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

INTRODUCTION: 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. METHODS: JAK2/MPL-non-mutated patients from the Belfast City Hospital (BCH) with either of the MPNs - ET or MF - and diagnosed between 1988 and 2014 were selected for CALR screen. All cases were validated according to the WHO 2008 classification for MPNs. Statistical analysis was performed with Minitab 16 Statistical Software package. Exon 9 of CALR was amplified by PCR using genomic DNA, and mutations were detected by fragment analysis. RESULTS: Of the 62 JAK2/MPL-non-mutated MPN patients screened, 57 had ET and 5 had MF; 34 patients (53.1%) carried CALR mutations. Three of 5 MF patients were CALR positive. Thirty-one ET patients (54.3%) harboured CALR mutation, whereas 26 (45.7%) were classified as 'triple negatives'. CONCLUSION: Detection of CALR mutations in a cohort of JAK2/MPL-non-mutated patients with suspected MPN confirmed the diagnosis of MPN in around 53% of cases. This is lower than initially reported, but similar to subsequent studies. However, a sizable cohort of patients remains lacking a specific molecular marker.

Myeloid sarcoma of the small bowel associated with a CBFbeta/MYH11 fusion and inv(16)(p13q22): a case report.

This report describes a case of aleukaemic myeloid sarcoma of the small intestine in a 50-year-old woman presenting with small bowel obstruction. Fluorescence in situ hybridisation analysis of interphase nuclei revealed a split CBFbeta signal, consistent with an underlying inversion of chromosome 16, inv(16)(p13q22). The resultant type A CBFbeta/MYH11 transcript was detected by reverse transcriptase PCR. Immunohistochemistry with the AH107 antibody to the CBFbeta-SMMHC chimeric protein showed strong nuclear staining of the tumour cell nuclei. This represents the first use of this antibody in the diagnosis of this subtype of myeloid sarcoma in the small intestine.

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

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.

ALK-positive diffuse large B-cell lymphoma of the stomach associated with a clathrin-ALK rearrangement.

ALK-positive diffuse large B-cell lymphoma is a rare, recently characterized lymphoma subtype that shows granular cytoplasmic ALK expression. This report describes a primary gastric ALK-positive B-lineage lymphoma in which a clathrin (CLTC)-ALK fusion was identified by RT-PCR and direct sequencing of the breakpoint. This confirmed the presence of t(2;17)(p23;q23) involving the CLTC gene and is only the 4th report of such a translocation in this lymphoma subtype and the first to describe this tumor within the stomach. As in previous reports, immunophenotyping showed the malignant cell to be a terminally differentiated B-lineage cell characterized by the absence of B-cell antigens and expression of antigens associated with plasma cell differentiation. This case confirms the existence of such a lymphoma subtype arising in extranodal locations and underscores the importance of detailed immunophenotyping and specialized molecular genetic investigations in confirming the diagnosis.

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(kappa) and Ig(lambda) 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.

Evaluation of an automated latex D-dimer immunoassay in the clinical assessment of suspected venous thromboembolism.

Because the reliability of clinical signs in venous thromboembolism (VTE) is poor, a highly sensitive, non-invasive test may improve the selection of patients requiring further investigation. We assessed the sensitivity and negative predictive value of an automated D-dimer latex immunoassay (IL-Test ) in 68 patients presenting with suspected VTE. The plasma D-dimer concentration was estimated and an appropriate diagnostic radiological investigation performed. Control values were obtained from healthy young and elderly volunteers. Using a cut-off value of 330 ng/ml, the assay had a sensitivity of 100% and negative predictive value of 100% for VTE. We conclude that the IL-Test. automated D-dimer assay has a suitably high sensitivity and adequate negative predictive value to be included in a pre-test clinical probability protocol for the evaluation of patients with suspected VTE.