Clinical significance of pathologic abnormalities in biopsy samples from the appendiceal orifice.

AIMS: Appendiceal orifice mucosa often appears inflamed endoscopically, even when other colonic segments appear normal. Histological findings in biopsy samples taken from endoscopically abnormal mucosa may simulate a variety of inflammatory colitides. We performed this study to evaluate the clinical implications of inflammatory changes isolated to the appendiceal orifice. METHODS AND RESULTS: In this double cohort study, biopsy samples from 26 histologically abnormal appendiceal orifices were reviewed. Twenty-five control cases were culled from endoscopically normal (n = 11) and abnormal (n = 14) appendiceal orifices that were histologically normal. Histological findings were correlated with presentation, medication history, findings at other colonic sites and clinical outcomes. Study cases displayed active inflammation (n = 12), chronic active inflammation (n = 13) or features simulating collagenous colitis (n = 1). Eighteen patients had biopsies taken from other colonic sites; these revealed benign polyps (n = 10) or displayed active (n = 4) or chronic active (n = 4) inflammation. All patients with findings isolated to the appendiceal orifice were asymptomatic at most recent clinical follow-up. Four of eight (50%) of the patients with inflammation in other biopsy samples were ultimately diagnosed with ulcerative colitis, in keeping with the well-established role of the appendix as a 'skip lesion' in that disorder. Control patients presented for screening colonoscopy (n = 19), iron deficiency anaemia (n = 3) or change in bowel habits (n = 3) and none reported gastrointestinal symptoms upon follow-up, regardless of the endoscopic appearance of the appendiceal orifice. CONCLUSION: Isolated inflammation of the appendiceal orifice mucosa should not be regarded as a feature of evolving inflammatory bowel disease or other types of chronic colitis.

SHROOM3-FYN Interaction Regulates Nephrin Phosphorylation and Affects Albuminuria in Allografts.

BACKGROUND: We previously showed that the presence of a CKD-associated locus in SHROOM3 in a donor kidney results in increased expression of SHROOM3 (an F-actin-binding protein important for epithelial morphogenesis, via rho-kinase [ROCK] binding); this facilitates TGF-b signaling and allograft fibrosis. However, other evidence suggests Shroom3 may have a protective role in glomerular development. METHODS: We used human data, Shroom3 knockdown podocytes, and inducible shRNA-mediated knockdown mice to study the role of Shroom3 in adult glomeruli. RESULTS: Expression data from the Nephroseq database showed glomerular and nonglomerular SHROOM3 had opposing associations with renal function in CKD biopsy samples. In human allografts, homozygosity at rs17319721, the SHROOM3 locus linked with lower GFR, was associated with reduced albuminuria by 2 years after transplant. Although our previous data showed reduced renal fibrosis with tubular Shroom3 knockdown, this study found that glomerular but not tubular Shroom3 knockdown induced albuminuria. Electron microscopy revealed diffuse foot process effacement, and glomerular RNA-sequencing showed enrichment of tyrosine kinase signaling and podocyte actin cytoskeleton pathways in knockdown mice. Screening SHROOM3-interacting proteins identified FYN (a src-kinase) as a candidate.We confirmed the interaction of endogenous SHROOM3 with FYN in human podocytes via a critical Src homology 3-binding domain, distinct from its ROCK-binding domain. Shroom3-Fyn interaction was required in vitro and in vivo for activation of Fyn kinase and downstream nephrin phosphorylation in podocytes. SHROOM3 knockdown altered podocyte morphology, cytoskeleton, adhesion, and migration. CONCLUSIONS: We demonstrate a novel mechanism that may explain SHROOM3's dichotomous associations in glomerular versus nonglomerular compartments in CKD.

Cytomorphometric Neutrophil and Monocyte Markers May Strengthen the Diagnosis of Sepsis.

BACKGROUND:: The diagnosis of sepsis is challenging in the absence of a gold standard test. Recent studies have explored the role of neutrophil and monocyte volume, conductivity, and scatter (VCS), derived from automated hematology analyzers, in diagnosing sepsis. We assessed the diagnostic accuracy of VCS parameters in critically ill patients with sepsis. METHODOLOGY:: In this prospective study, VCS parameters, procalcitonin, and C-reactive protein (CRP) were assessed in patients with proven sepsis (cases) and 2 control groups (intensive care unit [ICU] patients without sepsis and healthy blood donors). The diagnostic property of each test was explored by calculating sensitivity, specificity, negative and positive predictive values, and area under the curve (AUC). RESULTS:: The study included 65 patients with sepsis, 58 nonseptic ICU controls, and 98 blood donors. Procalcitonin and CRP were not significantly different ( P > .06) between patients with sepsis and nonseptic patients. Mean (95% confidence interval [CI]) neutrophil volume (MNV) was significantly higher ( P < .001) in patients with sepsis (165.5; 95%CI 161.6-169.4) than in nonseptic (157.3; 95%CI 154.6-160.1) patients and donors (148.9; 95%CI 147.9-150). A similar pattern was seen with mean monocyte volume (MMoV). Neutrophil and monocyte conductivity and scatter parameters were variably associated. The AUC was highest for MMoV (0.74) and lowest for CRP (0.62). Among all parameters, MNV and MMoV had the highest specificity of 85% and 80%, respectively. CONCLUSION:: In critically ill patients with suspected sepsis, VCS parameters may help strengthen the diagnostic probability of sepsis. Future studies may explore the role of serial monitoring of VCS to track response to antimicrobial therapy.

False negative single antigen bead assay: Is it always an effect of prozone?

BACKGROUND: Bead based flow cytometry and Luminex play a major role in identification of alloantibodies in renal transplant work-up. Strong sensitization events may lead to prozone phenomenon that can affect single antigen bead (SAB) assay and result in false negativity. However, this can also be due to high titer of other blocking antibodies. While methods like, heat inactivation, C1 inhibitor, Ethylene diamine tetra-acetic-acid and Dithio threitol treatment can remove interfering antibodies of complement and IgM, these methods are not optimal if false negativity is due to prozone effect, which is high titer of antibodies alone. METHODS: We hereby present a case of a highly sensitized renal transplant recipient with 64% panel reactive antibody positivity (PRA) and a subsequent negative SAB assay. This paradoxical finding hinted at SAB being a false negative result and serial dilutions were used to perform further tests. RESULTS: Serum dilutions lead to positive flow based panel reactive antibody (PRA) and flow cytometry crossmatch (FCXM), with an increasing trend in FCXM. CONCLUSIONS: In highly sensitized patients serial dilution should be considered during a transplant work-up to avoid missing any underlying antibodies. Serum dilution can be used as first option to circumvent prozone. Also, interference of other antibodies should not be labeled as prozone effect.

Upregulation of Enhancer of Zeste Homolog 2 (EZH2) with Associated pERK Co-Expression and PRC2 Complex Protein SUZ12 Correlation in Adult T-Cell Leukemia/Lymphoma.

EZH2, a subunit of the polycomb repressive complex 2 (PRC2), is an important methyltransferase that catalyzes the trimethylation of histone H3 at lysine 27 (H3K27me3). EZH2 is overexpressed in various malignancies. Here, we investigated EZH2 expression and potential signaling molecules that correlate with EZH2 expression in ATLL and other T-cell neoplasms. Immunohistochemical staining (IHC) was performed for EZH2, pERK, MYC, and pSTAT3 on 43 ATLL cases and 104 cases of other T-cell neoplasms. Further IHC studies were conducted for Ki-67, SUZ12, and H3K27me3 on ATLL cases. All ATLL cases showed EZH2 overexpression. In other T-cell neoplasms, a high prevalence of EZH2 overexpression was identified (86%), except for T-PLL (33%). In ATLL, EZH2 overexpression correlated with pERK co-expression (86%), while only a small subset of cases showed MYC (7%) or pSTAT3 (14%) co-expression. In the other T-cell neoplasms, there was a variable, but higher, co-expression of EZH2 with pERK, MYC, and pSTAT3. In ATLL, enhanced EZH2 expression correlated with higher Ki-67 staining, SUZ12 (another PRC2 subunit), and H3K27me3 co-expression. In conclusion, EZH2 is overexpressed in ATLL and is associated with pERK expression. It correlates with an increased proliferation index, indicating an aggressive clinical course. EZH2 also correlates with SUZ12 and H3K27me3 co-expression, suggesting its PRC2-dependent catalytic activity through trimethylation. Additionally, EZH2 is overexpressed in most T-cell neoplasms, suggesting that EZH2 could function as an oncogenic protein in T-cell tumorigenesis. EZH2 and pERK could serve as potential therapeutic targets for treating aggressive ATLL. EZH2 could also be targeted in other T-cell neoplasms.

MPO Expression of Background Neutrophils in MPO Negative Acute Promyelocytic Leukemia, An Easy Clue to Corroborate a Challenging Diagnosis: A Case Report and Review of Literature.

Acute promyelocytic leukemia (APL) is characterized by the pathogenic driver fusion transcript PML-RARA resulting from the t(15;17) translocation. Early recognition of APL with prompt ATRA induction has a decisive impact on the early death rate. The preliminary diagnosis of APL relies heavily on cytomorphology and flow cytometry. In APL with variant morphology, such as the microgranular variant, immunophenotype, especially the bright MPO positivity is the basis of diagnosis. Till date, only five cases of APL with reduced/absent MPO have been described in literature. The identification of MPO deficiency based on genetic testing would involve at the least a MPO gene scanning with NGS, followed by microarray to identify somatic uniparental disomy in heterozygotes. This testing is not only redundant given the scant clinical implications of heterozygous MPO deficiency but also time consuming. An easy way to identify background MPO deficiency confounding the immunophenotype of a myeloid neoplasm is the MPO expression in background neutrophils gated on the initial flow cytometry. A dim MPO in the background neutrophils, in the morphological setting of APL, can identify underlying MPO deficiency, clarifying the immunophenotypic ambiguity and thus establishing an unequivocal diagnosis as seen in the current case.

Cardiovascular Involvement of a CD138-Negative Anaplastic Myeloma: A Diagnostic Dilemma.

Anaplastic myeloma (AM) is an extremely rare and aggressive histological variant of myeloma. It is characterized by extramedullary presentation in the young and has a poor prognosis. It can be a diagnostic challenge when myeloma is not suspected and even more when the immunophenotype is unexpected. We present a rare presentation of anaplastic myeloma with cardiovascular involvement. Though the patient did not have the typical clinical features of myeloma, except lytic lesion in the femur, the cardiac biopsy showed sheets of anaplastic cells, and some with multinucleation. There were also some areas with a more plasmacytoid appearance. The initial immunohistochemical panel was negative for CD3, CD20, CD138, AE1/3, and kappa. It was positive for lambda. This led to an extended panel which showed positivity for CD79a and MUM1 and negative for LMP-1, HHV-8, CD43, CD117, CD56, and CD30. Even the flow cytometry on the bone marrow showed a small population of atypical cells positive for CD38 and negative for CD138 with lambda restriction. This is an unusual case of anaplastic myeloma with cardiovascular involvement and CD138 negativity. This case highlights the need to add a panel of plasma cell markers when myeloma is suspected, and it is pertinent to read flow cytometry with caution to avoid missing atypical plasma cells which maybe CD38+/CD138-.

CD138- Plasmablastic Lymphoma: A Multi-institutional Study and Review of the Literature.

CONTEXT.­: Plasmablastic lymphoma (PBL) is a rare aggressive lymphoma, usually positive for CD138 and frequently occurring in the oral cavity of human immunodeficiency virus (HIV) patients. Up to 10% of cases are negative for CD138 and diagnostically very challenging. OBJECTIVE.­: To investigate the appropriate approach to diagnose CD138- plasmablastic lymphoma and avoid misdiagnosis. DESIGN.­: We studied 21 cases of CD138- PBL from multiple large institutes in the United States and 21 cases from the literature. RESULTS.­: CD138- PBLs were positive for different B/plasma cell markers at various percentages: MUM1 (94.4%; 34 of 36), OCT2 (70.6%; 12 of 17), immunoglobulin light chains (68.8%; 22 of 32), CD38 (68.4%; 13 of 19), CD79a (34.2%; 13 of 38), and PAX5 (15.6%; 5 of 32), suggesting that MUM1, OCT2, immunoglobulin light chains, and CD38 are useful markers to help establish the lineage. A total of 83% of cases (30 of 36) were extraoral lesions. Extraoral lesions showed much lower Epstein-Barr virus (EBV) infection rates (16 of 30; 53.3%) and had worse prognosis. MYC was positive in 80% (8 of 10) of EBV+ cases and 40% (2 of 5) EBV- cases, indicating the importance of MYC in pathogenesis, especially in EBV+ cases. CONCLUSIONS.­: Our study emphasizes that CD138- PBLs tend to be extraoral lesions, with much lower EBV infection rates, and diagnostically very challenging. Accurate diagnosis requires a thorough investigation and workup by using appropriate markers.

Acute myeloid leukemias with JAK2/STAT mutations are associated with PD-L1 upregulation.

Even though overexpression of the immune checkpoint protein, programmed cell death ligand-1 (PD-L1), is observed in solid tumors, its expression patterns in acute myeloid leukemia remain understudied. As activation of the JAK/STAT pathway has been shown to enhance PD-L1 expression in preclinical models, we evaluated biopsies from AML patients with activating mutations in JAK2/STATs. PD-L1 expression was significantly upregulated in JAK2/STAT mutant cases when compared to JAK2 wildtype controls as demonstrated by PD-L1 immunohistochemistry staining and quantified using the combined positive score (CPS) system. There is significant overexpression of phosphorylated STAT3 expression in patients with oncogenic JAK2 activation and a positive correlation between p-STAT3 and PD-L1 expression. In conclusion, we demonstrate the CPS scoring system could be applied as a quantitative measure of PD-L1 expression in leukemias and that JAK2/STATs mutant AML can be potential candidates for checkpoint inhibitor trials.

Intermediate between Idiopathic Hypereosinophilia and Chronic Eosinophilic Leukemia: A Report of Two Hypereosinophilic Cases with Possible Novel Molecular Mutations.

To distinguish a reactive eosinophilia from its malignant counterpart is challenging. Establishing clonality of the eosinophils is crucial and considered the determining factor for establishing a diagnosis. Cases of hypereosinophilia without clear reactive etiologies, no evidence of end-organ damage, normal cytogenetics, and no molecular mutations are termed as "Idiopathic Hypereosinophilia (IHE)." For cases which lie between the spectrum of chronic eosinophilic leukemia (CEL) and IHE, identification of underlying molecular abnormalities might be helpful in better understanding the disease process and prognosis. Here, we report two cases of hypereosinophilia in which five possible novel molecular mutations were identified by targeted next-generation sequencing (NGS) analysis. They were FBXW7, KM2A, TCF3, ERBB4, and MET. With multiple genetic mutations, these cases could be classified as chronic eosinophilic leukemia. Both these young patients responded well to steroid therapy. While targeted NGS is a useful tool in identifying new molecular mutation associated with hypereosinophilia, our cases raise the question of further investigating this entity and if there is a possibility of an intermediate category lying between the spectrum of CEL and IHE. Defining hypereosinophilia with clonal molecular abnormality as a malignant process may need to be revisited. Even though attempts are being made to identify mutations in IHE, it might be more significant clinically to differentiate them based on response to steroid therapy and prognosis.

Features That Aid Identification of Autoimmune Gastritis in a Background of Active Helicobacter pylori Infection.

CONTEXT.­: Helicobacter pylori-associated and autoimmune gastritis may coexist in a subset of patients who require treatment for both disorders. OBJECTIVE.­: To delineate findings that identify autoimmune gastritis in the background of H pylori infection. DESIGN.­: We examined cases of (1) patients with H pylori-associated gastritis who had successful eradication therapy and subsequent biopsies diagnostic of autoimmune gastritis and (2) H pylori-associated gastritis wherein pathologists noted features of autoimmune gastritis during original interpretation. Control patients underwent H pylori eradication but lacked evidence of autoimmune gastritis or H pylori infection after 10 years of follow-up. RESULTS.­: Eight subjects had H pylori-associated gastritis followed by H pylori-negative sampling that showed autoimmune gastritis. Review of original samples showed full-thickness inflammation of oxyntic mucosa in 8 of 8 and oxyntic gland loss in 7 of 8 cases. Enterochromaffin-like (ECL) cell hyperplasia, pyloric metaplasia, and intestinal metaplasia were present in 4 of 8 (80% of 5 tested cases), 4 of 8, and 3 of 8 cases, respectively. Features of autoimmune gastritis were noted at the time of their original H pylori diagnosis in 11 study subjects. Ten of 11 samples displayed full-thickness inflammation of oxyntic mucosa and/or partial loss of oxyntic glands, 8 of 11 had ECL cell hyperplasia (all tested cases), 6 of 11 showed pyloric metaplasia, and 4 of 11 harbored intestinal metaplasia. Except for full-thickness oxyntic mucosa inflammation, these features were absent in control cases. CONCLUSIONS.­: Full-thickness inflammation combined with oxyntic gland loss and ECL cell hyperplasia may help to identify autoimmune gastritis in patients with concomitant H pylori infection.

A Rare CD4-CD8+ Adult T-Cell Leukemia/Lymphoma with Unique Molecular Mutations: A Case Report with Literature Review.

Adult T-cell leukemia/lymphoma (ATLL) is a mature T-cell neoplasm caused by infection of the human T-cell lymphotropic virus type 1 (HTLV-1). Most ATLL cases are CD4-positive and CD8-negative. Though rare, there are a few dual negative (CD4-CD8-), dual positive (CD4+CD8+), and CD4-CD8+ cases reported in literature. ATLL is associated with HTLV-1 infection, but HTLV-1 alone cannot cause the malignant transformation of infected T cells. Additional genetic and/or epigenetic events are required for the development of the disease. Here, we report an unusual CD4-CD8+ATLL in a 76-year-old male with a unique molecular genetic profile. Molecular studies revealed alterations in 10 genes. Three of them are predicted to be pathogenic by the computational models, including the frameshift change in ZFHX4 and missense mutations in RHOA and POT1. The specific mutations of POT1 (c.281A > G; p.Q94R), RHOA (c.47G > A; p.C16Y), and ZFHX4 (c.2871delC; p.F958Sfs ∗ 31) have never been previously reported in ATLL to the best of our knowledge. The clinical significance of other genetic alterations is unknown. Further research is warranted to correlate this patient's molecular findings with other ATLL cases. Correlation specifically with other cases of CD8+ ATLL could prove to be useful in understanding the pathogenesis of this rare variant of an already rare form of leukemia/lymphoma.

SARS-CoV-2 PCR cycle threshold at hospital admission associated with patient mortality.

BACKGROUND: Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) cycle threshold (Ct) has been suggested as an approximate measure of initial viral burden. The utility of cycle threshold, at admission, as a predictor of disease severity has not been thoroughly investigated. METHODS AND FINDINGS: We conducted a retrospective study of SARS-CoV-2 positive, hospitalized patients from 3/26/2020 to 8/5/2020 who had SARS-CoV-2 Ct data within 48 hours of admission (n = 1044). Only patients with complete survival data, discharged (n = 774) or died in hospital (n = 270), were included in our analysis. Laboratory, demographic, and clinical data were extracted from electronic medical records. Multivariable logistic regression was applied to examine the relationship of patient mortality with Ct values while adjusting for established risk factors. Ct was analyzed as continuous variable and subdivided into quartiles to better illustrate its relationship with outcome. Cumulative incidence curves were created to assess whether there was a survival difference in the setting of the competing risks of death versus patient discharge. Mean Ct at admission was higher for survivors (28.6, SD = 5.8) compared to non-survivors (24.8, SD = 6.0, P<0.001). In-hospital mortality significantly differed (p<0.05) by Ct quartile. After adjusting for age, gender, BMI, hypertension and diabetes, increased cycle threshold was associated with decreased odds of in-hospital mortality (0.91, CI 0.89-0.94, p<0.001). Compared to the 4th Quartile, patients with Ct values in the 1st Quartile (Ct <22.9) and 2nd Quartile (Ct 23.0-27.3) had an adjusted odds ratio of in-hospital mortality of 3.8 and 2.6 respectively (p<0.001). The discriminative ability of Ct to predict inpatient mortality was found to be limited, possessing an area under the curve (AUC) of 0.68 (CI 0.63-0.71). CONCLUSION: SARS-CoV-2 Ct was found to be an independent predictor of patient mortality. However, further study is needed on how to best clinically utilize such information given the result variation due to specimen quality, phase of disease, and the limited discriminative ability of the test.

Changing Trends in Therapeutic Plasmapheresis: An Indian Perspective.

Indications for therapeutic apheresis (TA) are dynamic; they keep changing and expanding because of introduction of newer treatment modalities and accumulating evidence in the form of case-reports, case-series and original articles. We evaluated our therapeutic plasmapheresis (TP) data and compared this data with an earlier published Indian report for indications, frequency, response rate and adverse reactions. We conducted a retrospective analysis of all TP procedures performed from January 2014 to June 2016 in department of Transfusion Medicine at a tertiary care hospital. All TP procedures performed for various clinical disorders including neurological, hematological, renal, hepatic and rheumatologic indications were included in the study. Analysis was performed with respect to demography, procedure details and response. 187 patients (118 Males and 69 females) underwent 683 TP procedures during study period. According to 2013 ASFA guidelines, 99, 59 and 29 patients belonged to category I, II and III respectively. In comparison with the earlier report, the number of patients and procedures have increased, indications have changed, response rate is comparable, and the frequency of adverse reactions have decreased. In the last decade there has been increase in number and spectrum of indications for therapeutic apheresis and frequency of procedures. The response rate and safety of these procedures have also improved.

A school-based intervention of screening a movie to increase hepatitis B vaccination levels among students in Uttar Pradesh, India: impact on knowledge, awareness, attitudes and vaccination levels.

BACKGROUND: India is home to one in 14 of all chronic hepatitis B virus (HBV) cases, meaning that it is important to develop HBV interventions that are applicable in the Indian context. Vaccination is the foremost tool for interrupting the HBV infection cycle. HBV vaccination was not included in India's government-sponsored expanded immunisation program until 2011, and many children born earlier remain unvaccinated. This study sought to observe the impact of the HOPE Initiative's school-based intervention to increase vaccination coverage by increasing HBV awareness among students in Lucknow, Uttar Pradesh. METHODS: At 430 schools in the administrative areas within and surrounding Lucknow, students viewed an educational documentary film on HBV and completed two questionnaires, one immediately before the screening and the other six weeks later. Both questionnaires asked the same 14 questions, which were organized into five domains: knowledge of the magnitude of the problem of HBV; knowledge of modes of HBV transmission; knowledge of consequences of HBV infection; awareness of HBV; and attitudes regarding HBV. The baseline questionnaire also asked students whether they had been vaccinated against HBV. At two-year follow-up, researchers measured vaccination levels at a subset of 30 intervention schools and six non-intervention schools to further assess the impact of the intervention. RESULTS: Baseline questionnaires were completed by 11,250 students, and post-intervention questionnaires, by 9698 students. Scores for knowledge about the magnitude of the HBV problem improved from 41% at baseline to 74% at follow-up, and scores for knowledge about modes of transmission, from 38% to 75% (p < 0.05 for both). The baseline HBV vaccination level among students receiving the intervention was 21%. Two years after the intervention, 45% of students (N = 4284) reported being vaccinated at intervention schools compared to 22% (N = 1264) at non-intervention schools. CONCLUSIONS: The observed increases in HBV awareness, knowledge and vaccination levels in this study indicate that school-based interventions can be used to achieve higher vaccination coverage among Indian children. The documentary film was found to be an affordable tool for reaching large audiences. More studies are needed to validate the impact of this intervention and to explore its applicability to other social causes.