A thorny matter: Spur cell anemia.

Spur cell anemia (SCA) is an acquired form of non-autoimmune hemolytic anemia that occurs in advanced liver disease. It is characterized by the presence of acanthocytes or spur cells, spiculated erythrocytes whose shortened life span causes anemia that is unresponsive to transfusion. SCA has been regarded as a rare condition with an ominous prognosis for which the only known cure is liver transplantation, but recent prospective studies have demonstrated the existence of a milder form of SCA in which there are smaller numbers of acanthocytes, but which is nevertheless associated with hemolysis and poor outcomes. This form of SCA appears to be considerably more common than the severe classical variant. The conventional understanding of the pathogenesis of SCA is that abnormalities of lipid metabolism are the primary event driving the formation of spur cells. However, the studies that underpin this theory are based on small numbers of patients with heterogeneous clinical features and inconsistent use of nomenclature for dysmorphic red blood cells. In this review, we discuss the evolution of the current understanding of SCA and therapeutic strategies that have been employed based on this understanding. Our goal is to raise awareness of this understudied condition that has significant implications for patient outcomes. Furthermore, we highlight the need for rigorous, contemporary research into the underlying cause or causes of SCA in order to develop an effective therapy for this disorder.

Correction to: Upregulation of FOXM1 leads to diminished drug sensitivity in myeloma.

As a result of an author oversight in the original article [1], the legend of Figure 5A and C is inaccurate and one panel in Figure 5C (FOXM1N H929 cells shown in the top row, left) is wrong.

Upregulation of FOXM1 leads to diminished drug sensitivity in myeloma.

BACKGROUND: Following up on previous work demonstrating the involvement of the transcription factor forkhead box M1 (FOXM1) in the biology and outcome of a high-risk subset of newly diagnosed multiple myeloma (nMM), this study evaluated whether FOXM1 gene expression may be further upregulated upon tumor recurrence in patients with relapsed multiple myeloma (rMM). Also assessed was the hypothesis that increased levels of FOXM1 diminish the sensitivity of myeloma cells to commonly used myeloma drugs, such as the proteasome inhibitor bortezomib (Bz) and the DNA intercalator doxorubicin (Dox). METHODS: FOXM1 message was evaluated in 88 paired myeloma samples from patients with nMM and rMM, using gene expression microarrays as measurement tool. Sources of differential gene expression were identified and outlier analyses were performed using statistical methods. Two independent human myeloma cell lines (HMCLs) containing normal levels of FOXM1 (FOXM1N) or elevated levels of lentivirus-encoded FOXM1 (FOXM1Hi) were employed to determine FOXM1-dependent changes in cell proliferation, survival, efflux-pump activity, and drug sensitivity. Levels of retinoblastoma (Rb) protein were determined with the assistance of Western blotting. RESULTS: Upregulation of FOXM1 occurred in 61 of 88 (69%) patients with rMM, including 4 patients that exhibited > 20-fold elevated expression peaks. Increased FOXM1 levels in FOXM1Hi myeloma cells caused partial resistance to Bz (1.9-5.6 fold) and Dox (1.5-2.9 fold) in vitro, using FOXM1N myeloma as control. Reduced sensitivity of FOXM1Hi cells to Bz was confirmed in vivo using myeloma-in-mouse xenografts. FOXM1-dependent regulation of total and phosphorylated Rb agreed with a working model of myeloma suggesting that FOXM1 governs both chromosomal instability (CIN) and E2F-dependent proliferation, using a mechanism that involves interaction with NIMA related kinase 2 (NEK2) and cyclin dependent kinase 6 (CDK6), respectively. CONCLUSIONS: These findings enhanced our understanding of the emerging FOXM1 genetic network in myeloma and provided preclinical support for the therapeutic targeting of the FOXM1-NEK2 and CDK4/6-Rb-E2F pathways using small-drug CDK and NEK2 inhibitors. Clinical research is warranted to assess whether this approach may overcome drug resistance in FOXM1Hi myeloma and, thereby, improve the outcome of patients in which the transcription factor is expressed at high levels.

Tim-3 is an inducible human natural killer cell receptor that enhances interferon gamma production in response to galectin-9.

NK-cell function is regulated by the integration of signals received from activating and inhibitory receptors. Here we show that a novel immune receptor, T-cell Ig and mucin-containing domain-3 (Tim-3), is expressed on resting human NK cells and is up-regulated on activation. The NK92 NK-cell line engineered to overexpress Tim-3 showed a marked increase in IFN-γ production in the presence of soluble rhGal-9 or Raji tumor cells engineered to express Gal-9. The Tim-3(+) population of low-dose IL-12/IL-18-activated primary NK cells significantly increased IFN-γ production in response to soluble rhGal-9, Gal-9 presented by cell lines, and primary acute myelogenous leukemia (AML) targets that endogenously express Gal-9. This effect is highly specific as Tim-3 Ab blockade significantly decreased IFN-γ production, and Tim-3 cross-linking induced ERK activation and degradation of IκBα. Exposure to Gal-9-expressing target cells had little effect on CD107a degranulation. Reconstituted NK cells obtained from patients after hematopoietic cell transplantation had diminished expression of Tim-3 compared with paired donors. This observation correlates with the known IFN-γ defect seen early posttransplantation. In conclusion, we show that Tim-3 functions as a human NK-cell coreceptor to enhance IFN-γ production, which has important implications for control of infectious disease and cancer.

Congenital dyserythropoietic anemia type I presenting as persistent pulmonary hypertension with pigeon chest deformity.

Congenital dyserythropoietic anemia (CDA) type-1 is a rare genetic disorder of ineffective erythropoiesis, which manifests in macrocytic anemia. We report a CDA1 patient who as a newborn presented with macrocytic anemia and persistent pulmonary hypertension of the newborn (PPHN) requiring mechanical ventilation. Post-infancy, the patient developed acral dysmorphism and pectus excavatum the latter rarely found in CDA1. Patient is a compound heterozygote for a known maternal-derived missense-mutation (c.1796A > G/p.Asn589Ser) and a novel paternal-derived deletion-mutation (c.1104_1106del/Phe365del) in CDAN1. This report highlights the importance of recognizing PPHN as a presenting symptom of CDA1 and expands the repertoire of the accompanying mutations and axial skeletal malformations.

Urologic Presentation of Unicentric Pediatric Castleman Disease in the Setting of Acute Renal Colic.

An 11-year-old otherwise healthy female presented with renal colic and during computed tomography imaging evaluation, she was found to have a right distal ureteral stone with associated hydroureteronephrosis, medially deviated ureter, and 4-cm solid retroperitoneal mass. The mass was palpable on physical exam and was further categorized with magnetic resonance imaging, ultrasound, and laboratory testing. A multidisciplinary team approach, including pediatric surgery, radiology, oncology, and urology, led to the patient undergoing a right retrograde pyelogram, ureteroscopy with stent placement, and laparoscopic excision of retroperitoneal mass. Her pathology revealed lymphoid hyperplasia with histologic features of Castleman disease.

Systematic Evaluation of Hematologic Parameters and Blood Smear Findings in Patients With SARS-CoV-2 vs Other Viral Respiratory Infections.

OBJECTIVES: We evaluated and compared the peripheral blood findings in patients with acute COVID-19 vs other viral respiratory infections. METHODS: We retrospectively reviewed peripheral blood counts and smear morphology in patients with a positive viral respiratory panel (VRP) or SARS-CoV-2 test. RESULTS: A total of 97 peripheral blood samples (COVID-19 infection, 53; VRP positive, 44) from 50 patients (mean [SD] age, 45.8 [20.8] years; females 52%) were reviewed. There were no statistically significant differences in the demographic characteristics between the 2 groups. The most common peripheral blood abnormalities were anemia, thrombocytopenia, absolute lymphopenia, and reactive lymphocytes. The following peripheral blood findings were significantly associated with other viral respiratory infections compared with COVID-19 infection: low red blood cell count, low hematocrit, high mean corpuscular volume, thrombocytopenia, low mean platelet volume, high red cell distribution width, band neutrophilia, and toxic granulation in neutrophils. CONCLUSIONS: Our study showed that there are several peripheral blood count and morphologic abnormalities seen in patients with COVID-19, but most of these findings lack specificity as they are also seen in the other viral respiratory infections.

Quantitative Epstein-Barr virus shedding and its correlation with the risk of post-transplant lymphoproliferative disorder.

We postulated that quantitative monitoring of Epstein-Barr virus (EBV) shedding after transplantation could distinguish EBV-associated illnesses and predict clinical outcome. EBV DNA was measured in solid organ (SOT) and hematopoietic cell transplants (HCT) using our own real-time TaqMan EBV PCR. The proportion of patients who had EBV DNAemia post-transplant was significantly lower in HCT vs. SOT (p < 0.001). Over a 7.5-yr period, post-transplant lymphoproliferative disorder (PTLD) occurred in 66 (5.8%) of 1131 patients who met adequate monitoring criteria. SOT recipients developed PTLD significantly later than HCT recipients (median, 2.8 yr vs. 121 d; p < 0.001). PTLD was documented in 53 (14%) of 376 patients who had EBV in ≥1 whole blood sample vs. 13 (2%) of 755 patients who had at least three EBV-negative blood samples and were never positive. PTLD risk in viremic patients increased with the peak quantity of EBV DNAemia (p < 0.001). PTLD occurred in 37/333 (11%) of patients with peak blood levels 10(3) -10(5) copies/mL vs. 16/43 (37%) of patients with levels >10(5) (p < 0.001). EBV PCR was predictive in 29 (78%) of 37 patients tested within three wk prior to tissue diagnosis of PTLD, and thus, we conclude that EBV PCR with careful attention paid to changes in EBV DNAemia could lead to earlier diagnosis and treatment of PTLD.

HSF1 is a driver of leukemia stem cell self-renewal in acute myeloid leukemia.

Acute myeloid leukemia (AML) is maintained by self-renewing leukemic stem cells (LSCs). A fundamental problem in treating AML is that conventional therapy fails to eliminate LSCs, which can reinitiate leukemia. Heat shock transcription factor 1 (HSF1), a central regulator of the stress response, has emerged as an important target in cancer therapy. Using genetic Hsf1 deletion and a direct HSF1 small molecule inhibitor, we show that HSF1 is specifically required for the maintenance of AML, while sparing steady-state and stressed hematopoiesis. Mechanistically, deletion of Hsf1 dysregulates multifaceted genes involved in LSC stemness and suppresses mitochondrial oxidative phosphorylation through downregulation of succinate dehydrogenase C (SDHC), a direct HSF1 target. Forced expression of SDHC largely restores the Hsf1 ablation-induced AML developmental defect. Importantly, the growth and engraftment of human AML cells are suppressed by HSF1 inhibition. Our data provide a rationale for developing efficacious small molecules to specifically target HSF1 in AML.

Markers of Follicular Helper T Cells Are Occasionally Expressed in T-Cell or Histiocyte-Rich Large B-Cell Lymphoma, Classic Hodgkin Lymphoma, and Atypical Paracortical HyperplasiaA Diagnostic Pitfall For T-Cell Lymphomas of T Follicular Helper Origin.

OBJECTIVES: Follicular helper T cell (TFH) markers are expressed in angioimmunoblastic T-cell lymphoma (AITL) and peripheral T-cell lymphoma of the TFH phenotype (PTCL-TFH). However, differential expression and coexpression of these markers in benign and other malignant lymphoid proliferations have not been well studied. METHODS: We performed programmed death-1 (PD-1), C-X-C motif chemokine ligand 13 (CXCL13), inducible costimulator (ICOS), CD10, and B-cell lymphoma 6 protein (BCL-6) immunohistochemistry on AITL, PTCL not otherwise specified (PTCL-NOS), PTCL-TFH, T-cell or histiocyte-rich large B-cell lymphoma (THRLBCL), classic Hodgkin lymphoma (CHL), atypical paracortical hyperplasia (PCH), progressive transformation of germinal centers (PTGC), and reactive follicular hyperplasia (RFH). RESULTS: CXCL13 and ICOS were more sensitive but less specific for AITL than PD-1, CD10, and BCL-6. Moreover, 74% of AITL (none of PTCL-NOS or PTCL-TFH) coexpressed more than 2 TFH markers. In background T cells of THRLBCL, 70% of cases coexpressed more than 1 marker. The background T cells of CHL expressed all TFH markers except CD10 in all cases. In addition, 13% of PCH cases coexpressed more than 1 marker. In RFH and PTGC, all markers were expressed mainly in germinal centers with rare extrafollicular staining. CONCLUSIONS: AITL, PTCL-NOS, and PTCL-TFH show differential expression of TFH markers. AITL frequently coexpresses more than 2 TFH markers. TFH markers can be expressed in PCH and in background T cells of THRLBCL and CHL. Consequently, caution should be used before a diagnosis of AITL is established, particularly with limited samples.

Utility of Flow Cytometry and Fluorescence In Situ Hybridization in Follow-up Monitoring of Plasma Cell Myeloma.

OBJECTIVES: We sought to investigate the clinical utility of flow cytometry (FC) and fluorescence in situ hybridization (FISH) in the workup of myeloma. METHODS: We retrospectively reviewed the reports of bone marrow biopsies received for myeloma evaluation between October 2015 and January 2019. RESULTS: A total of 1,708 biopsy specimens from 469 myeloma patients (mean age, 64.5 years [SD, 9.3]; female, 41.4%) were reviewed. Both FC and FISH had comparable detection rates at the time of initial diagnosis (97.6% vs 98.8%) and for follow-up cases (28.6% vs 28.2%). FC and FISH results were concordant in 98.8% of the initial diagnosis cases and 89.6% of the follow-up cases. The FISH-positive (FISH+)/FC-negative (FC-) discordance and FISH-/FC+ discordance occurred among 81 (5.0%) and 87 (5.4%) follow-up cases. In comparison with all concordant cases, FISH+/FC- discordant cases were more likely to have received treatment with daratumumab (P < .05). CONCLUSIONS: Plasma cell-enriched FISH and FC have comparable abnormal plasma cell detection rates, and approximately 10% of the follow-up cases have discordant FISH and FC results in which residual disease is detected by only one of these modalities. FISH testing should be considered for cases with negative FC, especially in patients who have received treatment with daratumumab or in cases in which there is concern about specimen adequacy.

Immunophenotypic Heterogeneity of Polytypic Plasma Cells and the Impact on Myeloma Minimal Residual Disease Detection by Multiparameter Flow Cytometry.

BACKGROUND: Flow cytometry is widely used for minimal residual disease (MRD) detection in plasma cell myeloma (PCM). Recently, an increasing number of studies have demonstrated that polytypic plasma cells (PPCs) display greater immunophenotypic variation than previously appreciated. Our aim was to further characterize the immunophenotype of PPC in this setting. METHODS: PPC in 102 bone marrow specimens (93 MRD-negative post-treatment PCM and 9 negative initial evaluations for plasma cell neoplasm) were evaluated by 10-color flow cytometry. Frequency of CD19, CD27, CD28, CD56, CD117, and CD138 expression was assessed. RESULTS: All cases showed CD27 and CD19 positivity. CD117 was uniformly negative. Percentage of CD138 expression was variable with one negative case (<20% expression). Percentage of CD28 and CD56 expression was variable and included 11 CD28+ cases as well as 38 CD56+ cases. Forty-two percent (43 of 102) cases showed atypical expression of at least one marker with 36 cases (35%) showing atypical expression of a single marker and 7 cases (7%) showing dual atypical marker expression (CD56+/CD28+). CONCLUSIONS: Considerable immunophenotypic variation exists in PPC. The assessment of cytoplasmic light chain distribution, in conjunction with surface marker expression, is recommended to avoid diagnostic inaccuracy in MRD evaluation. © 2019 International Clinical Cytometry Society.

Characterization of protein cross-links via mass spectrometry and an open-modification search strategy.

Protein-protein interactions are key to function and regulation of many biological pathways. To facilitate characterization of protein-protein interactions using mass spectrometry, a new data acquisition/analysis pipeline was designed. The goal for this pipeline was to provide a generic strategy for identifying cross-linked peptides from single LC/MS/MS data sets, without using specialized cross-linkers or custom-written software. To achieve this, each peptide in the pair of cross-linked peptides was considered to be "post-translationally" modified with an unknown mass at an unknown amino acid. This allowed use of an open-modification search engine, Popitam, to interpret the tandem mass spectra of cross-linked peptides. False positives were reduced and database selectivity increased by acquiring precursors and fragments at high mass accuracy. Additionally, a high-charge-state-driven data acquisition scheme was utilized to enrich data sets for cross-linked peptides. This open-modification search based pipeline was shown to be useful for characterizing both chemical as well as native cross-links in proteins. The pipeline was validated by characterizing the known interactions in the chemically cross-linked CYP2E1-b5 complex. Utility of this method in identifying native cross-links was demonstrated by mapping disulfide bridges in RcsF, an outer membrane lipoprotein involved in Rcs phosphorelay.

Flow cytometric aberrancies in plasma cell myeloma and MGUS - correlation with laboratory parameters.

BACKGROUND: Multiparametric flow cytometry is a useful tool for diagnosis of plasma cell (PC) dyscrasias and assessment of minimal residual disease in plasma cell myeloma (PCM). However, the immunophenotypic differences between the clonal PCs of PCM and those of monoclonal gammopathy of undetermined significance (MGUS) as well as the correlation of these flow cytometric markers with pertinent laboratory parameters have not been evaluated. METHODS: We retrospectively identified all newly diagnosed treatment-naive PCM and MGUS patients between 09/2014 and 06/2015 who underwent 10-color flow-cytometric evaluation: CD45, CD38, CD138, cKappa, cLambda, CD19, CD27, CD28, CD56, CD117. FACSDiva analysis was used to identify antigenic aberrancies and associations with pertinent laboratory parameters were evaluated. RESULTS: All cases demonstrated at least two aberrancies. There was a trend toward a greater number of aberrancies in PCM, with 68% showing >/= 4 aberrancies compared with 44% in MGUS (P = 0.11). The only marker more frequently aberrant in one disease class was CD19, aberrant in 68% of PCM and 25% of MGUS (P < 0.01). In PCM, significant associations were found for CD56 non-aberrancy (P = 0.05) and the presence of amyloid and CD27 aberrancy and normal serum albumin (P = 0.05). In MGUS, CD117 expression was associated with normal hemoglobin (P = 0.03). CONCLUSIONS: The PCs of PCM show a trend toward more antigenic aberrancy than those of MGUS. There is significant association between the antigenic profiles of PCM/MGUS and clinical parameters including amyloidosis, albumin level, and hemoglobin. © 2018 International Clinical Cytometry Society.

A virologic pilot study of valacyclovir in infectious mononucleosis.

BACKGROUND: Infectious mononucleosis decreases the productivity of many college students and Epstein-Barr virus (EBV) infection may result in long-term immune damage. OBJECTIVES: Evaluate the antiviral effect of valacyclovir during EBV-related acute infectious mononucleosis and explore potential clinical benefits. STUDY DESIGN: University students who presented during the first 7 days of illness were randomized to receive valacyclovir 3g/day for 14 days or not. The quantity of Epstein-Barr virus (EBV) DNA in oral and whole blood samples was determined by real-time (TaqMan) PCR. The primary outcome was the proportion of subjects with laboratory-confirmed primary EBV infection who had >or=2 log10 decrease in EBV copies/mL in oral washes during the treatment period. Secondary outcomes included clinical effects. RESULTS: Twenty subjects were studied. The proportion of valacyclovir recipients versus control subjects who had >or=2 log10 decrease in EBV copies was significantly greater for both oral wash fluid-derived cell pellet (P=0.03) and supernatant (P=0.001) samples. At the end of the treatment period, the number of reported symptoms (P=0.03) and the severity of illness (P=0.049) were reduced among valacyclovir recipients as compared with controls. CONCLUSIONS: Valacyclovir therapy caused a reduction of EBV excretion and possibly produced a clinical benefit in infectious mononucleosis. Because our study was small and not placebo-controlled, these results must be confirmed by a larger, placebo-controlled trial.

Leukemic Transdifferentiation of Follicular Lymphoma Into an Acute Histiocytic Leukemia in a 52-Year-Old Caucasian Woman.

We report an instructive case of acute myeloid leukemia with histiocytic differentiation (acute histiocytic leukemia) arising in a patient, a 52-year-old woman with a history of follicular lymphoma. The results of genetic studies proved a clonal relationship between the lymphoma and the leukemic cells. To our knowledge, this is the first report of leukemic transdifferentiation of follicular lymphoma into modified base 5-methylcytosine (M(5)c)-like acute histiocytic leukemia and the first reported karyotype on a transdifferentiated neoplasm.

CDKN1A and FANCD2 are potential oncotargets in Burkitt lymphoma and multiple myeloma.

BACKGROUND: Comparative genetic and biological studies on malignant tumor counterparts in human beings and laboratory mice may be powerful gene discovery tools for blood cancers, including neoplasms of mature B-lymphocytes and plasma cells such as Burkitt lymphoma (BL) and multiple myeloma (MM). METHODS: We used EMSA to detect constitutive NF-κB/STAT3 activity in BL- and MM-like neoplasms that spontaneously developed in single-transgenic IL6 (interleukin-6) or MYC (c-Myc) mice, or in double-transgenic IL6MYC mice. qPCR measurements and analysis of clinical BL and MM datasets were employed to validate candidate NF-κB/STAT3 target genes. RESULTS: qPCR demonstrated that IL6- and/or MYC-dependent neoplasms in mice invariably contain elevated mRNA levels of the NF-κB target genes, Cdkn1a and Fancd2. Clinical studies on human CDKN1A, which encodes the cell cycle inhibitor and tumor suppressor p21, revealed that high p21 message predicts poor therapy response and survival in BL patients. Similarly, up-regulation of FANCD2, which encodes a key member of the Fanconi anemia and breast cancer pathway of DNA repair, was associated with poor outcome of patients with MM, particularly those with high-risk disease. CONCLUSIONS: Our findings suggest that CDKN1A and FANCD2 are potential oncotargets in BL and MM, respectively. Additionally, the IL-6- and/or MYC-driven mouse models of human BL and MM used in this study may lend themselves to the biological validation of CDKN1A and FANCD2 as molecular targets for new approaches to cancer therapy and prevention.