Maeve Leahy

A multi-centre retrospective study of rituximab use in the treatment of relapsed or resistant warm autoimmune haemolytic anaemia.

This retrospective analysis assessed the response, safety and duration of response to standard dose rituximab 375 mg/m2 weekly for four weeks as therapy for patients with primary or secondary warm autoimmune haemolytic anaemia (WAIHA), who had failed initial treatment. Thirty‐four patients received rituximab for WAIHA in seven centres in the Republic of Ireland. The overall response rate was 70·6% (24/34) with 26·5% (9/34) achieving a complete response (CR). The time to response was 1 month post‐initiation of rituximab in 87·5% (21/24) and 3 months in 12·5% (3/24) of patients. The median duration of follow‐up was 36 months (range 6–90 months). Of the patients who responded, 50% (12/24) relapsed during follow up with a median time to next treatment of 16·5 months (range 6–60 months). Three patients were re‐treated with rituximab 375 mg/m2 weekly for four weeks at relapse and responded. There was a single episode of neutropenic sepsis. Rituximab is an effective and safe treatment for WAIHA but a significant number of patients will relapse in the first two years post treatment. Re‐treatment was effective in a small number of patients, suggesting that intermittent pulse treatment or maintenance treatment may improve long‐term response.

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

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.

Autologous stem cell transplantation in myeloma : the St james's Hospital experience, 1997-2003

BackgroundHigh-dose treatment with autologous stem cell transplantation (ASCT) has become the standard of care for patients with myeloma below the age of 65 years.AimsWe report an audit of 60 patients (median age: 52.5 years) who underwent ASCT in the National Bone Marrow Transplant centre in St James’s Hospital in Dublin between 1997 and 2003 inclusive.MethodsClinical and laboratory data were retrieved from patient medical records and hospital information management systems.ResultsThirty-six patients had IgG, 11 IgA, 1 IgD, 9 light chain and 3 non-secretory MM. Fifty-seven (95%) patients received anthracycline-corticosteroid combination chemotherapy prior to autografting. There was no transplant-related mortality (TRM). Complete (CR) and Partial Responses (PR) were seen in 16 (29.6%) and 29 (53.7%) of those evaluable (n = 54 (90%)). The actuarial Progression-Free (PFS) and Overall Survival (OS) rates at five years are 13% and 55% respectively.ConclusionCentre outcome is comparable to published international series and supports the use of ASCT in the treatment of this malignancy.

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.

Transient JAK2 V617F mutation in an aplastic anaemia patient with a paroxysmal nocturnal haemoglobinuria clone.

In 1969, William Dameshek proposed that paroxysmal nocturnal haemoglobinuria (PNH) should be considered a candidate myeloproliferative neoplasm (MPN) based on the evidence of red blood cell abnormalities in the context of bone marrow pancytopenia, low leucocyte alkaline phosphatase and the potential for leukaemic transformation (Dameshek, 1969). Further similarities between MPN and PNH include expansion of subsequent or simultaneous myeloid clones and an increased frequency of abdominal vein thrombosis. PNH is an acquired, stem cell disorder associated with mutations of the PIGA gene that encodes a protein required for the synthesis of glycosylphosphatidyl-inositol anchored proteins (GPI-AP). PNH blood cells are therefore deficient in cell surface proteins that use the GPI anchor and may be detected immunophenotypically by absent or reduced expression of CD55 and CD59 on red blood cells (RBC) and CD16, CD24, CD66b and FLAER (fluorescence labelled aerolysin) on granulocytes. PNH clones are also present in a significant number of patients with the related marrow failure syndromes aplastic anaemia (AA) and hypoplastic myelodysplastic syndrome (MDS). Although PNH originates from a multipotent haematopoietic stem cell, PIGA mutations are insufficient to drive clonal expansion and several models have attempted to explain this phenomenon: GPI-AP may be immunogenic and their deficiency enables evasion from immunological surveillance or the possibility that PIGA mutations themselves confer an intrinsic resistance to apoptosis (Brodsky, 2008). Another theory postulates the acquisition of a second genetic event that enables clonal expansion in co-operation with the PIGA mutation, some evidence for which comes from the deregulated expression of HMGA2 in a minority of PNH patients (Inoue et al, 2006). A recent report identified the JAK2 V617F mutation in GPI-AP deficient cells of three PNH patients and suggested this coexistence could explain clonal expansion (Sugimori et al, 2012). These findings prompted investigation into the frequency of the JAK2 V617F in other patients harbouring a PNH clone. Archival DNA samples from blood (n = 3) and bone marrow aspirate smears (n = 19) from 22 patients (PNH, n = 5; AA, n = 13; hypoplastic MDS, n = 4) with an immunophenotypically detectable PNH clone of both RBC and granulocytes were retrospectively analysed for JAK2 V617F by qualitative allele-specific polymerase chain reaction (PCR) (Baxter et al, 2005) with a sensitivity of 2% mutant alleles. The JAK2 V617F mutation was detected in one patient with AA. This female patient, who presented with severe AA and no evidence of a PNH clone (nor retrospectively the JAK2 V617F), was initially successfully treated with two courses of antithymocyte globulin (ATG) and cyclosporin. She relapsed in pregnancy 2 years later and was treated with immunoglobulin, and RBC and platelet transfusions. Post partum treatment included G-CSF, ATG (2 courses) and ciclosporin, the latter being discontinued after achieving RBC and platelet transfusion independence. Ten years after the initial presentation, progressive pancytopenia suggested relapse of AA with a PNH clone detected within the granulocytes (17 7%) and RBCs (5 4%). At this relapse, the JAK2 V617F was detected in the bone marrow and confirmed by quantitative PCR (qPCR; Larsen et al, 2007) at a reproducible level of 1 8% of total JAK2 alleles (Fig 1). Ciclosporin was recommenced with a good response attained. 3 years post-relapse, the PNH clone remained stable in granulocytes (18 9%) and RBCs (6 0%) with the JAK2 V617F not detected in the bone marrow by both allele-specific and qPCR. At no point during the clinical course did the patient have a persistent leucocytosis, erythrocytosis or thrombocytosis. The patient was subsequently diagnosed with malignant melanoma to which she succumbed. Diagnostic or pre-allogeneic haematopoietic stem cell transplantation samples from a further 38 AA patients without evidence of PNH were screened for the JAK2 V617F, none of which were found to harbour this mutation. We describe the hitherto unreported detection of the JAK2 V617F in a PNH-related bone marrow failure syndrome at a similarly low frequency to that previously described in classical PNH (Fouassier et al, 2009; Sugimori et al, 2012). As this case was identified retrospectively, existence of the JAK2 V617F in the PNH clone could not be verified by fluorescence-activated cell-sorting of the GPI-AP-negative population (Sugimori et al, 2012). However, the transient nature of the JAK2 mutation in the presence of a relatively stable PNH clone, taken together with the evidence of different JAK2 V617F and PNH clone sizes implies two distinct populations as opposed to the co-existence of mutations within one clone. The phenomenon of transient JAK2 V617F-positivity may be explained by the acquisition of the mutation in a more differentiated haematopoietic progenitor than that in patients with MPN. If this progenitor had reduced selfrenewal potential and capacity for differentiation then the clone generated might therefore be short-lived and not result in a clinical phenotype (Passamonti et al, 2007). Characterization of the role of JAK2 signalling in PNH and PNH-related disorders may provide some insight into Correspondence

Impact of the Introduction of Guidelines on Vitamin B12 Testing

To the Editor: In 2008, our laboratory received 42 546 requests for vitamin B12 testing, a 72% increase from 2003. We provide B12 testing to a population of 361 028 (according to the Irish census of 2006), a 6.3% increase from 2002 (1). This change in population cannot explain this substantial increase in B12 testing. This increasing laboratory workload is not unique to B12 testing or to our laboratory (2). We produced local guidelines to advise on appropriate testing for B12. The indications included in the guidelines were: hematologic (unexplained anemia/other cytopenias, unexplained macrocytosis), neurologic (peripheral neuropathy, dementia, unexplained neurology), pregnancy, glossitis, malabsorption, metformin therapy, and dialysis patients. These guidelines were introduced in May 2009. Subsequently, testing for B12 was performed if the clinical details were consistent with the guidelines. If the clinical indication was not consistent with local guidelines, the test was not performed, and the clinician was informed. Additionally, we suggested a minimum retest interval of 6 months, although we did …