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.

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.

Chromosomal defects in multiple myeloma.

Multiple myeloma is a plasma cell neoplasm driven by primary (e.g. hyperdiploidy; IGH translocations) and secondary (e.g. 1q21 gains/amplifications; del(17p); MYC translocations) chromosomal events. These are important to detect as they influence prognosis, therapeutic response and disease survival. Currently, cytogenetic testing is most commonly performed by interphase fluorescence in situ hybridisation (FISH) on aspirated bone marrow samples. A number of variations to FISH methodology are available, including prior plasma cell enrichment and incorporation of immunophenotypic plasma cell identification. Other molecular methods are increasingly being utilised to provide a genome-wide view at high resolution (e.g. single nucleotide polymorphism (SNP) microarray analysis) and these can detect abnormalities in most cases. Despite their wide application at diagnostic assessment, both FISH and SNP-array have relatively low sensitivity, limiting their use for identification of prognostically significant low-level sub-clones or for disease monitoring. Next-generation sequencing is increasingly being used to detect mutations and new FISH techniques such as by flow cytometry are in development and may address some of the current test limitations. Here we review the primary and secondary cytogenetic aberrations in myeloma and discuss the range of techniques available for their assessment.

Variability of bone marrow biopsy reporting affects accuracy of diagnosis of myeloproliferative neoplasms: data from the ALLG MPN01 registry.

The Philadelphia-negative myeloproliferative neoplasms (MPN) are a heterogeneous group of overlapping bone marrow disorders defined by characteristic peripheral blood counts and bone marrow morphological findings in conjunction with recurrent somatic mutations. The accurate diagnosis and subclassification of MPN relies upon careful reporting of bone marrow morphology combined with ancillary information in an integrated pathology report. This co-operative trial group study ALLG MPN01 (ANZCTR:12613000138785), led by the Australasian Leukaemia & Lymphoma Group (ALLG), aimed to describe the current approach to diagnosis of MPN in routine practice. Specifically, we assessed the frequency with which bone marrow biopsies were performed, and the adherence of reporting pathologists to recommendations contained in the revised 2016 WHO classification pertaining to MPN. We reviewed the diagnosis of 152 patients from eight institutions who were enrolled in a national MPN registry of the ALLG between 2010 and 2016. The ALLG MPN01 registry is now closed to recruitment. Key features were extracted from pathology reports provided to the registry. Bone marrow biopsies were performed in 112/152 cases (74%). The pathological information entered was concordant with the stated clinical diagnosis in 75/112 cases (67%). The main reasons for discordant results were incomplete descriptions of megakaryocyte topography and morphology, inconsistent grading of reticulin fibrosis, and failure to integrate the available morphological and ancillary clinicopathological information. In this retrospective audit, 26% of MPN patients did not undergo a diagnostic bone marrow biopsy. In those who did, the specific MPN subtype may not have been reported correctly in 33% of cases, as evidenced by inconsistent features reported or insufficient information to assess. A more standardised approach to bone marrow reporting is required to ensure accuracy of MPN diagnoses and consistent reporting to cancer registries and clinical trials.

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.

Chimeric antigen receptor T-cells: Properties, production, and quality control.

Chimeric antigen receptor (CAR) T-cell therapy is a novel adoptive T-cell immunotherapy for haematological malignancies. First introduced into clinical practice in 2017, CAR T-cell therapy is now finding its place in the management of lymphoid malignancies, primarily of B-cell lineage, including lymphoblastic leukaemia, non-Hodgkin lymphoma and plasma cell myeloma, with remarkable therapeutic outcomes. CAR T-cells are a customised therapeutic product for each patient. Manufacture commences with collection of autologous T-cells, which are then genetically engineered ex vivo to express transmembrane CARs. These chimeric proteins consist of an antibody-like extracellular antigen-binding domain, to recognise specific antigens on the surface of tumour cells (e.g. CD19), linked to the intracellular co-stimulatory signalling domains of a T-cell receptor (e.g. CD137). The latter is required for in vivo CAR T-cell proliferation, survival, and durable efficacy. Following reinfusion, CAR T-cells harness the cytotoxic capacity of a patient's immune system. They overcome major mechanisms of tumour immuno-evasion and have potential to generate robust cytotoxic anti-tumour responses. This review discusses the background to CAR T-cell therapies, including their molecular design, mechanisms of action, methods of production, clinical applications and established and emerging technologies for CAR T-cell evaluation. It highlights the need for standardisation, quality control and monitoring of CAR T-cell therapies, to ensure their safety and efficacy in clinical management.

Osteohematology: To be or Notch to be.

Osteohematology is an emerging research field that studies the crosstalk between hematopoietic and bone stromal cells, to elucidate the mechanisms of hematological and skeletal malignancies and diseases. The Notch is an evolutionary conserved developmental signaling pathway, with critical roles in embryonic development by controlling cell proliferation and differentiation. However, the Notch pathway is also critically involved in cancer initiation and progression, such as osteosarcoma, leukemia, and multiple myeloma. The Notch-mediated malignant cells dysregulate bone and bone marrow cells in the tumour microenvironment, resulting in disorders ranging from osteoporosis to bone marrow dysfunction. To date, the complex interplay of Notch signaling molecules in hematopoietic and bone stromal cells is still poorly understood. In this mini-review, we summarize the crosstalk between cells in bone and bone marrow and their influence under the Notch signaling pathway in physiological conditions and in tumour microenvironment.

Morphology of myeloproliferative neoplasms.

Myeloproliferative neoplasms (MPN) are a group of clonal haematological malignancies first described by Dameshek in 1957. The Philadelphia-negative MPN that will be described are polycythaemia vera (PV), essential thrombocythaemia (ET), pre-fibrotic myelofibrosis and primary myelofibrosis (PMF). The blood and bone marrow morphology are essential in diagnosis, for WHO classification, establishing a baseline, monitoring response to treatment and identifying changes that may indicate disease progression. The blood film changes may be in any of the cellular elements. The key bone marrow features are architecture and cellularity, relative complement of individual cell types, reticulin content and bony structure. Megakaryocytes are the most abnormal cell and key to classification, as their number, location, size and cytology are all disease-defining. Reticulin content and grade are integral to assignment of the diagnosis of myelofibrosis. Even with careful assessment of all these features, not all cases fit neatly into the diagnostic entities; there is frequent overlap reflecting the biological disease continuum rather than distinct entities. Notwithstanding this, an accurate morphologic diagnosis in MPN is crucial due to the significant differences in prognosis between different subtypes and the availability of different therapies in the era of novel agents. The distinction between "reactive" and MPN is also not always straightforward and caution needs to be exercised given the prevalence of "triple negative" MPN. Here we describe the morphology of MPN including comments on changes with disease evolution and with treatment.

"Immuno-FlowFISH": Applications for Chronic Lymphocytic Leukemia.

Imaging flow cytometry has the capacity to bridge the gap that currently exists between the diagnostic tests that detect important phenotypic and genetic changes in the clinical assessment of leukemia and other hematological malignancies or blood-based disorders. We have developed an "Immuno-flowFISH" method that leverages the quantitative and multi-parametric power of imaging flow cytometry to push the limits of single-cell analysis. Immuno-flowFISH has been fully optimized to detect clinically significant numerical and structural chromosomal abnormalities (i.e., trisomy 12 and del(17p)) within clonal CD19/CD5+ CD3- Chronic Lymphocytic Leukemia (CLL) cells in a single test. This integrated methodology has greater accuracy and precision than standard fluorescence in situ hybridization (FISH). We have detailed this immuno-flowFISH application with a carefully catalogued workflow, technical instructions, and a repertoire of quality control considerations to supplement the analysis of CLL. This next-generation imaging flow cytometry protocol may provide unique advancements and opportunities in the holistic cellular assessment of disease for both research and clinical laboratory settings.

IGH cytogenetic abnormalities can be detected in multiple myeloma by imaging flow cytometry.

AIMS: Cytogenetic abnormalities involving the IGH gene are seen in up to 55% of patients with multiple myeloma. Current testing is performed manually by fluorescence in situ hybridisation (FISH) on purified plasma cells. We aimed to assess whether an automated imaging flow cytometric method that uses immunophenotypic cell identification, and does not require cell isolation, can identify IGH abnormalities. METHODS: Aspirated bone marrow from 10 patients with multiple myeloma were studied. Plasma cells were identified by CD38 and CD138 coexpression and assessed with FISH probes for numerical or structural abnormalities of IGH. Thousands of cells were acquired on an imaging flow cytometer and numerical data and digital images were analysed. RESULTS: Up to 30 000 cells were acquired and IGH chromosomal abnormalities were detected in 5 of the 10 marrow samples. FISH signal patterns seen included fused IGH signals for IGH/FGFR3 and IGH/MYEOV, indicating t(4;14) and t(11;14), respectively. In addition, three IGH signals were identified, indicating trisomy 14 or translocation with an alternate chromosome. The lowest limit of detection of an IGH abnormality was in 0.05% of all cells. CONCLUSIONS: This automated high-throughput immuno-flowFISH method was able to identify translocations and trisomy involving the IGH gene in plasma cells in multiple myeloma. Thousands of cells were analysed and without prior cell isolation. The inclusion of positive plasma cell identification based on immunophenotype led to a lowest detection level of 0.05% marrow cells. This imaging flow cytometric FISH method offers the prospect of increased precision of detection of critical genetic lesions involving IGH and other chromosomal defects in multiple myeloma.

Hospital-Acquired Infection, Length of Stay, and Readmission in Elective Surgery Patients Transfused 1 Unit of Red Blood Cells: A Retrospective Cohort Study.

BACKGROUND: Most patients transfused red blood cells in elective surgery receive small volumes of blood, which is likely to be discretionary and avoidable. We investigated the outcomes of patients who received a single unit of packed red blood cells during their hospital admission for an elective surgical procedure when compared to those not transfused. METHODS: This retrospective cohort study included elective surgical admissions to 4 hospitals in Western Australia over a 6-year period. Participants were included if they were at least 18 years of age and were admitted for elective surgery between July 2014 and June 2020. We compared outcomes of patients who had received 1 unit of red blood cells to patients who had not been transfused. To balance differences in patient characteristics, we weighted our multivariable regression models using the inverse probability of treatment. In addition to propensity score weighting, our multivariable regression models adjusted for hemoglobin level, surgical procedure, patient age, gender, comorbidities, and the transfusion of fresh-frozen plasma or platelets. Outcomes studied were hospital-acquired infection, hospital length of stay, and all-cause emergency readmissions within 28 days. RESULTS: Overall, 767 (3.2%) patients received a transfusion of 1 unit of red blood cells throughout their admission. In the propensity score weighted analysis, the transfusion of a single unit of red blood cells was associated with higher odds of hospital-acquired infection (odds ratio, 3.94; 95% confidence interval [CI], 2.99-5.20; P < .001). Patients who received 1 unit of red blood cells throughout their admission were more likely to have a longer hospital stay (rate ratio, 1.57; 95% CI, 1.51-1.63; P < .001) and had 1.42 (95% CI, 1.20-1.69; P < .001) times higher odds of 28-day readmission. CONCLUSIONS: These results suggest that avoidance of even small volumes of packed red blood cells may prevent adverse clinical outcomes. This may encourage hospital administrators to implement strategies to avoid the transfusion of even small volumes of red blood cells by applying patient blood management practices.

CRLF3 plays a key role in the final stage of platelet genesis and is a potential therapeutic target for thrombocythemia.

The process of platelet production has so far been understood to be a 2-stage process: megakaryocyte maturation from hematopoietic stem cells followed by proplatelet formation, with each phase regulating the peripheral blood platelet count. Proplatelet formation releases into the bloodstream beads-on-a-string preplatelets, which undergo fission into mature platelets. For the first time, we show that preplatelet maturation is a third, tightly regulated, critical process akin to cytokinesis that regulates platelet count. We show that deficiency in cytokine receptor-like factor 3 (CRLF3) in mice leads to an isolated and sustained 25% to 48% reduction in the platelet count without any effect on other blood cell lineages. We show that Crlf3-/- preplatelets have increased microtubule stability, possibly because of increased microtubule glutamylation via the interaction of CRLF3 with key members of the Hippo pathway. Using a mouse model of JAK2 V617F essential thrombocythemia, we show that a lack of CRLF3 leads to long-term lineage-specific normalization of the platelet count. We thereby postulate that targeting CRLF3 has therapeutic potential for treatment of thrombocythemia.

Automated digital enumeration of plasma cells in bone marrow trephine biopsies of multiple myeloma.

AIMS: Determination of the number of plasma cells in bone marrow biopsies is required for the diagnosis and ongoing evaluation of plasma cell neoplasms. We developed an automated digital enumeration platform to assess plasma cells identified by antigen expression in whole bone marrow sections in multiple myeloma, and compared it with manual assessments. METHODS: Bone marrow trephine biopsy specimens from 91 patients with multiple myeloma at diagnosis, remission and relapse were stained for CD138 and multiple myeloma oncogene 1 (MUM1). Manual assessment and digital quantification were performed for plasma cells in the entire trephine section. Concordance rates between manual and digital methods were evaluated for each antigen by intraclass correlation analyses (ICC) with associated Spearman's correlations. RESULTS: The digital platform counted 16 484-1 118 868 cells and the per cent CD138 and MUM1-positive plasma cells ranged from 0.05% to 93.5%. Overall concordance between digital and manual methods was 0.63 for CD138 and 0.89 for MUM1. Concordance was highest with diffuse plasma cell infiltrates (MUM1: ICC=0.90) and lowest when in microaggregates (CD138: ICC=0.13). Manual counts exceeded digital quantifications for both antigens (CD138: mean=26.4%; MUM1: mean=9.7%). Diagnostic or relapse threshold counts, as determined by CD138 manual assessments, were not reached with digital counting for 16 cases (18%). CONCLUSIONS: Automated digital enumeration of the entire, immunohistochemically stained bone marrow biopsy section can accurately determine plasma cell burden, irrespective of pattern and extent of disease (as low as 0.05%). This increases precision over manual visual assessments which tend to overestimate plasma burden, especially for CD138, and when plasma cells are in clusters.

Mutation profile of acute myeloid leukaemia in a Chinese cohort by targeted next-generation sequencing.

BACKGROUND: Acute myeloid leukaemia (AML) results from the clonal expansion of blast cells of myeloid origin driven by genomic defects. The advances in next-generation sequencing (NGS) have allowed the identification of many mutated genes important in the pathogenesis of AML. AIMS: In this study, we aimed to assess the mutation types and frequency in a Chinese cohort presenting with de novo AML cohort using a targeted NGS strategy. METHODS: In total, we studied samples from 87 adult patients with de novo AML who had no prior history of cytotoxic chemotherapy. Samples were evaluated using a 120-gene targeted NGS panel to assess the mutation profile. RESULTS: Of the 87 AML patients, there were 60 (69%) with a normal karyotype. 89.7% of patients had variants, with an average of 1.9 mutations per patient (range: 0-5 mutations per patient). DNMT3A variants were the most common, being detected in 33 patients (37.9%). NPM1 (34.5%), IDH1/2 (24.1%) and FLT3-ITD (20.7%) mutations was the next most common. Of the patients with DNMT3A mutations, 24.2% also had mutations NPM1 and FLT3-ITD and 6.1% NPM1, FLT3-ITD and IDH mutations. CONCLUSION: Both DNMT3A and NPM1 mutations were more common than in other Chinese and Western AML cohorts that have been studied. DNMT3A mutations tended to co-occur with NPM1 and FLT3-ITD mutations and were most commonly seen with a normal karyotype.