Molecular diagnostics of myeloproliferative neoplasms

Since the discovery of the JAK2 V617F mutation in the majority of the myeloproliferative neoplasms (MPN) of polycythemia vera, essential thrombocythemia and primary myelofibrosis ten years ago, further MPN-specific mutational events, notably in JAK2 exon 12, MPL exon 10 and CALR exon 9 have been identified. These discoveries have been rapidly incorporated into evolving molecular diagnostic algorithms. While many of these mutations appear to have prognostic implications, establishing MPN diagnosis is of immediate clinical importance with selection, implementation and the continual evaluation of the appropriate laboratory methodology to achieve this diagnosis similarly vital. The advantages and limitations of these approaches in identifying and quantitating the common MPN-associated mutations is considered herein with particular regard to their clinical utility. The evolution of molecular diagnostic applications and platforms has occurred in parallel with the discovery of MPN-associated mutations and it therefore appears likely that emerging technologies such as next-generation sequencing and digital PCR will in the future, play an increasing role in the molecular diagnosis of MPN. 

Duration-dependent effects of the BDNF Val66Met polymorphism on anodal tDCS induced motor cortex plasticity in older adults: a group and individual perspective

The brain derived neurotrophic factor (BDNF) Val66Met polymorphism and stimulation duration are thought to play an important role in modulating motor cortex plasticity induced by non-invasive brain stimulation (NBS). In the present study we sought to determine whether these factors interact or exert independent effects in older adults. Fifty-four healthy older adults (mean age = 66.85 years) underwent two counterbalanced sessions of 1.5 mA anodal transcranial direct current stimulation (atDCS), applied over left M1 for either 10 or 20 min. Single pulse transcranial magnetic stimulation (TMS) was used to assess corticospinal excitability (CSE) before and every 5 min for 30 min following atDCS. On a group level, there was an interaction between stimulation duration and BDNF genotype, with Met carriers (n = 13) showing greater post-intervention potentiation of CSE compared to Val66Val homozygotes homozygotes (n = 37) following 20 min (p = 0.002) but not 10 min (p = 0.219) of stimulation. Moreover, Met carriers, but not Val/Val homozygotes, exhibited larger responses to TMS (p = 0.046) after 20 min atDCS, than following 10 min atDCS. On an individual level, two-step cluster analysis revealed a considerable degree of inter-individual variability, with under half of the total sample (42%) showing the expected potentiation of CSE in response to atDCS across both sessions. Intra-individual variability in response to different durations of atDCS was also apparent, with one-third of the total sample (34%) exhibiting LTP-like effects in one session but LTD-like effects in the other session. Both the inter-individual (p = 0.027) and intra-individual (p = 0.04) variability was associated with BDNF genotype. In older adults, the BDNF Val66Met polymorphism along with stimulation duration appears to play a role in modulating tDCS-induced motor cortex plasticity. The results may have implications for the design of NBS protocols for healthy and diseased aged populations.

Role of early visual cortex in trans-saccadic memory of object features

Early visual cortex (EVC) participates in visual feature memory and the updating of remembered locations across saccades, but its role in the trans-saccadic integration of object features is unknown. We hypothesized that if EVC is involved in updating object features relative to gaze, feature memory should be disrupted when saccades remap an object representation into a simultaneously perturbed EVC site. To test this, we applied transcranial magnetic stimulation (TMS) over functional magnetic resonance imaging-localized EVC clusters corresponding to the bottom left/right visual quadrants (VQs). During experiments, these VQs were probed psychophysically by briefly presenting a central object (Gabor patch) while subjects fixated gaze to the right or left (and above). After a short memory interval, participants were required to detect the relative change in orientation of a re-presented test object at the same spatial location. Participants either sustained fixation during the memory interval (fixation task) or made a horizontal saccade that either maintained or reversed the VQ of the object (saccade task). Three TMS pulses (coinciding with the pre-, peri-, and postsaccade intervals) were applied to the left or right EVC. This had no effect when (a) fixation was maintained, (b) saccades kept the object in the same VQ, or (c) the EVC quadrant corresponding to the first object was stimulated. However, as predicted, TMS reduced performance when saccades (especially larger saccades) crossed the remembered object location and brought it into the VQ corresponding to the TMS site. This suppression effect was statistically significant for leftward saccades and followed a weaker trend for rightward saccades. These causal results are consistent with the idea that EVC is involved in the gaze-centered updating of object features for trans-saccadic memory and perception.

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.

Clinical end points for drug treatment trials in BCR-ABL1-negative classic myeloproliferative neoplasms: consensus statements from European LeukemiaNET (ELN) and Internation Working Group-Myeloproliferative Neoplasms Research and Treatment (IWG-MRT)

The discovery of somatic mutations, primarily JAK2V617F and CALR, in classic BCR-ABL1-negative myeloproliferative neoplasms (MPNs) has generated interest in the development of molecularly targeted therapies, whose accurate assessment requires a standardized framework. A working group, comprised of members from European LeukemiaNet (ELN) and International Working Group for MPN Research and Treatment (IWG-MRT), prepared consensus-based recommendations regarding trial design, patient selection and definition of relevant end points. Accordingly, a response able to capture the long-term effect of the drug should be selected as the end point of phase II trials aimed at developing new drugs for MPNs. A time-to-event, such as overall survival, or progression-free survival or both, as co-primary end points, should measure efficacy in phase III studies. New drugs should be tested for preventing disease progression in myelofibrosis patients with early disease in randomized studies, and a time to event, such as progression-free or event-free survival should be the primary end point. Phase III trials aimed at preventing vascular events in polycythemia vera and essential thrombocythemia should be based on a selection of the target population based on new prognostic factors, including JAK2 mutation. In conclusion, we recommended a format for clinical trials in MPNs that facilitates communication between academic investigators, regulatory agencies and drug companies.

The role of the cerebral cortex in postural responses to externally induced perturbations

The ease with which we avoid falling down belies a highly sophisticated and distributed neural network for controlling reactions to maintain upright balance. Although historically these reactions were considered within the sub cortical domain, mounting evidence reveals a distributed network for postural control including a potentially important role for the cerebral cortex. Support for this cortical role comes from direct measurement associated with moments of induced instability as well as indirect links between cognitive task performance and balance recovery. The cerebral cortex appears to be directly involved in the control of rapid balance reactions but also setting the central nervous system in advance to optimize balance recovery reactions even when a future threat to stability is unexpected. In this review the growing body of evidence that now firmly supports a cortical role in the postural responses to externally induced perturbations is presented. Moreover, an updated framework is advanced to help understand how cortical contributions may influence our resistance to falls and on what timescale. The implications for future studies into the neural control of balance are discussed.

Timing of response differentiation in human motor cortex during a speeded Go/No-go task

We explored the brain's ability to quickly prevent a pre-potent but unwanted motor response. To address this, transcranial magnetic stimulation was delivered over the motor cortex (hand representation) to probe excitability changes immediately after somatosensory cues prompted subjects to either move as fast as possible or withhold movement. Our results showed a difference in motor cortical excitability 90 ms post-stimulus contingent on cues to either promote or prevent movement. We suggest that our study design emphasizing response speed coupled with well-defined early probes allowed us to extend upon similar past investigations into the timing of response inhibition.

The Calreticulin gene and myeloproliferative neoplasms

The Philadelphia negative myeloproliferative neoplasms include polycythaemia vera (PV), essential thrombocytopenia (ET) and primary myelofibrosis (PMF). Patients with these conditions were mainly thought to harbour JAK2V617F mutations or an Myeloproliferative leukaemia (MPL) substitution. In 2013, two revolutionary studies identified recurrent mutations in a gene that encodes the protein calreticulin (CALR). This mutation was detected in patients with PMF and ET with non-mutated JAK2 or MPL but was absent in patients with PV. The CALR gene encodes the calreticulin protein, which is a multifactorial protein, mainly located in the endoplasmic reticulum in chromosome 19 and regulates calcium homeostasis, chaperones and has also been implicated in multiple cellular processes including cell signalling, regulation of gene expression, cell adhesion, autoimmunity and apoptosis. Somatic 52 bp deletions and recurrent 52 bp insertion mutations in CALR were detected and all resulted in frameshift and clusters in exon 9 of the gene. This review will summarise the current knowledge on the CALR gene and mutation of the gene in pathological conditions and patient phenotypes.