Transcription factor 3 is dysregulated in megakaryocytes in myelofibrosis.

Transcription factor 3 (TCF3) is a DNA transcription factor that modulates megakaryocyte development. Although abnormal TCF3 expression has been identified in a range of hematological malignancies, to date, it has not been investigated in myelofibrosis (MF). MF is a Philadelphia-negative myeloproliferative neoplasm (MPN) that can arise de novo or progress from essential thrombocythemia [ET] and polycythemia vera [PV] and where dysfunctional megakaryocytes have a role in driving the fibrotic progression. We aimed to examine whether TCF3 is dysregulated in megakaryocytes in MPN, and specifically in MF. We first assessed TCF3 protein expression in megakaryocytes using an immunohistochemical approach analyses and showed that TCF3 was reduced in MF compared with ET and PV. Further, the TCF3-negative megakaryocytes were primarily located near trabecular bone and had the typical "MF-like" morphology as described by the WHO. Genomic analysis of isolated megakaryocytes showed three mutations, all predicted to result in a loss of function, in patients with MF; none were seen in megakaryocytes isolated from ET or PV marrow samples. We then progressed to transcriptomic sequencing of platelets which showed loss of TCF3 in MF. These proteomic, genomic and transcriptomic analyses appear to indicate that TCF3 is downregulated in megakaryocytes in MF. This infers aberrations in megakaryopoiesis occur in this progressive phase of MPN. Further exploration of this pathway could provide insights into TCF3 and the evolution of fibrosis and potentially lead to new preventative therapeutic targets.

What is the context? We investigated TCF3 (transcription factor 3), a gene that regulates megakaryocyte development, for genomic and proteomic changes in myelofibrosis.Myelofibrosis is the aggressive phase of a group of blood cancers called myeloproliferative neoplasms, and abnormalities in development and maturation of megakaryocytes is thought to drive the development of myelofibrosis.What is new? We report detection of three novel TCF3 mutations in megakaryocytes and decreases in TCF3 protein and gene expression in primary megakaryocytes and platelets from patients with myelofibrosis.This is the first association between loss of TCF3 in megakaryocytes from patients and myelofibrosis.What is the impact? TCF3 dysregulation may be a novel mechanism that is responsible for the development of myelofibrosis and better understanding of this pathway could identify new drug targets.

Imaging flow cytometric detection of del(17p) in bone marrow and circulating plasma cells in multiple myeloma.

BACKGROUND: Detection of del(17p) in myeloma is generally performed by fluorescence in situ hybridization (FISH) on a slide with analysis of up to 200 nuclei. The small cell sample analyzed makes this a low precision test. We report the utility of an automated FISH method, called "immuno-flowFISH", to detect plasma cells with adverse prognostic risk del(17p) in bone marrow and blood samples of patients with myeloma. METHODS: Bone marrow (n = 31) and blood (n = 19) samples from 35 patients with myeloma were analyzed using immuno-flowFISH. Plasma cells were identified by CD38/CD138-immunophenotypic gating and assessed for the 17p locus and centromere of chromosome 17. Cells were acquired on an AMNIS ImageStreamX MkII imaging flow cytometer using INSPIRE software. RESULTS: Chromosome 17 abnormalities were identified in CD38/CD138-positive cells in bone marrow (6/31) and blood (4/19) samples when the percent plasma cell burden ranged from 0.03% to 100% of cells. Abnormalities could be identified in 14.5%-100% of plasma cells. CONCLUSIONS: The "immuno-flowFISH" imaging flow cytometric method could detect del(17p) in plasma cells in both bone marrow and blood samples of myeloma patients. This method was also able to detect gains and losses of chromosome 17, which are also of prognostic significance. The lowest levels of 0.009% (bone marrow) and 0.001% (blood) for chromosome 17 abnormalities was below the detection limit of current FISH method. This method offers potential as a new means of identifying these prognostically important chromosomal defects, even when only rare cells are present and for serial disease monitoring.

PlateletSeq: A novel method for discovery of blood-based biomarkers.

Platelets are small circulating fragments of cells that play important roles in thrombosis, haemostasis, immune response, inflammation and cancer growth. Although anucleate, they contain a rich RNA repertoire which offers an opportunity to characterise changes in platelet gene expression in health and disease. Whilst this can be achieved with conventional RNA sequencing, a large input of high-quality RNA, and hence blood volume, is required (unless a pre-amplification step is added), along with specialist bioinformatic skills for data analysis and interpretation. We have developed a transcriptomics next-generation sequencing-based approach that overcomes these limitations. Termed PlateletSeq, this method requires very low levels of RNA input and does not require specialist bioinformatic analytical skills. Here we describe the methodology, from sample collection to processing and data analysis. Specifically, blood samples can be stored for up to 8 days at 4 °C prior to analysis. Platelets are isolated using multi-step centrifugation and a purity of ≤ 1 leucocyte per 0.26x106 platelets is optimal for gene expression analysis. We have applied PlateletSeq to normal adult blood samples and show there are no age-associated variations and only minor gender-associated differences. In contrast, platelets from patients with myeloproliferative neoplasms show differences in platelet transcript profiles from normal and between disease subtypes. This illustrates the potential applicability of PlateletSeq for biomarker discovery and studying platelet biology in patient samples. It also opens avenues for assessing platelet quality in other fields such as transfusion research.

Jumping translocation involving chromosome 13q in a patient with Crohn's Disease and inv(16)(p13.1q22)/CBFB-MYH11 acute myeloid leukemia.

Jumping translocations (JT) are rare chromosomal rearrangements caused by the translocation of one donor chromosome segment to two or more recipient chromosomes. In the setting of myeloid neoplasms, JT are typically associated with disease transformation to acute myeloid leukemia (AML), and studies to date have found JT to be associated with poor prognosis and short overall survival. However, JT have been only very rarely reported in AML associated with a favorable AML prognostic cytogenetic marker. Additionally, JT have infrequently been described in hematological malignancies associated with autoimmune diseases (AID) such as Crohn's Disease (CD). Here we describe a case of a 40-year-old female with a 24-year history of CD diagnosed with AML harbouring the inv(16)(p13.1q22)/CBFB-MYH11 rearrangement in conjunction with sideline clones containing trisomy 13, tetrasomy 13, and a JT of chromosome 13q12 jumping to 7q32 and 18p11.2. The patient attained molecular remission one month post diagnosis after induction 7 + 3 chemotherapy. Morphologic relapse of disease occurred 27 months post diagnosis. A second molecular remission was attained 3 months later after re-induction chemotherapy. The patient received a sibling bone marrow transplant 32 months post diagnosis and is currently in remission 7 months post allogeneic transplant. To the best of our knowledge, this case represents the first report of JT occurring in inv(16)(p13.1q22)/CBFB-MYH11 AML and the second of JT occurring in an AML patient with prior clinical history of CD. This case provides further insight into the rare occurrence of JT in AML, particularly AML with a favorable cytogenetic marker in conjunction with AID.

Optimal enoxaparin dosing strategies for venous thromboembolism prophylaxis and treatment of high body weight patients.

OBJECTIVE: Obesity is a risk factor for developing venous thromboembolism (VTE). Optimal dosage of enoxaparin has not been established in the obese population. We aimed to study clinical outcomes and complications with enoxaparin in obese patients. METHODS: A retrospective, single centre observational study of obese patients treated with enoxaparin for VTE (n = 47) using a body mass index (BMI)-stratified dosing, thromboprophylaxis (n = 46), and non-obese controls (n = 20) was performed. Anti-Xa was used to measure enoxaparin efficacy. RESULTS: Patients with a median BMI of 36.3 kg/m2 (range 30-52.7) with a median weight of 136 kg (range 68-240) received therapeutic enoxaparin at median 120 mg BID (range 60-200). A median targeted anti-Xa level of 0.79 (95% CI 0.72-1.03) IU/mL was achieved in 58% of patients. Dose reduction, or increase was needed in 25%, and 16% patients respectively. Mild or major haemorrhage, or VTE occurred in 10%, 2% and 2% patients respectively. Patients with a median weight of 160 kg (range 130-245) received thromboprophylaxis with 40 mg BID enoxaparin. Targeted median anti-Xa of 0.22 IU/mL (95% CI 0.19-0.24) was achieved in 59% patients. Mild haemorrhage was seen in 2%, while none developed major haemorrhage or VTE. Control patients who received enoxaparin 40 mg daily did not develop VTE; 5% had minor bleeding events. CONCLUSIONS: BMI-stratified therapeutic enoxaparin dosing regimen is safe and effective therapy in obese patients. Fixed dosing without monitoring may not be appropriate. Thromboprophylaxis with 40 mg BID in obese patients was efficacious in preventing VTE without excess bleeding compared to control patients.

Platelets in myeloproliferative neoplasms have a distinct transcript signature in the presence of marrow fibrosis.

Marrow fibrosis is a significant complication of myeloproliferative neoplasms (MPN) that affects up to 20% of patients and is associated with a poor prognosis. The pathological processes that lead to fibrotic progression are not well understood, but megakaryocytes have been implicated in the process. The aim of this study was to determine whether platelets, derived from megakaryocytes, have transcriptomic alterations associated with fibrosis. Platelets from MPN patients with and without fibrosis and non-malignant control individuals were assessed using next generation sequencing. Results from the initial training cohort showed discrete changes in platelet transcripts in the presence of marrow fibrosis. We identified more than 1000 differentially expressed transcripts from which a putative 3-gene fibrotic platelet signature (CCND1, H2AX [previously termed H2AFX] and CEP55) could be identified. This fibrosis-associated signature was assessed blinded on platelets from an independent test MPN patient cohort. The 3-gene signature was able to discriminate between patients with and without marrow fibrosis with a positive predictive value of 71% (93% specificity, 71% sensitivity). This demonstrates that assessment of dysregulated transcripts in platelets may be a useful monitoring tool in MPN to identify progression to marrow fibrosis. Further, sequential monitoring could have clinical applications for early prediction of progression to fibrosis.

"Immuno-flowFISH" for the Assessment of Cytogenetic Abnormalities in Chronic Lymphocytic Leukemia.

Imaging flow cytometry is emerging as a diagnostic tool for the assessment of leukemia. It has the functionality of standard flow cytometry and generates high-resolution digital images of each cell with quantifiable numerical data. We demonstrate the use of an automated high-throughput method for performing fluorescence in situ hybridization (FISH) on immunophenotyped whole cells in suspension and analyzed by imaging flow cytometry, a technique called "Immuno-flowFISH". The aim of this study was to demonstrate the application of immuno-flowFISH for the detection of chromosomal abnormalities in CLL, specifically trisomy 12 and del(17p). Mononuclear cells were isolated and immunophenotyped with fluorescently conjugated CD3, CD5, and CD19 monoclonal antibodies. Following fixation, cells were permeabilized, dsDNA denatured and hybridized with chromosome 12 or 17 enumeration (CEP 12 and CEP17) and 17p12 locus-specific FISH probes. Cells were analyzed on the Amnis ImageStream®X Mark II to assess the number and percent FISH-positive CLL cells and the ratio of FISH spot counts for CD5/CD19-positive CLL cells to CD3/CD5-positive T cells (FISH "mean spot ratio"). Deletion of 17p was detected in about 8% of cases to date, with del(17p) ranged from 3.5-22.8% and the FISH "mean spot ratio" 0.86-0.96. Immuno-flowFISH also detected a minimal residual disease case with +12 with a limit of detection of 0.13% and a rare case that presented with atypical phenotype and cytogenetics. Immuno-flowFISH could detect del(17p) in phenotypically identified CD5/CD19-positive B-cells. The 100-fold increase in analyzed cells, as well as the addition of cell phenotype increased the sensitivity and specificity over current clinical FISH testing. Furthermore, immuno-flowFISH analysis demonstrated specific utility in unique clinical scenarios such as residual disease and atypical biology cases which may be of significant benefit with regards to prognostication and MRD analysis. The method will assist in therapeutic decision making and disease monitoring for many hematological malignancies. © 2019 International Society for Advancement of Cytometry.

Imaging flow cytometry to assess chromosomal abnormalities in chronic lymphocytic leukaemia.

Chronic Lymphocytic Leukaemia (CLL), the most common leukaemia in the Western world, has a characteristic phenotype and prognosis largely defined by the presence of cytogenetic aberrations. The gold standard for detecting these cytogenetic abnormalities is interphase fluorescence in situ hybridisation (FISH) performed on cell smears or tissue sections on glass slides. Fluorescently labelled DNA probes bind to specific chromosomal regions and the signal detected by fluorescent microscopy. Generally only 200 cells are assessed and the limit of sensitivity is 3% positive cells. Here we report the development and use of imaging flow cytometry to assess chromosomes by FISH in phenotyped CLL cells in suspension. Thousands of CLL cells, identified by their phenotype, are assessed for specific FISH probe signals using an automated, high throughput imaging flow cytometer. The "extended depth of field" capability of the imaging flow cytometer enables FISH probe signals ("spots") to be resolved and localised within the (stained) nucleus of the immunophenotyped cells. We report the development of the automated "immuno-flowFISH" on normal blood using the Amnis ImageStreamX mark II platform and illustrate the clinical application of the method for the assessment of chromosome 12 in CLL. It is a powerful new method which has potential to be applied at diagnosis for disease stratification, and following treatment to assess residual disease. These applications will assist clinicians in optimising therapeutic decision making and thereby improve patient outcome.

Blood use in patients receiving intensive chemotherapy for acute leukemia or hematopoietic stem cell transplantation: the impact of a health system-wide patient blood management program.

BACKGROUND: Little is published on patient blood management (PBM) programs in hematology. In 2008 Western Australia announced a health system-wide PBM program with PBM staff appointments commencing in November 2009. Our aim was to assess the impact this program had on blood utilization and patient outcomes in intensive chemotherapy for acute leukemia or hematopoietic stem cell transplantation. STUDY DESIGN AND METHODS: A retrospective study of 695 admissions at two tertiary hospitals receiving intensive chemotherapy for acute leukemia or undergoing hematopoietic stem cell transplantation between July 2010 and December 2014 was conducted. Main outcomes included pre-red blood cell (RBC) transfusion hemoglobin (Hb) levels, single-unit RBC transfusions, number of RBC and platelet (PLT) units transfused per admission, subsequent day case transfusions, length of stay, serious bleeding, and in-hospital mortality. RESULTS: Over the study period, the mean RBC units transfused per admission decreased 39% from 6.1 to 3.7 (p < 0.001), and the mean PLT units transfused decreased 35% from 6.3 to 4.1 (p < 0.001), with mean RBC and PLT units transfused for follow-up day cases decreasing from 0.6 to 0.4 units (p < 0.001). Mean pre-RBC transfusion Hb level decreased from 8.0 to 6.8 g/dL (p < 0.001), and single-unit RBC transfusions increased 39% to 67% (p < 0.001). This reduction represents blood product cost savings of AU$694,886 (US$654,007). There were no significant changes in unadjusted or adjusted length of stay, serious bleeding events, or in-hospital mortality over the study. CONCLUSION: The health system-wide PBM program had a significant impact, reducing blood product use and costs without increased morbidity or mortality in patients receiving intensive chemotherapy for acute leukemia or hematopoietic stem cell transplantation.

Megakaryocytes in Myeloproliferative Neoplasms Have Unique Somatic Mutations.

Myeloproliferative neoplasms (MPNs) are a group of related clonal hemopoietic stem cell disorders associated with hyperproliferation of myeloid cells. They are driven by mutations in the hemopoietic stem cell, most notably JAK2V617F, CALR, and MPL. Clinically, they have the propensity to progress to myelofibrosis and transform to acute myeloid leukemia. Megakaryocytic hyperplasia with abnormal features are characteristic, and it is thought that these cells stimulate and drive fibrotic progression. The biological defects underpinning this remain to be explained. In this study we examined the megakaryocyte genome in 12 patients with MPNs to determine whether there are somatic variants and whether there is any association with marrow fibrosis. We performed targeted next-generation sequencing for 120 genes associated with myeloid neoplasms on megakaryocytes isolated from aspirated bone marrow. Ten of the 12 patients had genomic defects in megakaryocytes that were not present in nonmegakaryocytic hemopoietic marrow cells from the same patient. The greatest allelic burden was in patients with increased reticulin deposition. The megakaryocyte-unique mutations were predominantly in genes that regulate chromatin remodeling, chromosome alignment, and stability. These findings show that genomic abnormalities are present in megakaryocytes in MPNs and that these appear to be associated with progression to bone marrow fibrosis.

Angiotensin-converting enzyme inhibitor and visceral angio-oedema.

Abdominal pain can be a challenging presenting complaint with a broad differential diagnosis. Medication side-effect must always be considered. Visceral angio-oedema secondary to angiotensin-converting enzyme inhibitor use can cause abdominal pain. The association of angiotensin-converting enzyme inhibitor and visceral angio-oedema is not well recognized partly because the onset of angio-oedema might be delayed for months or years after commencement of an angiotensin-converting enzyme inhibitor. The epidemiology of angio-oedema is now changing in parallel with the increasing use of angiotensin-converting enzyme inhibitors. We present a case of visceral angio-oedema secondary to perindopril. This diagnosis requires a high index of suspicion because if not recognized early patients undergo extensive and expensive negative evaluation.

Topology optimisation of spinal interbody cage for reducing stress shielding effect.

The stress shielding effect is an event in which the replacement implant limits the load transferred to bone and the ineffective stress in the vertebrae causes bony growth to cease. In the present study, a 3D finite element L4-L5 model was developed and subjected to a 1200 N compression preload. Five groups of muscle forces were applied on L4 under flexion-extension, lateral bending and axial rotation. Topology optimisation was employed for reducing the stress shielding effect by removing the ineffective material from the design domain. The optimised design was designed with polyaryletheretherketone (PEEK) titanium and cortical materials to encounter the shielding response. The stress responses show that the new design increased the stress magnitude by at least 17.10, 18.11 and 18.43% in 4 Nm of flexion-extension, lateral bending and axial rotation, respectively. In conclusion, the material factor did not significantly alter the stress magnitude, but volume was the key factor in reducing the stress shielding effect.