Robert Cuthbert

Insight into the pathogenesis of chronic lymphocytic leukemia (CLL) through analysis of IgVH gene usage and mutation status in familial CLL

To address the proposition that familial B-cell chronic lymphocytic leukemia (CLL) may exhibit a more restricted phenotype than sporadic CLL with respect to immunoglobulin gene usage or ontogenic development, we compared immunoglobulin (Ig) heavy chain variable region (VH) gene usage and IgVH mutation status in 327 patients with CLL from 214 families with 724 patients with sporadic cases. The frequency of mutated CLL was higher in familial CLL (P < .001), and there was evidence of intrafamilial concordance in mutation status (P < .001). The repertoire and frequency of IgVH usage was, however, not significantly different between familial and sporadic CLL. Furthermore, IgVH usage was not correlated between affected members of the same family. These observations provide evidence that familial CLL is essentially indistinguishable from sporadic CLL, favoring a genetic basis to disease development in general rather than a simple environmental etiology.

Comparison of diagnostic criteria for polycythaemia vera

Abstract Three sets of diagnostic criteria for polycythaemia vera (PY); the Polycythaemia Vera Study Group (PVSG) criteria (1975), the British Committee for Standards in Haematology (BCSH) criteria (1996) and the World Health Organisation (WHO) criteria (2001) have been described. We compared the ability of each set of criteria to accurately diagnose PV and differentiate it from secondary, apparent and idiopathic erythrocytosis. A cohort was drawn from a clinical database of erythrocytosis patients currently attending the Belfast City Hospital and the relevant information from the time of diagnosis for each patient was assessed according to each set of criteria, with the BCSH criteria used as a comparator. Sufficient data was available on 71 patients: 46 PV, 8 idiopathic, 8 apparent and 9 secondary erythrocytosis. The BCSH criteria classified 34 of 46 patients (73.9%) as PV and the WHO criteria had a sensitivity and specificity of 100% for classifying PV. For idiopathic and apparent erythrocytosis, the specificity of the WHO criteria, compared to the BCSH criteria, declined to 66.7 and 87.5%, respectively. The PVSG criteria were limited by the unavailability of required data for some patients resulting in a sensitivity and specificity of 50% for PV and specificity of 100% for all other groups. The Janus kinase 2 (JAK2) V617F mutation was present in 34 (85.3%) PV, 2 (50%) IE, 1 (50%) apparent and no secondary erythrocytosis cases. We concluded that the BCSH criteria were the most accurate diagnostic criteria for PV as they had an acceptable level of sensitivity and could differentiate between PV and other erythrocytoses.

Integration of conventional cytogenetics, comparative genomic hybridisation and interphase fluorescence in situ hybridisation for the detection of genomic rearrangements in acute leukaemia

Aims: To screen for genomic imbalances in patients with acute leukaemia using conventional (G-banding) and molecular (comparative genomic hybridisation (CGH) and fluorescence in situ hybridisation (FISH)) methods to determine whether an integrative screening approach increases abnormality detection rate. Methods: G-banded analysis was performed on unstimulated bone marrow (BM) or peripheral blood (PB) cells after short-term (24-hour) culture. CGH was performed on reference (control) and neoplastic (test patient) genomic DNA extracted from BM or PB samples. Interphase FISH (i-FISH) was selectively carried out at disease diagnosis on patients with acute lymphoblastic leukaemia and acute myeloid leukaemia using conventional methods. Results: Genomic rearrangements were detected in 4, 7 and 6 patients using G-banding, CGH and i-FISH respectively. Discordance in results between G-banding, CGH and/or i-FISH was found in 7 of the 12 patients screened. G-banding and CGH, when used individually, detected a genomic imbalance/rearrangement in 33.3% and 58.3%, respectively, of the patients screened. However, when both screening methods were integrated, the abnormality detection rate increased to 66.7%. This detection rate increased further to 75.0% with the use of i-FISH screening. Conclusions: The advantages and disadvantages of using G-banding, CGH and i-FISH as either stand-alone or integrated screening methods for the detection and characterisation of genomic imbalances in acute leukaemia are clearly demonstrated. Abnormality detection rate significantly increased when an integrated screening approach was employed which could potentially provide valuable information for risk stratification in patients with acute leukaemia.

Immunoglobulin Gene Rearrangement Investigations in the Diagnosis of Lymphoid Malignancies from Formaldehyde-fixed Biopsies

Determination of the biologic potential of lymphoid proliferations in biopsies can be difficult by standard histological or even immunohistochemical examination. Polymerase chain reaction (PCR) has been used with increasing frequency to detect clonal rearrangements of the immunoglobulin heavy chain (IgH) in formaldehyde fixed, paraffin wax embedded tissues. Sensitivity ranges between 50 and 80%, and therefore at least 20% of neoplasms remain undetected by these approaches. Few investigators have attempted to detect immunoglobulin light chain (IgL) gene rearrangements by PCR using paraffin wax embedded samples. We studied 29 cases of B-cell neoplasms, along with 21 cases with equivocal histology and 4 reactive biopsies, using degenerate oligoprimers to amplify Ig κ and Ig   light chain genes, along with IgH (Fr 1, 2 and 3) gene rearrangement analysis. The combination of these methods detected clonality in 93% of cases (27/29) with histological diagnosis of B-NHL. Fr2 and Fr3 primers detected clonality in 79% (23/29) of cases. IgL chain rearrangements detected 4 cases (14%), negative for IgH rearrangements, improving sensitivity from 79 to 93%. Clonality was detected in 52% (11/21) of histologically equivocal lymphoid proliferations, including one case detected by IgL rearrangements which was negative for IgH rearrangements. Archival material from 4 cases with reactive histology produced polyclonal results. These results confirm that PCR based immunoglobulin gene rearrangement is a sensitive and specific method for demonstrating B-cell clonality in paraffin-wax embedded sections. The addition of IgL analysis to the IgH assay allows the detection of greater than 90% of B-cell lymphoproliferative disorders from routine histological specimens with poor preservation of genomic DNA.

Use of imatinib mesylate in elderly patients in Northern Ireland: evidence of comparable haematological and molecular responses to younger patients

Abstract Advanced age is an indicator of poor prognosis in chronic myeloid leukaemia (CML). Since obtaining its UK licence in 2001, the tyrosine kinase inhibitor imatinib mesylate has effected a paradigm shift in the treatment of CML. We compared survival and molecular response rates in elderly patients to younger patients presenting with CML since the introduction of imatinib. Twenty-five patients aged >60 years were identified. No significant survival difference was found when this group was compared with younger patients. In the elderly group, 53% of those with molecular data (36% of all elderly patients) had a major molecular response as assessed by real time quantitative PCR (RT-PCR). The advent of imatinib therapy appears to have ameliorated much of the negative impact of advancing age on survival in patients with CML.

Dramatic resolution of respiratory symptoms with imatinib mesylate in patients with chronic myeloid leukemia presenting with lower airway symptoms resembling asthma

Recent observations show that imatinib mesylate induces early normalization of leukotrine C4 synthase expression in patients with chronic myeloid leukemia (CML) [1].There is a strong association between increased leukotrine C4 levels and exerciseinduced asthma [2,3]. Normalization of leukotrine C4 levels with imatinib mesylate treatment can lead to resolution of asthma-like symptoms. We describe two cases supporting this who presented with lower airway symptoms resembling asthma and CML in whom treatment with imatinib mesylate led to complete resolution of lower airway symptoms.

Urinary bladder infiltration with chronic B-lymphocytic leukemia:Two cases with unusual presentation

Chronic lymphocytic leukemia (CLL) is the most common type of leukemia in the western world. About 70% of cases are diagnosed incidentally on a routine full blood count [1]. Patients with CLL may present with lymphadenopathy, splenomegaly, hepatomegaly, constitutional symptoms or symptoms of bone marrow failure or immunosuppression. Infiltration of solid organs has been reported but is rare and usually asymptomatic [2,3]. Skin is the most commonly involved non-lymphoid organ. CLL is generally an indolent disease and there is a considerable variation in disease progression with survival from initial diagnosis ranging from 2 – 20 years. In the past, staging systems (Rai and Binet) were used to predict the overall survival. More recently, IgVH gene mutational status and CD38 expression has shown a correlation with the overall survival. Patients with mutated IgVH gene and 530% CD38 surface expression appear to have longer survival compared to patients with unmutated IgVH gene and 430% CD38 expression [4,5]. ZAP-70 expression can predict IgVH mutation status; therefore, it is a useful marker to predict time to disease progression and overall survival [6]. We report two female patients presented with urinary symptoms and treated as recurrent UTI for several years. Case 1 is a 60-year-old patient complaining of dysuria and microscopic hematuria since 1995. She has a history of rheumatoid arthritis and was treated as recurrent UTI with different courses of antibacterials without clinical improvement. She has had persistently negative urine cultures and microscopic examination revealed only red blood cells. In 1998, she was referred to the hematology department with a lymphocyte count of 306 10 l. Peripheral blood lymphocyte immunophenotyping showed cells positive for CD5, CD19, CD23, with weak expression of CD22 and smIg k and the diagnosis of B-CLL, Binet stage A was established. CD38 was expressed on 60% of lymphocytes. In 2000, she became iron deficient and anemic due to persistent hematuria. Her hemoglobin was 9.4 g dl with normal platelet and neutrophil count and low ferritin at 15 mg ml (20 – 300). Further evaluation revealed no evidence of hemolytic anemia. Her lymphocyte count increased from 32 – 706 10 l over a 4-month period. She then received six courses of chlorambucil and prednisone. The treatment has resulted in a remarkable improvement in her urinary symptoms. In 2002, she presented with frank hematuria. Platelet count, prothrombin time and partial thromboplastin time were normal. Abdominal ultrasound was normal. Intravenous urogram showed filling defects in the left superolateral aspect of her urinary bladder. Cystoscopy revealed patchy erythematous areas. Histological examination with hematoxylin and eosin stain revealed heavy lymphoid infiltration (Figure 1 (a,b)). On immunohistochemistry, lymphocytes coexpressed CD5 & CD20 in keeping with infiltration with B-CLL (Figure 1 (c,d)). She then received another six courses of chlorambucil therapy. This resulted into a remarkable improvement and disappearance of hematuria which lasted for 30 months. Case 2 is a 67-year-old patient referred with lymphocytosis in 2002. She has a history of type II diabetes mellitus and epilepsy. Full blood count revealed hemoglobin of 11 g dl, white cell count 90.76 10 l, platelets 1986 10 l and

The prevalence of CALR mutations in a cohort of patients with myeloproliferative neoplasms.

To investigate the prevalence of calreticulin (CALR) mutations in JAK2‐ and MPL‐non‐mutated patients with suspected myeloproliferative neoplasm (MPN) from a large MPN clinic and confirm a diagnosis of MPN.

A novel mutation, Ile289Thr, in the ALAS2 gene in a family with pyridoxine responsive sideroblastic anaemia

X-linked sideroblastic anaemia (XLSA; OMIM 301 300) is characterised by accumulation of inorganic iron in erythroblast mitochondria, visualised on staining as distinctive perinuclear rings. It arises from a deficiency of the erythroid specific isoenzyme of δ-aminolaevulinate synthase (ALAS2; E.C. 2.3.1.3.7), caused mainly by mutations affecting the catalytic or substrate-binding domains.1,2 ALAS2 uses pyridoxal-5-phosphate as a cofactor to catalyse the first, rate-limiting step of erythroid haem synthesis and pyridoxine treatment can alleviate anaemia in many cases, although the response is variable and affected by factors such as mutation, age and iron load.3,4 …

Absence of CALR Mutations in Idiopathic Erythrocytosis Patients with Low Serum Erythropoietin Levels

a Clinical Haematology, Belfast City Hospital, Belfast Health and Social Care Trust, Belfast, UK; b Ecole Pratique des Hautes Etudes (EPHE), PSL Research University, Paris, France; c INSERM U892, CNRS 6299, Université de Nantes, Nantes, France; d Service d’Hématologie Biologique CHU Dijon, Dijon, France; e INSERM, UMR866, University of Burgundy Franche-Comté, Dijon, France; f Wessex Regional Genetics Laboratory, Salisbury, UK; g Centre for Medical Education (CME), Queen’s University Belfast, Belfast, UK Received: March 28, 2018 Accepted: March 30, 2018 Published online: May 30, 2018