Contribution of immunoglobulin lambda light chain gene rearrangement analysis in the diagnosis of B-cell neoplasms.

Identification of clonal IGH, IGK and IGL gene rearrangements offers diagnostic adjunct in suspected B-cell neoplasms. However, many centres omit IGL analysis as its value is uncertain. A review of 567 cases with IGH, IGK and IGL rearrangement assessed using BIOMED-2 assays showed clonal immunoglobulin gene rearrangement in 54% of cases, of which 24% had a clonal IGL rearrangement. In two cases, the clonal rearrangement was detected exclusively by IGL analysis. This finding demonstrates the added value of IGL analysis for clonality assessment, especially in suspected B-cell neoplasms in which a clonal IGH and/or IGK rearrangement is not detected or is equivocal.

Leukemia-associated somatic mutations drive distinct patterns of age-related clonal hemopoiesis.

Clonal hemopoiesis driven by leukemia-associated gene mutations can occur without evidence of a blood disorder. To investigate this phenomenon, we interrogated 15 mutation hot spots in blood DNA from 4,219 individuals using ultra-deep sequencing. Using only the hot spots studied, we identified clonal hemopoiesis in 0.8% of individuals under 60, rising to 19.5% of those ≥90 years, thus predicting that clonal hemopoiesis is much more prevalent than previously realized. DNMT3A-R882 mutations were most common and, although their prevalence increased with age, were found in individuals as young as 25 years. By contrast, mutations affecting spliceosome genes SF3B1 and SRSF2, closely associated with the myelodysplastic syndromes, were identified only in those aged >70 years, with several individuals harboring more than one such mutation. This indicates that spliceosome gene mutations drive clonal expansion under selection pressures particular to the aging hemopoietic system and explains the high incidence of clonal disorders associated with these mutations in advanced old age.

Hairy cell leukemia - immunotargets and therapies.

Hairy cell leukemia (HCL) is an indolent low-grade B-cell lymphoproliferative disorder that is reasonably sensitive to standard first-line purine analog therapy. However, in many cases, repeat relapses occur, requiring multiple courses of purine analog therapy, promoting eventual drug resistance. This, coupled with the concerning side effects of repeated purine analog exposure, has prompted the search for alternative targets and therapies that may provide deeper remissions. Novel strategies employing immune-mediated targeting via monoclonal antibody therapies and recombinant immunotoxins appear promising in HCL and are currently under investigation. More recently, the concept of targeted kinase inhibition using small-molecule inhibitors in HCL has emerged as another potentially viable option. As a deeper understanding of the aberrant molecular pathways contributing to the pathogenesis of HCL develops, the landscape of management for HCL, particularly in the relapse setting, may change significantly in the future as a result of these promising immunotargets and therapies.

Pitfalls in the diagnosis of anaplastic large cell lymphoma with a small cell pattern.

Anaplastic large cell lymphoma with a small cell pattern is a rare T-cell lymphoma. This condition is more frequently seen in younger patients and should be considered when patients present with leucocytosis and constitutional symptoms. In this report, we describe our diagnostic work-up for one such case using blood, lymph node, and bone marrow aspirate samples, highlighting the variability of antigen expression seen in different sample types and methodologies. This case shows the importance of having a high index of suspicion and assessing CD30 and anaplastic lymphoma kinase expression in all suspected T-cell neoplasms even though this rare condition is not necessarily expected.

Detection of cytoplasmic nucleophosmin expression by imaging flow cytometry.

Mutations within the nucleophosmin NPM1 gene occur in approximately one-third of cases of acute myeloid leukemia (AML). These mutations result in cytoplasmic accumulation of the mutant NPM protein. NPM1 mutations are currently detected by molecular methods. Using samples from 37 AML patients, we investigated whether imaging flow cytometry could be a viable alternative to this current technique. Bone marrow/peripheral blood cells were stained with anti-NPM antibody and DRAQ5 nuclear stain, and data were acquired on an ImageStream imaging flow cytometer (Amnis Corp., Seattle, USA). Using the similarity feature for data analysis, we demonstrated that this technique could successfully identify cases of AML with a NPM1 mutation based on cytoplasmic NPM protein staining (at similarity threshold of 1.1 sensitivity 88% and specificity 90%). Combining data of mean fluorescence intensity and % dissimilar staining in a 0-2 scoring system further improved the sensitivity (100%). Imaging flow cytometry has the potential to be included as part of a standard flow cytometry antibody panel to identify potential NPM1 mutations as part of diagnosis and minimal residual disease monitoring. Imaging flow cytometry is an exciting technology that has many possible applications in the diagnosis of hematological malignancies, including the potential to integrate modalities.

High resolution melting analysis for detection of BRAF exon 15 mutations in hairy cell leukaemia and other lymphoid malignancies.

The BRAF V600E mutation has recently been described in all cases of hairy cell leukaemia (HCL). We have developed and validated a rapid and sensitive high-resolution melting analysis (HRMA) assay that detects BRAF exon 15 mutations when hairy cells are as low as 5-10% in a sample. All 48 HCL patients were positive for the BRAF V600E mutation, while 114 non-HCL cases were all V600E negative. Interestingly, we detected a novel BRAF D594N mutation in one patient with multiple myeloma. The HRMA assay offers a useful tool to aid the laboratory diagnosis of HCL.

PML protein analysis using imaging flow cytometry.

Acute promyelocytic leukaemia (APML) can be promptly diagnosed by detecting abnormal diffuse staining patterns of PML bodies in abnormal promyelocytes using immunofluorescence microscopy. However, this technique is subjective, with low sensitivity. Using samples from 18 patients with acute myeloid leukaemia (AML) (including four with APML), the authors investigated whether imaging flow cytometry could be a viable alternative to this current technique and improve sensitivity levels. Bone marrow/peripheral blood cells were stained with an antibody to PML, and data were acquired on an ImageStream (Amnis Corporation, Seattle, Washington, USA). Using the modulation feature for data analysis, the authors demonstrated that this technique could successfully identify cases of APML. Imaging flow cytometry, by analysing greater numbers of cells and with the potential to include disease-specific antigens, increases the sensitivity of the current immunofluorescence technique. Imaging flow cytometry is an exciting technology that has many possible applications in the diagnosis of haematological malignancies, including the potential to integrate modalities.

AT9283, a potent inhibitor of the Aurora kinases and Jak2, has therapeutic potential in myeloproliferative disorders.

Constitutive activation of Janus kinase (Jak) 2 is the most prevalent pathogenic event observed in the myeloproliferative disorders (MPD), suggesting that inhibitors of Jak2 may prove valuable in their management. Inhibition of the Aurora kinases has also proven to be an effective therapeutic strategy in a number of haematological malignancies. AT9283 is a multi-targeted kinase inhibitor with potent activity against Jak2 and Aurora kinases A and B, and is currently being evaluated in clinical trials. To investigate the therapeutic potential of AT9283 in the MPD we studied its activity in a number of Jak2-dependent systems. AT9283 potently inhibited proliferation and Jak2-related signalling in Jak2-dependent cell lines as well as inhibiting the formation of erythroid colonies from haematopoietic progenitors isolated from MPD patients with Jak2 mutations. The compound also demonstrated significant therapeutic potential in vivo in an ETV6-JAK2 (TEL-JAK2) murine leukaemia model. Inhibition of both Jak2 and Aurora B was observed in the model systems used, indicating a dual mechanism of action. Our results suggest that AT9283 may be a valuable therapy in patients with MPD and that the dual inhibition of Jak2 and the Aurora kinases may potentially offer combinatorial efficacy in the treatment of these diseases.

Clinical utility of routine MPL exon 10 analysis in the diagnosis of essential thrombocythaemia and primary myelofibrosis.

Approximately 50% of essential thrombocythaemia and primary myelo-fibrosis patients do not have a JAK2 V617F mutation. Up to 5% of these are reported to have a MPL exon 10 mutation but testing for MPL is not routine as there are multiple mutation types. The ability to routinely assess both JAK2 and MPL mutations would be beneficial in the differential diagnosis of unexplained thrombocytosis or myelofibrosis. We developed and applied a high resolution melt (HRM) assay, capable of detecting all known MPL mutations in a single analysis, for the detection of MPL exon 10 mutations. We assessed 175 ET and PMF patients, including 67 that were JAK2 V617F-negative by real time polymerase chain reaction (PCR). Overall, 19/175 (11%) patients had a MPL exon 10 mutation, of whom 16 were JAK2 V617F-negative (16/67; 24%). MPL mutation types were W515L (11), W515K (4), W515R (2) and W515A (1). One patient had both W515L and S505N MPL mutations and these were present in the same haemopoietic colonies. Real time PCR for JAK2 V617F analysis and HRM for MPL exon 10 status identified one or more clonal marker in 71% of patients. This combined genetic approach increases the sensitivity of meeting the World Health Organization diagnostic criteria for these myeloproliferative neoplasms.

Phospho-STAT5 and phospho-Akt expression in chronic myeloproliferative neoplasms.

The majority of Myeloproliferative Neoplasms (MPNs) are characterised by mutations in genes encoding molecules or receptors involved in cell signalling, the most common being the JAK2 V617F mutation. This mutation leads to ligand-independent activation of downstream signalling pathways by constitutive phosphorylation. The signalling pathways affected include the Janus kinase-signal transducers and activators of transcription (JAK-STAT) and phosphotidylinositide-3 kinase (PI3K) pathways, which regulate cell survival and apoptosis respectively. Monoclonal antibodies to phospho-STAT5 and phospho-Akt were generated and assessed by immunocytochemistry on bone marrow biopsies of MPN patients with JAK2 V617F, JAK2 exon 12, MPL exon 10 and KIT D816V mutations. JAK2 V617F mutation was associated with significantly increased levels of phosphorylated STAT5 and Akt in haemopoietic cells, most marked in megakaryocytes. In contrast, JAK2 exon 12 and MPL exon 10 mutations did not affect the level of phosphorylation. In systemic mastocytosis with KIT D618V mutation there was significantly increased expression of phosphorylated STAT5 and Akt in neoplastic mast cells although there was no change in the expression in other haemopoietic cells. JAK2 V617F is associated with upregulated phosphorylation of STAT5 and Akt in megakaryocytes, and to a lesser extent in other haemopoietic cells. Immunocytochemistry of bone marrow trephines for these phospho-proteins can be used as a supplementary diagnostic test with a high negative predictive value.

A practical strategy for the routine use of BIOMED-2 PCR assays for detection of B- and T-cell clonality in diagnostic haematopathology.

BIOMED-2 polymerase chain reaction (PCR) assays for clonality analysis of immunoglobulin (IG) and T-cell receptor (TCR) gene rearrangements were evaluated in routine haematopathological practice where paraffin-embedded tissues constitute the majority of specimens. One hundred and twenty-five fresh/frozen and 316 paraffin specimens were analysed for DNA quality and clonality. Seventy-nine per cent of paraffin specimens yielded PCR products of over 300 bp. These specimens and all fresh/frozen specimens were analysed with the complete set of BIOMED-2 reactions for IG (8 reactions) and/or TCR (6 reactions) gene rearrangements. The rate of detection of clonality was 96% in mature B-cell neoplasms and 98% in mature T-cell neoplasms and there were no significant differences in these rates between paraffin and fresh/frozen specimens. As the value of sole use of any individual BIOMED-2 reaction in clonality detection was limited, we assessed combinations of reactions that gave the greatest sensitivity with fewest reactions and were applicable for both fresh/frozen and paraffin specimens. For IG gene rearrangements, three reactions combining one targeting the IG heavy chain framework-2 region and two targeting the IG kappa locus achieved a 91% detection rate. For TCR gene rearrangements, the two TCR gamma reactions gave a 94% detection rate. We therefore recommend this strategy as the first-line assays for routine B- and T-cell clonality analysis in diagnostic haematopathology.

The incidence, indications and outcome for the non-operative management of breast cancer.

BACKGROUND AND OBJECTIVES: This study aimed to identify the proportion of patients with breast cancer who do not undergo primary operative treatment, to identify the reasons surgery is not performed, and to determine the outcome for this group of patients. METHODS: Data was obtained from the Bedford Breast Cancer Registry for all non-metastatic patients presenting between January 1990 and December 2004 who were initially treated non-operatively. Robust diagnostic, therapeutic, and follow-up data on all patients was collected prospectively during this period. RESULTS: One hundred and eighty-five out of 2110 episodes of breast cancer were treated non-operatively during this period. Sixty-eight percent of patients were unfit for surgery, 15% had inoperable tumours, and 17% refused surgical intervention. Median survival and 5-year survival rate for all non-operative patients were 3.7 years and 41.2%. Median survival for inoperable patients was 3.7 years, compared with 3.5 years for those unfit for surgery and 4.2 years for those who refused surgery. The 5-year survival rate for patients refusing surgery was 43%, compared with 61% for a matched group of patients undergoing standard surgical therapy. CONCLUSIONS: This study provides useful data on the reasons for, and outcome of, the non-operative management of breast cancer.

JAK2 exon 12 mutations in polycythemia vera and idiopathic erythrocytosis.

BACKGROUND: The V617F mutation, which causes the substitution of phenylalanine for valine at position 617 of the Janus kinase (JAK) 2 gene (JAK2), is often present in patients with polycythemia vera, essential thrombocythemia, and idiopathic myelofibrosis. However, the molecular basis of these myeloproliferative disorders in patients without the V617F mutation is unclear. METHODS: We searched for new mutations in members of the JAK and signal transducer and activator of transcription (STAT) gene families in patients with V617F-negative polycythemia vera or idiopathic erythrocytosis. The mutations were characterized biochemically and in a murine model of bone marrow transplantation. RESULTS: We identified four somatic gain-of-function mutations affecting JAK2 exon 12 in 10 V617F-negative patients. Those with a JAK2 exon 12 mutation presented with an isolated erythrocytosis and distinctive bone marrow morphology, and several also had reduced serum erythropoietin levels. Erythroid colonies could be grown from their blood samples in the absence of exogenous erythropoietin. All such erythroid colonies were heterozygous for the mutation, whereas colonies homozygous for the mutation occur in most patients with V617F-positive polycythemia vera. BaF3 cells expressing the murine erythropoietin receptor and also carrying exon 12 mutations could proliferate without added interleukin-3. They also exhibited increased phosphorylation of JAK2 and extracellular regulated kinase 1 and 2, as compared with cells transduced by wild-type JAK2 or V617F JAK2. Three of the exon 12 mutations included a substitution of leucine for lysine at position 539 of JAK2. This mutation resulted in a myeloproliferative phenotype, including erythrocytosis, in a murine model of retroviral bone marrow transplantation. CONCLUSIONS: JAK2 exon 12 mutations define a distinctive myeloproliferative syndrome that affects patients who currently receive a diagnosis of polycythemia vera or idiopathic erythrocytosis.

Identifying gene regulatory elements by genomic microarray mapping of DNaseI hypersensitive sites.

The identification of cis-regulatory elements is central to understanding gene transcription. Hypersensitivity of cis-regulatory elements to digestion with DNaseI remains the gold-standard approach to locating such elements. Traditional methods used to identify DNaseI hypersensitive sites are cumbersome and can only be applied to short stretches of DNA at defined locations. Here we report the development of a novel genomic array-based approach to DNaseI hypersensitive site mapping (ADHM) that permits precise, large-scale identification of such sites from as few as 5 million cells. Using ADHM we identified all previously recognized hematopoietic regulatory elements across 200 kb of the mouse T-cell acute lymphocytic leukemia-1 (Tal1) locus, and, in addition, identified two novel elements within the locus, which show transcriptional regulatory activity. We further validated the ADHM protocol by mapping the DNaseI hypersensitive sites across 250 kb of the human TAL1 locus in CD34+ primary stem/progenitor cells and K562 cells and by mapping the previously known DNaseI hypersensitive sites across 240 kb of the human alpha-globin locus in K562 cells. ADHM provides a powerful approach to identifying DNaseI hypersensitive sites across large genomic regions.