Venous Stasis Ulceration due to Massive Splenomegaly Causing Iliac Vein Compression From Secondary Myelofibrosis.

Venous stasis ulcers are nonhealing lesions due to venous hypertension secondary to valvular dysfunction or deep venous outflow obstruction. We describe a case of a 71-year-old male with a history of polycythemia vera, secondary myelofibrosis, and massive splenomegaly up to 38 cm who presented with chronic, perimalleolar venous stasis ulcers and pain on the left lower extremity. CT showed significant compression of the left common iliac vein due to mass effect from the spleen. He was managed medically while being evaluated for partial splenic artery embolization but expired due to other chronic conditions before any intervention could be performed. Partial splenic artery embolization may be considered as a treatment option for patients with symptomatic iliac vein compression due to massive splenomegaly secondary to myelofibrosis, as long as extramedullary hematopoiesis is not compromised.

Clinical characterization of the mutational landscape of 24,639 real-world samples from patients with myeloid malignancies.

Myeloid neoplasms represent a broad spectrum of hematological disorders for which somatic mutation status in key driver genes is important for diagnosis, prognosis and treatment. Here we summarize the findings of a targeted, next generation sequencing laboratory developed test in 24,639 clinical myeloid samples. Data were analyzed comprehensively and as part of individual cohorts specific to acute myeloid leukemia (AML), myelodysplastic syndrome (MDS), and myeloproliferative neoplasms (MPN). Overall, 48,015 variants were detected, and variants were found in all 50 genes in the panel. The mean number of mutations per patient was 1.95. Mutation number increased with age (Spearman's rank correlation coefficient, ρ = 0.29, P < 0.0001) and was higher in patients with AML than MDS or MPN (Student's t-test, P < 0.0001). TET2 was the most common mutation detected (19.1% of samples; 4,695/24,639) including 7.7% (1,908/24,639) with multi-hit TET2 mutations. Mutation frequency was correlated between patients with cytopenias and MDS (Spearman's, ρ = 0.97, P < 2.2×10-16) with the MDS diagnostic gene SF3B1 being the only notable outlier. This large retrospective study shows the utility of NGS testing to inform clinical decisions during routine clinical care and highlights the mutational landscape of a broad population of myeloid patients.

Poloxamer 407 Induces Hypertriglyceridemia but Decreases Atherosclerosis in Ldlr-/- Mice.

BACKGROUND: Hypertriglyceridemia (HTG) increases the risk for atherosclerotic cardiovascular disease, but underlying mechanisms are incompletely understood. Circulating monocytes play an important role in atherogenesis by infiltrating arterial walls, where they differentiate into macrophages. We tested the hypothesis that HTG is mechanistically linked to atherogenesis by altering the monocyte phenotype and infiltration into atherosclerotic lesions in a model of diet-induced atherogenesis in Ldlr-/- mice. METHODS: HTG was induced in male Ldlr-/- mice, fed a Western, high-fat high-cholesterol diet, by daily injection of poloxamer 407 (P407), a lipoprotein lipase inhibitor, for seven weeks. Atherosclerosis, monocyte phenotypes, and monocyte migration into atherosclerotic lesions were determined by well-validated methods. RESULTS: Compared with the saline control, P407 injection in Ldlr-/- mice rapidly induced profound and persistent HTG, modestly elevated plasma cholesterol levels, and increased levels of triglyceride and cholesterol carried in very-low-density lipoprotein and low-density lipoprotein. Unexpectedly, mice receiving P407 versus saline control showed less atherosclerosis. Following induction of HTG by P407, CD36+ (also CD11c+), but not CD36- (CD11c-), monocytes showed early increases in lipid accumulation, but the number of CD36+ (not CD36-) monocytes was dramatically decreased afterwards in the circulation until the end of the test. Concurrently, CD36+ (CD11c+) monocyte migration into atherosclerotic lesions was also reduced in mice receiving P407 versus controls. CONCLUSIONS: P407 induced severe HTG, but reduced atherosclerosis, in Ldlr-/- mice, possibly because of profound reductions of circulating CD36+ (CD11c+) monocytes, leading to decreased monocyte migration into atherosclerotic lesions.

Detection of mind wandering using EEG: Within and across individuals.

Mind wandering is often characterized by attention oriented away from an external task towards our internal, self-generated thoughts. This universal phenomenon has been linked to numerous disruptive functional outcomes, including performance errors and negative affect. Despite its prevalence and impact, studies to date have yet to identify robust behavioral signatures, making unobtrusive, yet reliable detection of mind wandering a difficult but important task for future applications. Here we examined whether electrophysiological measures can be used in machine learning models to accurately predict mind wandering states. We recorded scalp EEG from participants as they performed an auditory target detection task and self-reported whether they were on task or mind wandering. We successfully classified attention states both within (person-dependent) and across (person-independent) individuals using event-related potential (ERP) measures. Non-linear and linear machine learning models detected mind wandering above-chance within subjects: support vector machine (AUC = 0.715) and logistic regression (AUC = 0.635). Importantly, these models also generalized across subjects: support vector machine (AUC = 0.613) and logistic regression (AUC = 0.609), suggesting we can reliably predict a given individual's attention state based on ERP patterns observed in the group. This study is the first to demonstrate that machine learning models can generalize to "never-seen-before" individuals using electrophysiological measures, highlighting their potential for real-time prediction of covert attention states.

Discrepant Cytogenetic and Interphase Fluorescence In Situ Hybridization (I-FISH) Results from Bone Marrow Specimens of Patients with Hematologic Neoplasms.

Conventional cytogenetic and routine I-FISH (interphase fluorescence in-situ hybridization) studies periodically present discrepant results on the same sample calling into question their validity. Generally it is expected that these tests confirm each other, otherwise there is concern that they may represent laboratory error. We present data showing that these discrepant results are rarely due to laboratory error, and that M-FISH (metaphase fluorescence in-situ hybridization) can usually reconcile them by identifying the nature of these differences. This report includes 32 bone marrow (BM) samples from patients with hematologic neoplasms that showed incongruent cytogenetic/I-FISH results. M-FISH was selectively applied for further clarification of these discrepancies when deemed necessary. This study evaluated BM samples in our laboratory (Integrated Oncology, Phoenix, AZ) that represented 5 major categories of hematologic disorders (MDS/AML, MPN, NHL, CLL, & PCN). Five general categories of these cases were identified: 1) laboratory error (clerical), 2) limited resolution of testing methods, 3) cellular response to culture/preparative conditions, 4) cytogenetic bi-clonality and 5) failed hybridizations due to cover-slipping. Our results suggest that the majority of discrepant results are related to the intrinsic nature of the malignant cells (and how they respond to their growth environment) evaluated by these two testing methods.

SMAD3/Stat3 Signaling Mediates ß-Cell Epithelial-Mesenchymal Transition in Chronic Pancreatitis-Related Diabetes.

Many patients with chronic pancreatitis develop diabetes (chronic pancreatitis-related diabetes [CPRD]) through an undetermined mechanism. Here we used long-term partial pancreatic duct ligation (PDL) as a model to study CPRD. We found that long-term PDL induced significant ß-cell dedifferentiation, followed by a time-dependent decrease in functional ß-cell mass-all specifically in the ligated tail portion of the pancreas (PDL-tail). High levels of transforming growth factor ß1 (TGFß1) were detected in the PDL-tail and were mainly produced by M2 macrophages at the early stage and by activated myofibroblasts at the later stage. Loss of ß-cell mass was then found to result from TGFß1-triggered epithelial-mesenchymal transition (EMT) by ß-cells, rather than resulting directly from ß-cell apoptosis. Mechanistically, TGFß1-treated ß-cells activated expression of the EMT regulator gene Snail in a SMAD3/Stat3-dependent manner. Moreover, forced expression of forkhead box protein O1 (FoxO1), an antagonist for activated Stat3, specifically in ß-cells ameliorated ß-cell EMT and ß-cell loss and prevented the onset of diabetes in mice undergoing PDL. Together, our data suggest that chronic pancreatitis may trigger TGFß1-mediated ß-cell EMT to lead to CPRD, which could substantially be prevented by sustained expression of FoxO1 in ß-cells.

Forkhead Box Protein 1 (FoxO1) Inhibits Accelerated ß Cell Aging in Pancreas-specific SMAD7 Mutant Mice.

The mechanisms underlying the effects of exocrine dysfunction on the development of diabetes remain largely unknown. Here we show that pancreatic depletion of SMAD7 resulted in age-dependent increases in ß cell dysfunction with accelerated glucose intolerance, followed by overt diabetes. The accelerated ß cell dysfunction and loss of proliferation capacity, two features of ß cell aging, appeared to be non-cell-autonomous, secondary to the adjacent exocrine failure as a "bystander effect." Increased Forkhead box protein 1 (FoxO1) acetylation and nuclear retention was followed by progressive FoxO1 loss in ß cells that marked the onset of diabetes. Moreover, forced FoxO1 expression in ß cells prevented ß cell dysfunction and loss in this model. Thus, we present a model of accelerated ß cell aging that may be useful for studying the mechanisms underlying ß cell failure in diabetes. Moreover, we provide evidence highlighting a critical role of FoxO1 in maintaining ß cell identity in the context of SMAD7 failure.

Evolution of hepatic steatosis to fibrosis and adenoma formation in liver-specific growth hormone receptor knockout mice.

BACKGROUND: Non-alcoholic fatty liver disease (NAFLD) is one of the most common forms of chronic liver diseases closely associated with obesity and insulin resistance; deficient growth hormone (GH) action in liver has been implicated as a mechanism. Here, we investigated the evolution of NAFLD in aged mice with liver-specific GHR deletion. METHODS: We examined glucose tolerance, insulin responsiveness, and lipid profiles in aged male mice (44-50 weeks) with GHRLD. We performed proteomics analysis, pathway-based Superarray assay, as well as quantitative RT-PCR to gain molecular insight into the mechanism(s) of GHR-deficiency-mediated NAFLD. In addition, we examined the pathological changes of livers of aged GHRLD male mice. RESULTS: The biochemical profile was consistent with that of the metabolic syndrome: abnormal glucose tolerance, impaired insulin secretion, and hyperlipidemia. RT-qPCR analysis of key markers of inflammation revealed a three- to fivefold increase in TNFα and CCL3, confirming the presence of inflammation. Expression of fibrotic markers (e.g., Col1A2 and Col3A1) was significantly increased, together with a two- to threefold increase in TGFß transcripts. Proteomics analyses showed a marked decrease of Mup1 and Selenbp2. In addition, pathway-analysis showed that the expression of cell cycle and growth relevant genes (i.e., Ccnd1, Socs2, Socs3, and Egfr) were markedly affected in GHRLD liver. Microscopic analyses (H&E) of GHRLD livers revealed the presence of hepatic adenomas of different stages of malignancy. CONCLUSION: Abrogation of GH signaling in male liver leads to metabolic syndrome, hepatic steatosis, increased inflammation and fibrosis, and development of hepatic tumor. Since obesity, a common precursor of NAFLD, is a state of deficient GH secretion and action, the GHRLD model could be used to unravel the contribution of compromised hepatic GH signaling in these pathological processes, and help to identify potential targets for intervention.

Diagnosis of paroxysmal nocturnal hemoglobinuria in peripheral blood and bone marrow with six-color flow cytometry.

AIM: Identification of paroxysmal nocturnal hemoglobinuria (PNH) by detecting a glycophosphatidylinositol-anchored defect by flow cytometry is presently the standard method of choice for diagnosing PNH. However, the selection of suitable markers will be critical and significantly affect the determination and quantification of PNH clones in various cell lineages. MATERIALS & METHODS: In this study, we investigated the performance of various immunophenotypic markers including CD59, GPHA (a clustered antigen, CD235a), CD33, CD15 and fluorescent aerolysin (FLAER) combined with CD16, CD24 and CD14 in a PNH panel using six-color flow cytometry. RESULTS: The results strongly indicate that these markers can collectively and effectively identify and quantify PNH clones in erythrocyte, granulocyte and monocyte populations derived from peripheral blood and bone marrow (BM). A sensitivity threshold as low as 0.01% in identifying PNH clones in erythrocyte and granulocyte populations from peripheral blood is achieved by this panel in a series dilution assay. In addition, a direct side-by-side comparison between BM and peripheral blood from the same patients suggests that the FLAER PNH test is capable of identifying to PNH clones in BM specimens. CONCLUSION: The data support the premise that a six-color flow cytometry PNH panel using the combination of CD59, CD235a, CD33, CD15, FLAER, CD16, CD24 and CD14 can enhance and improve the current methods used in diagnosis and management of PNH by specifically identifying PNH clones in the erythrocyte, granulocyte and monocyte population.

Risk stratification of plasma cell neoplasm: insights from plasma cell-specific cytoplasmic immunoglobulin fluorescence in situ hybridization (cIg FISH) vs. conventional FISH.

UNLABELLED: We directly compared the results of routine fluorescence in situ hybridization (FISH) and plasma cell-specific cytoplasmic immunoglobulin (cIg) FISH from 75 paired samples for myeloma risk stratification. CIg FISH improves test specificity and sensitivity and tends to eliminate borderline results. It proves that most plasma cells (PCs) consistently carry the abnormality in myelomas with an IGH translocation, whereas routine FISH detects these cells only at variably low levels. BACKGROUND: Routine cytogenetic analysis of plasma cell neoplasms (PCNs) has a low sensitivity. Conventional fluorescence in situ hybridization (FISH) is not plasma cell (PC) specific and results are diluted by other cells in the sample. Although PC-specific FISH testing has been recommended for multiple myeloma (MM) risk stratification, eg, by combining cytoplasmic immunoglobulin (cIg) staining with FISH, the benefits of cIg FISH have never been directly demonstrated in a controlled study. PATIENTS AND METHODS: Seventy-five samples from patients with PCNs were analyzed by concomitant conventional FISH and cIg FISH with probes for t(4;14), t(11;14), t(14;16), -13, 17p-, and +3. The results were compared for their reliability, specificity, and consistency. RESULTS: Apart from marginally improving detection threshold in samples with low PC burden, cIg FISH identified more abnormal cases (50 vs. 47 cases) and more chromosome abnormalities (113 vs. 103 events) than did conventional FISH. It differentiated del(13q) in myelodysplasia from MM. Remarkably, cIg FISH consistently identified a high percentage of abnormal PCs in all cases. It detected IGH translocation in 78% to 100% of PCs in all but 2 positive cases, whereas conventional FISH detected 0% to 46% in these cases (median, 91% vs. 9%). The abnormal cells found in patients with 17p- were 19% to 96% by cIg FISH vs. 0% to 13% by conventional FISH (median, 54% vs. 9%). Cases with insufficient PCs for cIg FISH had only normal conventional FISH results. CONCLUSION: CIg FISH improves reliability of FISH testing for PCNs by eliminating borderline results. In myelomas with an IGH translocation, myeloma cells invariably carry the abnormality.

Reactive oxygen species suppress cardiac NaV1.5 expression through Foxo1.

Na(V)1.5 is a cardiac voltage-gated Na(+) channel αsubunit and is encoded by the SCN5a gene. The activity of this channel determines cardiac depolarization and electrical conduction. Channel defects, including mutations and decrease of channel protein levels, have been linked to the development of cardiac arrhythmias. The molecular mechanisms underlying the regulation of Na(V)1.5 expression are largely unknown. Forkhead box O (Foxo) proteins are transcriptional factors that bind the consensus DNA sequences in their target gene promoters and regulate the expression of these genes. Comparative analysis revealed conserved DNA sequences, 5'-CAAAACA-3' (insulin responsive element, IRE), in rat, mouse and human SCN5a promoters with the latter two containing two overlapping Foxo protein binding IREs, 5'-CAAAACAAAACA-3'. This finding led us to hypothesize that Foxo1 regulates Na(V)1.5 expression by directly binding the SCN5a promoter and affecting its transcriptional activity. In the present study, we determined whether Foxo1 regulates Na(V)1.5 expression at the transcriptional level and also defined the role of Foxo1 in hydrogen peroxide (H(2)O(2))-mediated Na(V)1.5 suppression in HL-1 cardiomyocytes using chromatin immunoprecipitation (ChIP), constitutively nuclear Foxo1 expression, and RNAi Foxo1 knockdown as well as whole cell voltage-clamp recordings. ChIP with anti-Foxo1 antibody and follow-up semi-quantitative PCR with primers flanking Foxo1 binding sites in the proximal SCN5a promoter region clearly demonstrated enrichment of DNA, confirming Foxo1 recruitment to this consensus sequence. Foxo1 mutant (T24A/S319A-GFP, Foxo1-AA-GFP) was retained in nuclei, leading to a decrease of Na(V)1.5 expression and Na(+) current, while silencing of Foxo1 expression by RNAi resulted in the augmentation of Na(V)1.5 expression. H(2)O(2) significantly reduced Na(V)1.5 expression by promoting Foxo1 nuclear localization and this reduction was prevented by RNAi silencing Foxo1 expression. These studies indicate that Foxo1 negatively regulates Na(V)1.5 expression in cardiomyocytes and reactive oxygen species suppress Na(V)1.5 expression through Foxo1.

Foxo1 mediates insulin-like growth factor 1 (IGF1)/insulin regulation of osteocalcin expression by antagonizing Runx2 in osteoblasts.

In this study, we determined the molecular mechanisms whereby forkhead transcription factor Foxo1, a key downstream signaling molecule of insulin-like growth factor 1 (IGF1)/insulin actions, regulates Runx2 activity and expression of the mouse osteocalcin gene 2 (Bglap2) in osteoblasts in vitro. We showed that Foxo1 inhibited Runx2-dependent transcriptional activity and osteocalcin mRNA expression and Bglap2 promoter activity in MC-4 preosteoblasts. Co-immunoprecipitation assay showed that Foxo1 physically interacted with Runx2 via its C-terminal region in osteoblasts or when co-expressed in COS-7 cells. Electrophoretic mobility shift assay demonstrated that Foxo1 suppressed Runx2 binding to its cognate site within the Bglap2 promoter. IGF1 and insulin prevented Foxo1 from inhibiting Runx2 activity by promoting Foxo1 phosphorylation and nuclear exclusion. In contrast, a neutralizing anti-IGF1 antibody decreased Runx2 activity and osteocalcin expression in osteoblasts. Chromatin immunoprecipitation assay revealed that IGF1 increased Runx2 interaction with a chromatin fragment of the proximal Bglap2 promoter in a PI3K/AKT-dependent manner. Conversely, knockdown of Foxo1 increased Runx2 interaction with the promoter. This study establishes that Foxo1 is a novel negative regulator of osteoblast-specific transcription factor Runx2 and modulates IGF1/insulin-dependent regulation of osteocalcin expression in osteoblasts.

CD71 is selectively and ubiquitously expressed at high levels in erythroid precursors of all maturation stages: a comparative immunochemical study with glycophorin A and hemoglobin A.

Histology assessment of erythroid precursors in bone marrow biopsies can be challenging under pathologic conditions and often requires ancillary studies. CD71 (transferring receptor-1) is known to be expressed in the earliest erythroid precursors, and has been useful for flow cytometry. However, CD71 is also regarded as a proliferation marker, and its lineage specificity has not been systemically investigated by immunohistochemistry in detail. In this study, we found that CD71 was strongly expressed in all erythroid precursors in normal and dyspoietic marrows. Staining of CD71 effectively highlighted pronormoblasts in all 4 cases of parvovirus infection and erythroblasts in all 6 cases of acute erythroleukemia, for which staining of glycophorin A and hemoglobin A was either absent or unreliable. CD71 was absent in the background mature red blood cells in general, nonerythroid elements in the normal marrow, myeloid precursors in myeloproliferative disorders, and blasts in nearly all acute myeloid leukemia encompassing all common French-American-British subtypes. Benign lymphoid infiltrates and low-grade lymphomas involving the marrow also lacked detectable CD71. Although weak CD71 expression was found in acute lymphoblastic leukemia, diffuse large B-cell lymphoma, and 1 of 2 cases of acute megakaryoblastic leukemia, it had little impact on interpretation due to a high signal-to-noise ratio of the staining intensities when compared with erythroid precursors in the same section. We conclude that CD71 is selectively expressed at high levels in erythroid precursors, including those at early maturation stages. It can be reliably used as an independent erythroid marker for immunohistochemical analysis of the marrow.

Flow cytometry rapidly identifies all acute promyelocytic leukemias with high specificity independent of underlying cytogenetic abnormalities.

Acute promyelocytic leukemia (APL) is a highly aggressive disease requiring prompt diagnosis and specific early intervention. Immunophenotyping by flow cytometry (FCM) facilitates a rapid diagnosis, but commonly used criteria are neither sufficiently sensitive nor specific. With an antibody panel for diagnostic screening in routine practice, we found all 149 APL cases in this study exhibited a unique immunophenotypic profile, ie, a characteristic CD11b- myeloid population and absent CD11c expression in all myeloid populations; 96.6% of cases also lacked HLA-DR expression. These distinctive features allowed recognition of all unusual cases phenotypically resembling the regular myeloblasts (CD34+/HLA-DR+) or granulocytes (CD117-/CD34-/HLA-DR-). FCM effectively identified all 19 APL cases with variant translocations, including cases with a normal karyotype due to a cryptic submicroscopic t(15;17)(q22;q21), t(11;17)(q23;q21) that escaped the detection by fluorescence in situ hybridization for t(15;17) and der(15)ider(17)(q10) that lacked a simple reciprocal t(15;17). When APL-associated profiles were validated against 107 AML cases of non-APL subtypes, including 51 HLA-DR- cases, the diagnostic specificity and positive predictive value were 98%. FCM effectively provides independent detection of APL during diagnostic workup and harmonizes with the subsequent molecular cytogenetic diagnosis.

Immunophenotypic analysis of CD103+ B-lymphoproliferative disorders: hairy cell leukemia and its mimics.

CD103 is characteristically expressed in hairy cell leukemia (HCL), a B-lymphoproliferative disorder highly responsive to treatment with purine analogs. Other CD103+ diseases are rare and do not respond well to the same therapy, including HCL variant (HCLv) and splenic marginal zone B-cell lymphoma (SMZL) variants. We analyzed 215 cases of CD103+ B-lymphoproliferative disorders to further delineate their immunophenotypic features. Flow cytometric analysis revealed that 78.6% of all cases expressed CD25 and CD103, characteristic of classical HCL. Cases analyzed immunohistochemically were also invariably positive for annexin-A1; a subset coexpressed CD10 (33/169 [19.5%]) or BCL1 (26/65 [36.9%]). In contrast, 21.4% of cases lacked CD25, a subset of which was analyzed and was invariably negative for annexin-A1, CD10, and BCL1. The CD25- cases had variable morphologic features ranging from HCLv and SMZL to prolymphocytic leukemia and diffuse large B-cell lymphoma. Clinically, patients with CD25- disease tended to be older (P= .001), typically had leukocytosis (P= .014), and did not respond well to cladribine or pentostatin. We suggest categorizing CD103+ B-lymphoproliferative disorders into 2 groups. While HCL coexpresses CD25 and annexin-A1, diseases lacking CD25 and annexin-A1 behave clinically differently and can be separated from HCL on diagnosis.

Laboratory practice guidelines for detecting and reporting BCR-ABL drug resistance mutations in chronic myelogenous leukemia and acute lymphoblastic leukemia: a report of the Association for Molecular Pathology.

The BCR-ABL tyrosine kinase produced by the t(9;22)(q34;q11) translocation, also known as the Philadelphia chromosome, is the initiating event in chronic myeloid leukemia (CML) and Ph+ acute lymphoblastic leukemia (ALL). Targeting of BCR-ABL with tyrosine kinase inhibitors (TKIs) has resulted in rapid clinical responses in the vast majority of patients with CML and Philadelphia chromosome+ ALL. However, long-term use of TKIs occasionally results in emergence of therapy resistance, in part through the selection of clones with mutations in the BCR-ABL kinase domain. We present here an overview of the current practice in monitoring for such mutations, including the methods used, the clinical and laboratory criteria for triggering mutational analysis, and the guidelines for reporting BCR-ABL mutations. We also present a proposal for a public database for correlating mutational status with in vitro and in vivo responses to different TKIs to aid in the interpretation of mutation studies.