Amjad Hayat

CD38 expression level and pattern of expression remains a reliable and robust marker of progressive disease in chronic lymphocytic leukemia

Chronic lymphocytic leukemia (CLL) follows a variable clinical course which is difficult to predict at diagnosis. We assessed somatic mutation (SHM) status, CD38 and ZAP-70 expression in 87 patients (49 male, 38 female) with stage A CLL and known cytogenetic profile to compare their role in predicting disease progression, which was assessed by the treatment free interval (TFI) from diagnosis. Sixty (69%) patients were SHM+, 24 (28%) were CD38+ and ten (12%) were ZAP-70+. The median TFI for: (i) SHM + versus SHM− patients was 124 versus 26 months; hazard ratio (HR) = 3.6 [95% confidence interval (CI) = 1.8 – 7.3; P = 0.001]: (ii) CD38− versus CD38+ patients was 120 versus 34 months; HR = 2.4 (95% CI = 1.4 – 5.3; P = 0.02); and (iii) ZAP70− versus ZAP70+ was 120 versus 16 months; HR = 3.4 (95% CI = 1.4 – 8.7; P = 0.01). SHM status and CD38 retained prognostic significance on multivariate analysis whereas ZAP-70 did not. We conclude that ZAP-70 analysis does not provide additional prognostic information in this group of patients.

The Novel Tubulin-Targeting Agent Pyrrolo-1,5-Benzoxazepine-15 Induces Apoptosis in Poor Prognostic Subgroups of Chronic Lymphocytic Leukemia

Pyrrolo-1,5-benzoxazepine-15 (PBOX-15) is a novel microtubule depolymerization agent that induces cell cycle arrest and subsequent apoptosis in a number of cancer cell lines. Chronic lymphocytic leukemia (CLL) is characterized by clonal expansion of predominately nonproliferating mature B cells. Here, we present data suggesting PBOX-15 is a potential therapeutic agent for CLL. We show activity of PBOX-15 in samples taken from a cohort of CLL patients (n = 55) representing both high-risk and low-risk disease. PBOX-15 exhibited cytotoxicity in CLL cells (n = 19) in a dose-dependent manner, with mean IC(50) of 0.55 micromol/L. PBOX-15 significantly induced apoptosis in CLL cells (n = 46) including cells with poor prognostic markers: unmutated IgV(H) genes, CD38 and zeta-associated protein 70 (ZAP-70) expression, and fludarabine-resistant cells with chromosomal deletions in 17p. In addition, PBOX-15 was more potent than fludarabine in inducing apoptosis in fludarabine-sensitive cells. Pharmacologic inhibition and small interfering RNA knockdown of caspase-8 significantly inhibited PBOX-15-induced apoptosis. Pharmacologic inhibition of c-jun NH(2)-terminal kinase inhibited PBOX-15-induced apoptosis in mutated IgV(H) and ZAP-70(-) CLL cells but not in unmutated IgV(H) and ZAP-70(+) cells. PBOX-15 exhibited selective cytotoxicity in CLL cells compared with normal hematopoietic cells. Our data suggest that PBOX-15 represents a novel class of agents that are toxic toward both high-risk and low-risk CLL cells. The need for novel treatments is acute in CLL, especially for the subgroup of patients with poor clinical outcome and drug-resistant disease. This study identifies a novel agent with significant clinical potential.

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

Home administration of bortezomib: Making a difference to myeloma patients' lives

IntroductionMultiple myeloma is a clonal malignancy of plasma cells, char-acterized by anaemia, renal dysfunction, lytic bone lesions and thepresence of excess monoclonal immunoglobulin. It is the secondmost common hematological disorder (Devenney and Erickson,2004). It remains a complex disease to diagnose and treat.However, our understanding of the biology of myeloma continuesto develop, and hence a number of new potential therapies havebeen identified, with improved outcomes and survival (Kumaret al., 2008).The introduction of novel agents, such as immunomodulatorydrugsorproteasomeinhibitors,eitheraloneorincombinationwithtraditional agents for the treatment of myeloma has led to a majorimprovement in patient outcomes, including survival, in the pastdecade. Based on significant improvements in response rates andoverallsurvival in elderlypatients when combined with melphalanin elderly patients (San Miguel et al., 2008), Bortezomib, a protea-someinhibitor,isnowlicensedasfrontlinetreatmentformyeloma.Bortezomib combined with dexamethasone has also proven to bea very effective induction therapy in younger patients prior toautologous stem cell transplant (Harousseau et al., 2006) and isnow viewed by some as the new standard for initial therapy ofyounger patients. Younger patients are those less than 65 years ofage and eligible for stem cell transplant. However, they must alsohave good performance status and without other co-morbidities.Initiatives in the home administration of chemotherapy areevident internationally (e.g. Lashlee and O’Hanlon Curry, 2007).With regard to the home administration of bortezomib, a pilotfeasibilityprojectofhomeadministrationofbortezomibtopatientswith myeloma has recently been reported from BournmouthHospital in England (McCarthy et al., 2009). However, that pilotprogram only included patients with relapsed disease and patientshadtolivewithina12 mileradiusofthehospital.Inaddition,Day1and Day 4 doses were administered in the hospital, and bloodsamples were taken on each visit. Three of our patient group werenewly diagnosed, receiving initial treatment for their myeloma.Furthermore, all patients on our program received first doses ofbortezomib safely at home. In addition, bloods on our program areonly taken on Day 8; the platelet nadir is day 11 so checking on day8 detects any significant drop prior to this. Finally, our patients liveas far away as 100 miles from the hospital.Why the initiative was startedGalway University Hospital (GUH) is a regional Irish hospitalserving a local urban and widely dispersed rural population. Giventhe emerging data supporting its use in all categories of patientswith multiple myeloma, there has been a major increase in the useofbortezomibastreatmentofmultiplemyelomapatientsattendingGUH. Since bortezomib requires frequent intravenous administra-tion(usuallytwiceaweek,fortwoconsecutiveweekswitha10dayrest period) this has impacted significantly on the hospital’s hae-matology day unit facility, which has severe capacity issues. Theadministration of bortezomib only takes ten seconds. However,

Effective use of imatinib-mesylate in the treatment of relapsed chronic myeloid leukemia after allogeneic transplantation.

Despite recent advances in the treatment of chronic myeloid leukemia (CML), allogeneic stem-cell transplantation (SCT) remains the only curative option. The success of SCT is limited because of relapse in 20–30% of patients.[1][1],[2][2] As the graft-versus-leukemia (GvL) effect contributes to

Molecular heterogeneity of familial myeloproliferative neoplasms revealed by analysis of the commonly acquired JAK2, CALR and MPL mutations

The myeloproliferative neoplasms (MPN) are clonal, hematological malignancies that include polycythemia vera, essential thrombocythemia and primary myelofibrosis. While most cases of MPN are sporadic in nature, a familial pattern of inheritance is well recognised. The phenotype and status of the commonly acquired JAK2 V617F, CALR exon 9 and MPL W515L/K mutations in affected individuals from a consecutive series of ten familial MPN (FMPN) kindred are described. Affected individuals display the classical MPN phenotypes together with one kindred identified suggestive of hereditary thrombocytosis. In affected patients the JAK2 V617F mutation is the most commonly acquired followed by CALR exon nine mutations with no MPL W515L/K mutations detected. The JAK2 V617F and CALR exon 9 mutations appear to occur at approximately the same frequency in FMPN as in the sporadic forms of these diseases. The familial nature of MPN may often be overlooked and accordingly more common than previously considered. Characterisation of these FMPN kindred may allow for the investigation of molecular events that contribute to this inheritance.

Risk adjusted therapy in chronic lymphocytic leukemia: a phase II cancer trials Ireland (CTRIAL-IE [ICORG 07-01]) study of fludarabine, cyclophosphamide, and rituximab therapy evaluating response adapted, abbreviated frontline therapy with FCR in non-del(17p) CLL

Abstract Minimal residual disease negative complete response (MRD-negative CR) provides an early marker for time to treatment failure (TTF) in CLL treated with fludarabine, cyclophosphamide, and rituximab (FCR). MRD was assessed after four FCR cycles (FCR4); MRD-negative CR patients discontinued treatment. Fifty-two patients (35M; 17F) were enrolled. Eighteen (18/52; 34.6%) patients reached MRD-negative CR after FCR4 and 29/52 (55.8%) were MRD-negative CR at end of treatment (EOT). Median TTF was 71.1 months (95% CI 61.3–84.1 months), with median overall survival not reached. Mutated immunoglobulin heavy chain gene rearrangements (IGHV) were associated with early MRD-negative remissions, translating into prolonged TTF. Unmutated-IGHV, mutated-SF3B1 and mutated-NOTCH1 were associated with shortened TTF. No TTF difference was observed between patients in MRD-negative CR after four versus six cycles (82.2 versus 85.3 months, p = .6306). Abbreviated FCR therapy is effective for patients achieving early MRD-negative remissions. Interim MRD assessment assists in personalizing therapy and reducing chemotherapy-associated toxicity.

A novel molecular assay using hybridisation probes and melt curve analysis for CALR exon 9 mutation detection in myeloproliferative neoplasms

Aims Somatic insertions/deletions in exon 9 of the calreticulin gene have been identified in patients with essential thrombocythemia and primary myelofibrosis. Over 55 mutations have been discovered, 80% of which consist of either type 1 52-bp deletion or type 2 5-bp insertion. Other mutations (types 3–5) in conjunction with types 1 and 2 account for >87% of identified mutations. The aim of this study was development of a rapid PCR-based assay using LightCycler Hybridisation Probes for the detection of type 1–5 CALR mutations. Method A real-time PCR assay using a novel HybProbe set was developed for use on the LightCycler 480 Instrument II. The acceptor probe was labelled with LC640 and Faststart DNA Master HybProbe kit was used for PCR reactions. Results Assay limit of detection was determined to be seven target copies with a probability of 95%. The specificity of the assay was determined by using synthetic constructs of CALR wild-type and CALR mutation types 1–5 with no non-specific detection observed. Samples from 21 patients with essential thrombocythemia (ET) and 12 patients with primary myelofibrosis (PMF), together with 29 control samples from patients diagnosed with various conditions, were screened using the assay. Of these, 24 were found to have mutations in CALR exon 9, with the assay detecting 8 type 1 mutations, 12 type 2 mutations, 2 type 24 mutations, 1 type 20 mutation and 1 31-bp deletion. Conclusions The novel assay described has potential for application as a rapid, sensitive, high-throughput screening method in the clinical diagnostics setting.

Tumour Lysis Syndrome and Partial Remission Occurring After Administration of a Test Dose of Obinutuzumab

Chronic lymphocytic leukaemia (CLL) is one of the most common haematological malignancies worldwide, with an increasing prevalence in the elderly population. Obinutuzumab is a type II anti-CD20 monoclonal antibody which showed superiority over rituximab in combination chemotherapy with chlorambucil for the treatment of CLL in the CLL11 trial (NCT01010061) and is becoming part of standard first line treatment for CLL in the elderly based on its potent efficacy and benign safety profile. We report the case of a chemotherapy naive patient who develop tumour lysis syndrome despite appropriate prophylaxis, and had partial remission of her disease after receiving only the initial test dose of obinutuzumab. LEARNING POINTS Chemotherapy-induced tumour lysis syndrome is a serious complication that can occur after even a small dose of chemotherapy. Chronic lymphocytic leukaemia patients with strongly positive CD20 cells can be more sensitive to obinutuzumab chemotherapy.

Molecular Profiling: A Case of ZBTB16-RARA Acute Promyelocytic Leukemia

Several variant RARA translocations have been reported in acute promyelocytic leukemia (APL) of which the t(11;17)(q23;q21), which results in a ZBTB16-RARA fusion, is the most widely identified and is largely resistant to therapy with all-trans retinoic acid (ATRA). The clinical course together with the cytogenetic and molecular characterization of a case of ATRA-unresponsive ZBTB16-RARA APL is described. Additional mutations potentially cooperating with the translocation fusion product in leukemogenesis have been hitherto unreported in ZBTB16-RARA APL and were sought by application of a next-generation sequencing approach to detect those recurrently found in myeloid malignancies. This technique identified a solitary, low level mutation in the CEBPA gene. Molecular profiling of additional mutations may provide a platform to individualise therapeutic management in patients with this rare form of APL.