) in a significant proportion of patients with aCML and chronic neutrophilic leukaemia (CNL). Whereas the functional significance of SETBP1 mutations in aCML and CNL are not yet fully understood, the characterization of CSF3R mutations has revealed a possibility for targeted therapeutic intervention with tyrosine kinase inhibitors. Approximately 50–60% of CNL and aCML patients harbour an acquired CSF3R mutation that can be either membrane proximal or in the cytoplasmic tail of the receptor. These two classes of mutations exhibit different downstream signalling patterns: membrane proximal mutations activate the JAK/STAT pathway and are therefore potentially amenable to inhibition with JAK inhibitors such as ruxolitinib, whereas truncation mutations predominantly signal through SRC family kinases and therefore may be targeted by SRC inhibitors such as dasatinib. The most prevalent CSF3R mutation in aCML and CNL is the membrane proximal T618I. In addition to this potential for targeted therapy, identification of aCML-specific CSF3R mutations affords the opportunity for evaluation of these mutations as disease-specific markers of residual disease in those patients undergoing allo-SCT. Mutations of SETBP1 and CSF3R exons 14 and 17 were sought by Sanger sequencing in a retrospective series of eight patients with aCML, CNL or atypical MPN. The clinical courses of two aCML patients are described, both of whom possessed a heterozygous CSF3R p. Thr618Ile (T618I) mutation (c.15482C>T, reference sequence NG_016270.1). A 60-year-old woman (Patient #1) presented in 2002 with what appeared to be morphologically CML but had a normal karyotype with no evidence of BCR-ABL1 by fluorescence in situ hybridization (FISH) or reverse transcription-PCR. She had no haematological response to 1 month of imatinib and commenced hydroxyurea. The pre-transplant work-up revealed worsening constitutional symptoms and adenopathy. Retrospectively, a CSF3R T618I mutation was detected 3 months pre-transplant (Figure 1a, upper). A reduced-intensity conditioning, sibling donor (CSF3R wild type) allo-SCT was performed. The initial post-allo-SCT course was unremarkable; however by day 72, whole blood donor chimerism was only 20% with the T618I mutation retrospectively still evident (Figure 1a, centre). Full donor chimerism was not achieved and she relapsed 9 months post allo-SCT with increasing BM myeloblasts. Two doses of DLI and a subsequent course of daunorubicin, cytarabine, thioguanine (3+10) did not improve the chimerism status; the marrow remained hypoplastic with 10–15% blasts and continued evidence of the CSF3R T618I (Figure 1a, lower). The patient became red cell and platelet transfusion dependent, deteriorated and died 17 months post allo-SCT. No constitutional material was available from Patient #1 to demonstrate the somatic nature of the CSF3R T618I mutation. A 48-year-old man (Patient #2) presented with an elevated white cell count and a hypercellular BM consistent with aCML. The patient had a normal karyotype, no evidence of BCR-ABL1, PDGFRA or PDGFRB rearrangements by FISH, with molecular studies demonstrating the absence of the JAK2 V617F and BCR-ABL1 transcripts but the presence of the CSF3R T618I mutation (Figure 1b, upper). He commenced treatment with hydroxyurea and 6 weeks before allo-SCT the CSF3R T618I clone remained evident but at a reduced level (Figure 1b, centre). He underwent a myeloablative allo-SCT with a sibling donor (CSF3R wild type). At day 30 post allo-SCT, he developed skin GvHD. The T618I mutation was not detected by sequencing on day 46 in whole peripheral blood (Figure 1b, lower). He remains well on a tapering steroid dose with improving GvHD. In other forms of MPN, allele-specific monitoring of diseaseassociated mutations, such as the JAK2 V617F and those within MPL exon 10 at defined time points, has been shown to be of value in predicting response to allo-SCT and may also aid in

Targeted next-generation sequencing of familial platelet disorder with predisposition to acute myeloid leukaemia

Familial platelet disorder with propensity to acute myeloid leukaemia (FPD-AML) is a rare, autosomal dominant disorder characterized by quantitative and qualitative platelet abnormalities with a propensity to develop a myelodysplastic syndrome (MDS) or AML. FPD-AML kindred are defined by germ-line mutations of RUNX1 (Song et al, 1999), which encodes a transcription factor essential for definitive haematopoiesis and myeloid cell differentiation, commonly dysregulated by translocations, mutations or amplification in de novo and secondary MDS and acute leukaemias. Most germ line RUNX1 mutations are unique to the individual FPDAML pedigree with variability observed in the MDS or AML phenotype and the incidence of leukaemic transformation of affected individuals (Nickels et al, 2013). The spectrum of somatic genetic events associated with progression to MDS or AML have not been fully appreciated but acquisition of cytogenetic abnormalities, single gene defects that occur in de novo MDS and AML, and bi-allelic RUNX1 mutations have all been demonstrated (Minelli et al, 2004; Preudhomme et al, 2009; Shiba et al, 2012). More recently, mutations of CDC25C have been identified in approximately half of affected FPD-AML patients. CDC25C mutations appear to disrupt a critical cell cycle check point in pre-leukaemic clones, allowing subsequent acquisition of further sub-clonal mutations (Yoshimi et al, 2014). Emerging next-generation sequencing (NGS) technologies, platforms and diseasetargeted panels allow the simultaneous identification of numerous mutational events. Such a targeted NGS approach was applied to a known RUNX1 mutated FPD-AML kindred to identify additional molecular events that co-operate with the germ line RUNX1 mutation in driving leukaemic transformation. A 56-year-old male and a 45-year-old female sibling both presented with AML with myelodysplastic features (Fig 1, I–3 and I–7 respectively). At diagnosis, I-3 had trisomy 8 and I-7 had monosomy 7. The eldest son of patient I-7 (II-1, Fig 1) has thrombocytopenia. The history of familial thrombocytopenia coupled with development of AML suggested a diagnosis of FPD-AML, which was confirmed by Sanger sequencing identification of a heterozygous RUNX1 p.Arg166X mutation in the leukaemic blasts and constitutional buccal scrapes of both affected patients. Patient I-3 underwent a reduced intensity conditioning allogeneic stem cell transplant (ASCT) from RUNX1 wild type sibling donor I-5. Patient I-7 underwent a myeloblative ASCT from RUNX1 wild type sibling donor I-6. Both patients achieved 100% donor chimerism by day-100 post-ASCT. For NGS, amplicon libraries were generated from AML diagnostic bone marrow DNA of I-3 and I-7 using the Ion Ampliseq AML Panel (Thermo Fisher Scientific, Life Technologies, Paisley, UK), a four primer-pool panel that generates 237 amplicons to allow interrogation of 19 commonly mutated genes implicated in AML. Amplicons cover the entire coding region of DNMT3A, CEBPA, GATA2, TET2, TP53 and mutational hot spot regions of ASXL1, BRAF, CBL, FLT3, IDH1, IDH2, JAK2, KIT, KRAS, NPM1, NRAS, PTPN11, RUNX1 and WT1. Sequencing was performed on an Ion PGM with data analysed and reviewed using Torrent Browser and Ion Reporter 4 2 software (Thermo Fisher Scientific, Life Technologies). Criteria to allow confident calling of somatic mutations were a minimum target coverage of 500X, the presence of a mutation at >5% and a predicted change in amino acid sequence. Sanger sequencing was also performed of exon 8 of CDC25C, encompassing the mutation hotspot previously described (Yoshimi et al, 2014). In addition to confirmation of the heterozygous RUNX1 p.Arg166X mutation, targeted NGS demonstrated the presence of further mutations in the genes known to disrupt epigenetic (ASXL1, IDH1, TET2) and transcription factor (CEBPA, RUNX1) function in AML (Table I). Although

Evaluation of a JAK2 V617F quantitative PCR to monitor residual disease post-allogeneic hematopoietic stem cell transplantation for myeloproliferative neoplasms.

*Corresponding author: Karl Haslam, Cancer Molecular Diagnostics, Central Pathology Laboratory, St. James’s Hospital, Dublin 8, Ireland, Phone: +353 1 4103575, Fax: +353 1 4103513, E-mail: khaslam@stjames.ie Karen M. Molloy and Stephen E. Langabeer: Cancer Molecular Diagnostics, Central Pathology Laboratory, St. James’s Hospital, Dublin, Ireland Eibhlin Conneally: Department of Haematology, St. James’s Hospital, Dublin, Ireland

Monitoring Minimal Residual Disease in the Myeloproliferative Neoplasms: Current Applications and Emerging Approaches

The presence of acquired mutations within the JAK2, CALR, and MPL genes in the majority of patients with myeloproliferative neoplasms (MPN) affords the opportunity to utilise these mutations as markers of minimal residual disease (MRD). Reduction of the mutated allele burden has been reported in response to a number of therapeutic modalities including interferon, JAK inhibitors, and allogeneic stem cell transplantation; novel therapies in development will also require assessment of efficacy. Real-time quantitative PCR has been widely adopted for recurrent point mutations with assays demonstrating the specificity, sensitivity, and reproducibility required for clinical utility. More recently, approaches such as digital PCR have demonstrated comparable, if not improved, assay characteristics and are likely to play an increasing role in MRD monitoring. While next-generation sequencing is increasingly valuable as a tool for diagnosis of MPN, its role in the assessment of MRD requires further evaluation.

Acute Lymphoblastic Leukaemia with an e1a3 BCR-ABL1 Fusion

inter-phase fluorescence in situ hybridization with dual labelled BCR and ABL1 probes detected hybridization signals in 66% of nuclei inspected, consistent with the presence of a t(9; 22). A standard RT-PCR methodology [9] detected the sole presence of e1a3 BCR-ABL1 transcripts. The patient commenced induction therapy according to the UK ALL XII protocol [10] but had 60% residual leukaemic blasts after induction with 54% of inter-phase nuclei examined, containing a BCR-ABL1 hybridization signal. The patient then received FLAG-Ida (fludarabine, cytarabine, granulocyte colony-stimulating factor, idarubucin) and achieved a morphological and cytogenetic remission with e1a3 BCR-ABL1 transcripts still detectable by RT-PCR. She then proceeded to consolidation with three courses of high-dose methotrexate, subsequently receiving continuous imatinib maintenance of 400 mg escalated to 600 mg. The patient relapsed 18 months after presentation with a karyotype of 44,XX, –3,–7,t(9; 22)(q34;q11),add(15)(q22–24) in 6 evaluable metaphases. She received re-induction with vincristine and prednisolone and subsequent imatinib maintenance (600 mg). Her clinical course over the next few months was complicated by Mycobacterium chelonae infection of the skin. The patient had a central nervous system relapse at 27 months after presentation and died soon after. MinApproximately 25–30% of adult patients with acute lymphoblastic leukaemia have evidence of the Philadelphia chromosome (Ph+ ALL). Identification of BCRABL1 transcripts is vital as these patients have a relatively adverse prognosis necessitating intensive therapies including allogeneic transplantation in eligible patients. More recently, it has been demonstrated that incorporation of a tyrosine kinase inhibitor into treatment regimens significantly improves prognosis [1] . Approximately 70% of Ph+ ALL patients express e1a2 BCR-ABL1 transcripts and 25% express either e13a2 or e14a2 BCR-ABL1 transcripts, with a number of other less common, variant transcripts reported that usually involve fusion of alternate exons [2] . The e1a3 BCR-ABL1 variant fusion transcript, lacking ABL1 exon a2 that partially encodes the src homology 3 (SH3) domain, has been described in three cases of chronic myeloid leukaemia (CML), all with a relatively indolent clinical course [3, 4] . In Ph+ ALL, this BCR-ABL1 genotype has only been reported previously in 8 adult patients [2, 5–7] and 1 paediatric case [8] , making any relationships between phenotype and outcome difficult to ascertain. We describe the clinical course of a further case of Ph+ ALL with an e1a3 BCR-ABL1 fusion. A 62-year-old female presented with pre-B ALL by immunophenotype. Although G-banded karyotype failed, Received: June 28, 2011 Accepted after revision: July 20, 2011 Published online: September 16, 2011