Nicola J. Weston-Bell

Report of the European Myeloma Network on multiparametric flow cytometry in multiple myeloma and related disorders

In multiple myeloma, the use of multiparametric flow cytometry in many laboratories is currently restricted to clinical research studies and the differential diagnosis of unusual cases. This article report the indications of the European Myeloma Network for flow cytometry in patients with monoclonal gammopathies, and the technical recommendations for the analysis of plasma cells. The European Myeloma Network (EMN) organized two flow cytometry workshops. The first aimed to identify specific indications for flow cytometry in patients with monoclonal gammopathies, and consensus technical approaches through a questionnaire-based review of current practice in participating laboratories. The second aimed to resolve outstanding technical issues and develop a consensus approach to analysis of plasma cells. The primary clinical applications identified were: differential diagnosis of neoplastic plasma cell disorders from reactive plasmacytosis; identifying risk of progression in patients with MGUS and detecting minimal residual disease. A range of technical recommendations were identified, including: 1) CD38, CD138 and CD45 should all be included in at least one tube for plasma cell identification and enumeration. The primary gate should be based on CD38 vs. CD138 expression; 2) after treatment, clonality assessment is only likely to be informative when combined with immunophenotype to detect abnormal cells. Flow cytometry is suitable for demonstrating a stringent complete remission; 3) for detection of abnormal plasma cells, a minimal panel should include CD19 and CD56. A preferred panel would also include CD20, CD117, CD28 and CD27; 4) discrepancies between the percentage of plasma cells detected by flow cytometry and morphology are primarily related to sample quality and it is, therefore, important to determine that marrow elements are present in follow-up samples, particularly normal plasma cells in MRD negative cases.

Exome sequencing in tracking clonal evolution in multiple myeloma following therapy

Sequencing the tumor genome using next-generation sequencing (NGS) is providing an unparalleled insight into the pathogenesis and progression of the disease. Much of the current focus of NGS in cancer is on defining mutations in the tumor genome at disease presentation, and these findings are central to understanding the molecular mechanisms that underlie pathogenesis.

Defining origins of malignant B cells: a new circulating normal human IgM(+)D(+) B-cell subset lacking CD27 expression and displaying somatically mutated IGHV genes as a relevant memory population.

In probing the cell of origin in malignant B cells, an imprint of somatic hypermutation (SHM) in immunoglobulin (Ig) variable (V) region genes delineates antigen encounter, and identifying the precise pathway generating SHM in the normal B-cell counterpart becomes relevant. SHM remains the definitive memory imprint in normal human B cells, but CD27 expression also delineates memory. Recently, dye extrusion adenosine triphosphate-binding transporter assays identified circulating isotype-switched memory B cells that lacked CD27, yet exhibited low levels of SHM. To extend findings, we report a pre-switched CD27−ve circulating memory B-cell population in normal blood using comparable assays, and isolated CD19+IgM+D+CD27−ve cells (>99% purity) for the analysis of IGHV5/IGHV3-IGHM transcripts. Of these (n=334), ∼78% were germ line and naive B cell derived. Strikingly, 21.9% of the transcripts were mutated. They showed 3–5 mutations (13.5% of sequences) and >5 mutations (8.4% of sequences) per transcript. Accrual of mutations in a subset of CD19+IgM+D+CD27−ve cells define a new circulating pre-switched memory B-cell pool, present in substantial numbers in the population harboring naive B cells. These CD19+IgM+D+CD27−ve memory B cells may have a distinct lineage and function, and seem relevant to understanding origins of malignant B cells, in particular those of hairy cell leukemia cells, which display mutated V genes yet lack CD27 expression.

Differential mRNA splicing and precursor processing of neurokinin B in neuroendocrine tissues

The tachykinin neurokinin B which is encoded on the tachykinin 3 precursor, has prominent roles in both neuronal and endocrine systems, yet little is known about its evolution, potential splice variants and the manner in which it is processed. Here, we deduce the diversity within the vertebrate tachykinin 3 precursors, and identify novel tachykinin 3 splice variants and precursors. A total of 35 different tachykinin 3 precursors were identified in mammals, birds and reptiles. Nine additional alternatively spliced tachykinin 3 mRNA transcripts were also discovered in humans leading to the formation of three tachykinin 3 precursors (named alpha, beta and gamma tachykinin 3), but no novel tachykinin. gamma tachykinin 3, albeit rarer, was not found to encode neurokinin B. Differential processing of the tachykinin 3 precursor in the human placenta leads to the formation of potential NH2-terminally extended forms of neurokinin B. Moreover, we found increased proteolytic cleavage of the tachykinin 3 precursor during the pregnancy syndrome of pre-eclampsia. We have established neurokinin B to be an evolutionarily conserved peptide, nonetheless the significance of the three different tachykinin 3 precursors is not clear, but could represent an evolutionarily redundant splicing mechanism once employed by an ancestral gene that encoded two tachykinins. Our results indicate that differential mRNA splicing and precursor processing is likely to play an important role in differentiating the actions of the tachykinin 3 gene products in both neuronal and endocrine tissues.

Hairy cell leukemia cell lines expressing annexin A1 and displaying B-cell receptor signals characteristic of primary tumor cells lack the signature BRAF mutation to reveal unrepresentative origins

Hairy cell leukemia cell lines expressing annexin A1 and displaying B-cell receptor signals characteristic of primary tumor cells lack the signature BRAF mutation to reveal unrepresentative origins

CD27 in Defining Memory B-Cell Origins in Waldenström's Macroglobulinemia

CD27 is a tumor necrosis factor receptor family glycoprotein, identified in seminal studies as an apparently robust marker for normal memory B cells. Somatic hypermutation (SHM) in immunoglobulin variable (V) region genes, however, remains the definitive memory imprint. In Waldenströms macroglobulinemia (WM), SHM defines a predominant mutated (MUT) subset and a minor unmutated subset indicative of naive B-cell origin. In MUT-WM, tumor cells can lack CD27 expression, raising suggestions of unusual memory B-cell origins. We recently identified such normal IgM+D+CD27-ve memory B-cells, with low levels of SHM in VH genes. While these could seed WM, the possibility remains that WM could derive from classical memory B cells that shed CD27. The utility of CD27 expression in defining memory in MUT-WM origins, then, is uncertain, but SHM unequivocally defines memory B-cell derivation in most WM. Patterns of SHM and additional IgH locus events furthermore reveal ongoing intra-tumoral diversification in WM.

Variant B cell receptor isotype functions differ in hairy cell leukemia with mutated BRAF and IGHV genes.

A functional B-cell receptor (BCR) is critical for survival of normal B-cells, but whether it plays a comparable role in B-cell malignancy is as yet not fully delineated. Typical Hairy Cell Leukemia (HCL) is a rare B-cell tumor, and unique in expressing multiple surface immunoglobulin (sIg) isotypes on individual tumor cells (mult-HCL), to raise questions as to their functional relevance. Typical mult-HCL also displays a mutated BRAF V(600)E lesion. Since wild type BRAF is a primary conduit for transducing normal BCR signals, as revealed by deletion modelling studies, it is as yet not apparent if mutated BRAF alters BCR signal transduction in mult-HCL. To address these questions, we examined BCR signalling in mult-HCL cases uniformly displaying mutated BRAF and IGHV genes. Two apparent functional sets were delineated by IgD co-expression. In sIgD+ve mult-HCL, IgD mediated persistent Ca2+ flux, also evident via >1 sIgH isotype, linked to increased ERK activation and BCR endocytosis. In sIgD−ve mult-HCL however, BCR-mediated signals and downstream effects were restricted to a single sIgH isotype, with sIgM notably dysfunctional and remaining immobilised on the cell surface. These observations reveal discordance between expression and function of individual isotypes in mult-HCL. In dual sIgL expressing cases, only a single sIgL was fully functional. We examined effects of anti-BCR stimuli on mult-HCL survival ex-vivo. Significantly, all functional non-IgD isotypes increased ERK1/2 phosphorylation but triggered apoptosis of tumor cells, in both subsets. IgD stimuli, in marked contrast retained tumor viability. Despite mutant BRAF, BCR signals augment ERK1/2 phosphorylation, but isotype dictates functional downstream outcomes. In mult-HCL, sIgD retains a potential to transduce BCR signals for tumor survival in-vivo. The BCR in mult-HCL emerges as subject to complex regulation, with apparent conflicting signalling by individual isotypes when co-expressed with sIgD. This suggests the possibility that mutant BRAF by-passes BCR constraints in mult-HCL.

Exome Sequencing in Classic Hairy Cell Leukaemia Reveals Widespread Variation in Acquired Somatic Mutations between Individual Tumours Apart from the Signature BRAF V(600)E Lesion.

In classic Hairy cell leukaemia (HCLc), a single case has thus far been interrogated by whole exome sequencing (WES) in a treatment naive patient, in which BRAF V(600)E was identified as an acquired somatic mutation and confirmed as occurring near-universally in this form of disease by conventional PCR-based cohort screens. It left open however the question whether other genome-wide mutations may also commonly occur at high frequency in presentation HCLc disease. To address this, we have carried out WES of 5 such typical HCLc cases, using highly purified splenic tumour cells paired with autologous T cells for germline. Apart from BRAF V(600)E, no other recurrent somatic mutation was identified in these HCLc exomes, thereby excluding additional acquired mutations as also prevalent at a near-universal frequency in this form of the disease. These data then place mutant BRAF at the centre of the neoplastic drive in HCLc. A comparison of our exome data with emerging genetic findings in HCL indicates that additional somatic mutations may however occur recurrently in smaller subsets of disease. As mutant BRAF alone is insufficient to drive malignant transformation in other histological cancers, it suggests that individual tumours utilise largely differing patterns of genetic somatic mutations to coalesce with BRAF V(600)E to drive pathogenesis of malignant HCLc disease.

Immunochemical Methods for the Peptidomic Analysis of Tachykinin Peptides and Their Precursors

The tachykinins represent the largest known peptide family and are responsible for a range of pleiotropic functions in both vertebrates and invertebrates. Recent research has shown a diversity of mechanisms such as mRNA splicing, precursor processing and post-translation modification that can lead to a complex and continually expanding repertoire of tachykinin peptides. The peptidomic analysis of the tachykinins has been hindered by the lack of specific methodologies to capture, purify and characterise each tachykinin. This chapter summarises some of the methods that have been developed in order to further purify and characterise individual groups of tachykinin peptides from the peptidome.

Single cell exomes in an index case of amp1q21 multiple myeloma reveals more diverse mutanomes than the whole population

TO THE EDITOR: A focus on defining genomic mutations at the whole population level in cancer may underestimate clonal plasticity in the tumor genome and how it generates genetic variation between individual single cells (SCs). This may have an impact on understanding cancer evolution and biological