Predicting cytopenias, progression, and survival in patients with clonal cytopenia of undetermined significance: a prospective cohort study.

BACKGROUND: Somatic mutations are frequently reported in individuals with cytopenia but without a confirmed haematological diagnosis (clonal cytopenia of undetermined significance; CCUS). These patients have an increased risk of progression to a myeloid malignancy and worse overall survival than those with no such mutations. To date, studies have been limited by retrospective analysis or small patient numbers. We aimed to establish the natural history of CCUS by prospectively investigating outcome in a large, well defined patient cohort. METHODS: This prospective cohort study was conducted at the Haematological Malignancy Diagnostic Service, a diagnostic laboratory in Leeds, UK. Patients aged at least 18 years who were referred for investigation of cytopenia were eligible for inclusion; those with a history of myeloid malignancy were not eligible. Targeted sequencing was conducted alongside routine clinical testing. Baseline mutation analysis was then correlated with the main study outcomes: longitudinal blood counts, disease progression to a myeloid malignancy, and overall survival with a median follow-up of 4·54 years (IQR 4·03-5·04). Data were collected manually from hospital records or extracted from laboratory or clinical outcome databases. FINDINGS: Bone marrow samples from 2348 patients were received at the Haematological Malignancy Diagnostic Service between July 1, 2014, and July 31, 2016. Of these, 2083 patients (median age 72 years [IQR 63-80, range 18-99]; 854 [41·0%] female and 1229 [59·0%] male) met the inclusion criteria and had samples of sufficient quality for further analysis. 598 (28·7%) patients received a diagnosis on the basis of their biopsy sample, whereas 1485 (71·3%) samples were classified as non-diagnostic; of these, CCUS was confirmed in 400 (26·9%) patients (256 [64·0%] male and 144 [36·0%] female). TET2, SRSF2, and DNMT3A were the most frequently mutated genes in patients with CCUS, with 320 (80%) of 400 patients harbouring a mutation in at least one of these genes. Age (p<0·0001), sex (p=0·0027), and mutations in ASXL1 (p=0·0009), BCOR (p=0·0056), and TP53 (p=0·0055) correlated with a worse overall survival; however, the number of mutations was the strongest predictor for progression to a myeloid malignancy (two mutations, p=0·0024; three or more mutations, p=0·0004). Extended sequencing of samples from a subgroup of patients with sequential samples and no mutations in the initial myeloid gene panel showed recurrent mutations in both DDX41 and UBA1, suggesting that these genes should be included in clinical test panels. INTERPRETATION: Mutation analysis is advised in patients who have undergone bone marrow examination and have an otherwise-unexplained cytopenia. High-risk genetic mutations and increased numbers of mutations are predictive of both survival and progression within 5 years of presentation, warranting clinical surveillance and, when necessary, intervention. FUNDING: MDS Foundation.

Somatic Mutations and the Risk of Undifferentiated Autoinflammatory Disease in MDS: An Under-Recognized but Prognostically Important Complication.

Objectives: We theorized that myelodysplastic syndrome (MDS) with somatic mutations and karyotype abnormalities are associated with autoinflammation, and that the presence of autoinflammatory disease affected prognosis in MDS. Methods: One hundred thirty-four MDS patients were assessed for the prevalence of autoinflammatory complications and its link with karyotypes and somatic mutation status. Autoinflammatory complications were described either as well-defined autoinflammatory diseases (AD) or undifferentiated "autoinflammatory disease" (UAD) (defined as CRP over 10.0 mg/L on five consecutive occasions, taken at separate times and not explained by infection). Several patient characteristics including demographic, clinical, laboratory, cytogenetics charts, and outcomes, were compared between different groups. Results: Sixty-two (46.3%) patients had an autoinflammatory complication manifesting as arthralgia (43.5% vs. 23.6%, p = 0.0146), arthritis (30.6% vs. 15.3%, p = 0.0340), skin rash (27.4% vs. 12.5%, p = 0.0301), pleuritis (14.5% vs. 4.2%, p = 0.0371) and unexplained fever (27.4% vs. 0%, p < 0.0001). AD were found in 7.4% of MDS patients (with polymyalgia rheumatic being the most frequently one). Classical autoimmune diseases were found only in 4 MDS patients (3.0%). Transcription factor pathway mutations (RUNX1, BCOR, WTI, TP53) (OR 2.20 [95%CI 1.02-4.75], p = 0.0451) and abnormal karyotypes (OR 2.76 [95%CI 1.22-6.26], p = 0.0153) were associated with autoinflammatory complications. Acute leukaemic transformation was more frequent in MDS patients with autoinflammatory features than those without (27.4% vs. 9.7%, p = 0.0080). Conclusions: Autoinflammatory complications are common in MDS. Somatic mutations of transcription factor pathways and abnormal karyotypes are associated with greater risk of autoinflammatory complications, which are themselves linked to malignant transformation and a worse prognosis.

Comparative analysis of gene expression platforms for cell-of-origin classification of diffuse large B-cell lymphoma shows high concordance.

Cell-of-origin subclassification of diffuse large B cell lymphoma (DLBCL) into activated B cell-like (ABC), germinal centre B cell-like (GCB) and unclassified (UNC) or type III by gene expression profiling is recommended in the latest update of the World Health Organization's classification of lymphoid neoplasms. There is, however, no accepted gold standard method or dataset for this classification. Here, we compare classification results using gene expression data for 68 formalin-fixed paraffin-embedded DLBCL samples measured on four different gene expression platforms (Illumina wG-DASLTM arrays, Affymetrix PrimeView arrays, Illumina TrueSeq RNA sequencing and the HTG EdgeSeq DLBCL Cell of Origin Assay EU using an established platform agnostic classification algorithm (DAC) and the classifier native to the HTG platform, which is CE marked for in vitro diagnostic use (CE-IVD). Classification methods and platforms show a high level of concordance, with agreement in at least 80% of cases and rising to much higher levels for classifications of high confidence. Our results demonstrate that cell-of-origin classification by gene expression profiling on different platforms is robust, and that the use of the confidence value alongside the classification result is important in clinical applications.

Financial Effect of Unbundling Moderate Sedation from Procedural Codes in Radiology.

PURPOSE: To assess and quantify the financial effect of unbundling newly unbundled moderate sedation codes across major payors at an academic radiology practice. MATERIALS AND METHODS: Billing and reimbursement data for 23 months of unbundled moderate sedation codes were analyzed for reimbursement rates and trends. This included 10,481 and 28,189 units billed and $443,257 and $226,444 total receipts for codes 99152 (initial 15 minutes of moderate sedation) and 99153 (each subsequent 15 minute increment of moderate sedation), respectively. Five index procedures-(i) central venous port placement, (ii) endovascular tumor embolization, (iii) tunneled central venous catheter placement, (iv) percutaneous gastrostomy placement, and (v) percutaneous nephrostomy placement-were identified, and moderate sedation reimbursements for Medicare and the dominant private payor were calculated and compared to pre-bundled reimbursements. Revenue variation models across different patient insurance mixes were then created using averages from 4 common practice settings among radiologists (independent practices, all hospitals, safety-net hospitals, and non-safety-net hospitals). RESULTS: Departmental reimbursement for unbundled moderate sedation in FY2018 and FY2019 totaled $669,701.34, with high per-unit variability across payors, especially for code 99153. Across the 5 index procedures, moderate sedation reimbursement decreased 1.3% after unbundling and accounted for 3.9% of procedural revenue from Medicare and increased 11.9% and accounted for 5.5% of procedural revenue from the dominant private payor. Between different patient insurance mix models, estimated reimbursement from moderate sedation varied by as much as 29.9%. CONCLUSIONS: Departmental reimbursement from billing the new unbundled moderate sedation codes was sizable and heterogeneous, highlighting the need for consistent and accurate reporting of moderate sedation. Total collections vary by case mix, patient insurance mix, and negotiated reimbursement rates.

Exploratory Study of MYD88 L265P, Rare NLRP3 Variants, and Clonal Hematopoiesis Prevalence in Patients With Schnitzler Syndrome.

OBJECTIVE: To assess the prevalence of the MYD88 L265P mutation and variants within NLRP3 and evaluate the status of oligoclonal hematopoiesis in 30 patients with Schnitzler syndrome (SchS). METHODS: Thirty patients with SchS were recruited from 3 clinical centers. Six patients with known acquired cryopyrin-associated periodic syndromes (aCAPS) were included as controls. Allele-specific oligonucleotide-polymerase chain reaction was used for the detection of the MYD88 L265P variant, next-generation sequencing was applied to analyze NLRP3 and 28 genes associated with myelodysplastic syndrome, and gene scanning was performed for the detection of X chromosome inactivation. RESULTS: Activating NLRP3 mutations were not present in 11 SchS patients who had not been sequenced for this gene previously. The MYD88 L265P variant was present in 9 of 30 SchS patients, and somatic mutations associated with clonal hematopoiesis were identified in 1 of 30 patients with SchS and 1 of 6 patients with aCAPS. Evidence of nonrandom X chromosome inactivation was detected in 1 female patient with SchS and 1 female patient with aCAPS. CONCLUSION: A shared molecular mechanism accounting for the pathogenesis of inflammation in SchS remains elusive. Clonal hematopoiesis is not associated with other somatic mutations found in individuals with SchS or aCAPS.

The use of targeted sequencing and flow cytometry to identify patients with a clinically significant monocytosis.

The diagnosis of chronic myelomonocytic leukemia (CMML) remains centered on morphology, meaning that the distinction from a reactive monocytosis is challenging. Mutational analysis and immunophenotyping have been proposed as potential tools for diagnosis; however, they have not been formally assessed in combination. We aimed to investigate the clinical utility of these technologies by performing targeted sequencing, in parallel with current gold standard techniques, on consecutive samples referred for investigation of monocytosis over a 2-year period (N = 283). Results were correlated with the morphological diagnosis and objective outcome measures, including overall survival (OS) and longitudinal blood counts. Somatic mutations were detected in 79% of patients, being invariably identified in those with a confirmed diagnosis (99%) but also in 57% of patients with nondiagnostic bone marrow features. The OS in nondiagnostic mutated patients was indistinguishable from those with CMML (P = .118) and significantly worse than in unmutated patients (P = .0002). On multivariate analysis, age, ASXL1, CBL, DNMT3A, NRAS, and RUNX1 mutations retained significance. Furthermore, the presence of a mutation was associated with a progressive decrease in hemoglobin/platelet levels and increasing monocyte counts compared with mutation-negative patients. Of note, the immunophenotypic features of nondiagnostic mutated patients were comparable to CMML patients, and the presence of aberrant CD56 was highly specific for detecting a mutation. Overall, somatic mutations are detected at high frequency in patients referred with a monocytosis, irrespective of diagnosis. In those without a World Health Organization-defined diagnosis, the mutation spectrum, immunophenotypic features, and OS are indistinguishable from CMML patients, and these patients should be managed as such.

Prospective Safety Evaluation of a Cardiovascular Health Dietary Supplement in Adults with Prehypertension and Stage I Hypertension.

OBJECTIVE: To prospectively examine the long-term safety of a cardiovascular health dietary supplement by assessing a comprehensive set of safety measures. DESIGN: Single-arm, open-label study. LOCATION: National University of Natural Medicine, Portland, OR. SUBJECTS: Thirty adults with screening blood pressure readings consistent with prehypertension or stage I hypertension. INTERVENTION: One caplet per day of a dietary supplement for 6 months. The investigated herbal-mineral supplement contains several ingredients, most notably Rauwolfia serpentina. OUTCOME MEASURES: Primary measures included b-type natriuretic peptide (NT-proBNP), aspartate aminotransferase (AST), alanine aminotransferase (ALT), estimated glomerular filtration rate (eGFR), electrolytes, and the Patient Health Questionnaire (PHQ-9). Exploratory measures included physical vital signs, cholesterol levels, high-sensitivity cardiac troponin-I, cystatin C, endothelin, interleukin (IL)-6, IL-17a, tumor necrosis factor-α, high-sensitivity C-reactive protein, blood counts, and the Patient Reported Outcome Measure Information System (PROMIS) Sleep Disturbance Short Form 8b. RESULTS: NT-proBNP, AST, ALT, eGFR, sodium, calcium, magnesium, PHQ-9 score, and the majority of exploratory measures did not change. However, serum potassium increased (p < 0.05), systolic blood pressure decreased (p < 0.0001), and diastolic blood pressure decreased (p < 0.0001). There were no serious adverse events, but 30% of participants withdrew citing potential side effects, most commonly nasal congestion or fatigue; most participants who reported nasal congestion also reported concomitant seasonal allergies. Adherence to the supplement was 90.9%. CONCLUSIONS: The findings of this study suggest that the investigated dietary supplement is safe for long-term use in adults with prehypertension and stage I hypertension. Additional results of this study, particularly the increase in serum potassium and decreases in systolic and diastolic blood pressure, are promising and suggest that future research on this dietary supplement, or its ingredients, should further explore effects on blood pressure and biologic mechanisms of action, which may involve potassium-sparing and diuretic effects.

Metabolic Rate of Diploid and Triploid Edible Frog Pelophylax esculentus Correlates Inversely with Cell Size in Tadpoles but Not in Frogs.

In multicellular organisms, cell size may have crucial consequences for basic parameters, such as body size and whole-body metabolic rate (MR). The hypothesis predicts that animals composed of smaller cells (a higher membrane surface-to-cell volume ratio) should have a higher mass-specific MR because a large part of their energy is used to maintain cell membranes and ionic gradients. In this article, we investigated the link between cell size and MR in diploid and triploid tadpoles and froglets of the hybridogenetic frog Pelophylax esculentus. In our previous study, we showed that triploids had significantly larger cells (erythrocytes, hepatocytes, and epidermal cells were measured). Therefore, we hypothesized that triploid tadpoles and froglets would have a lower standard metabolic rate (SMR). Our study demonstrated for the first time two distinct effects of polyploidy/cell size on MR within a single species developing in both aquatic and terrestrial habitats. As we hypothesized, diploid tadpoles had a higher SMR than triploids, whereas in froglets, ploidy did not affect the SMR. We also found that the water temperatures in which tadpoles were reared had no effect on the SMR of froglets after metamorphosis. Based on our results and other reports, we suggest that cell size may have more consequences for whole-body MR in aquatic habitats than in terrestrial habitats because oxygen is less available in water and its availability in relation to oxygen demand decreases with temperature.

Low Temperature and Polyploidy Result in Larger Cell and Body Size in an Ectothermic Vertebrate.

Previous studies reported that low temperatures result in increases in both cell size and body size in ectotherms that may explain patterns of geographic variation of their body size across latitudinal ranges. Also, polyploidy showed the same effect on body size in invertebrates. In vertebrates, despite their having larger cells, no clear effect of polyploidy on body size has been found. This article presents the relationship between temperature, cell size, growth rate, and body size in diploid and polyploid hybridogenetic frog Pelophylax esculentus reared as tadpoles at 19° and 24°C. The size of cells was larger in both diploid and triploid tadpoles at 19°C, and triploids had larger cells at both temperatures. In diploid and triploid froglets, the temperature in which they developed as tadpoles did not affect the size of their cells, but triploids still had larger cells. Triploid tadpoles grew faster than diploids at 19°C and had larger body mass; there was no clear difference between ploidies in growth rate at 24°C. This indicates better adaptation of triploid tadpoles to cold environment. This is the first report on the increase of body mass of a polyploid vertebrate caused by low temperature, and we showed relationship between increase in cell size and increased body mass. The large body mass of triploids may provide a selective advantage, especially in colder environments, and this may explain the prevalence of triploids in the northern parts of the geographic range of P. esculentus.

Reproduction is not costly in terms of oxidative stress.

One of the core assumptions of life-history theory is the negative trade-off between current and future reproduction. Investment in current reproduction is expected to decrease future reproductive success or survival, but the physiological mechanisms underlying these costs are still obscure. To test for a role of oxidative stress, we measured oxidative damage to lipids and proteins in liver, heart, kidneys and muscles, as well as the level of antioxidants (total glutathione and catalase), in breeding and non-breeding bank voles. We used females from lines selected for high aerobic metabolism and non-selected control lines and manipulated their reproductive investment by decreasing or increasing litter size. Unlike in most previous studies, the females reared four consecutive litters (the maximum possible during a breeding season). Contrary to predictions, oxidative damage in reproducing females was decreased or not changed, and did not differ between the selected and control lines. Oxidative damage to lipids and proteins in the liver was lower in females that weaned enlarged litters than in non-breeding ones, and was intermediate in those with reduced litters. Oxidative damage to proteins in the heart also tended to be lower in breeding females than in non-breeding ones. A negative relationship between the level of oxidative damage and activity of catalase in kidneys indicated a protective action of antioxidants. In conclusion, our study falsified the hypothesis that oxidative stress is a part of the proximate physiological mechanism underlying the fundamental life-history trade-off between current and future reproduction.

Is reproduction costly? No increase of oxidative damage in breeding bank voles.

According to life-history theory, investment in reproduction is associated with costs, which should appear as decreased survival to the next reproduction or lower future reproductive success. It has been suggested that oxidative stress may be the proximate mechanism of these trade-offs. Despite numerous studies of the defense against reactive oxygen species (ROS) during reproduction, very little is known about the damage caused by ROS to the tissues of wild breeding animals. We measured oxidative damage to lipids and proteins in breeding bank vole (Myodes glareolus) females after rearing one and two litters, and in non-breeding females. We used bank voles from lines selected for high maximum aerobic metabolic rates (which also had high resting metabolic rates and food intake) and non-selected control lines. The oxidative damage was determined in heart, kidneys and skeletal muscles by measuring the concentration of thiobarbituric acid-reactive substances, as markers of lipid peroxidation, and carbonyl groups in proteins, as markers of protein oxidation. Surprisingly, we found that the oxidative damage to lipids in kidneys and muscles was actually lower in breeding than in non-breeding voles, and it did not differ between animals from the selected and control lines. Thus, contrary to our predictions, females that bred suffered lower levels of oxidative stress than those that did not reproduce. Elevated production of antioxidant enzymes and the protective role of sex hormones may explain the results. The results of the present study do not support the hypothesis that oxidative damage to tissues is the proximate mechanism of reproduction costs.