Parvez M. Lokhandwala

Mitotic count by phosphohistone H3 immunohistochemical staining predicts survival and improves interobserver reproducibility in well-differentiated neuroendocrine tumors of the pancreas.

Well-differentiated neuroendocrine tumors (WDNETs) of the pancreas are graded on the basis of mitotic count or Ki67 index. Mitotic count has a narrow cutoff; its assessment is time consuming and carries poor interobserver reproducibility. Phosphohistone H3 (PHH3) is a mitosis-specific marker whose value has been validated in several tumor types. We sought to assess the utility of PHH3 in histologic grading of pancreatic WDNETs. Sixty-three cases of surgically resected primary pancreatic WDNETs were retrieved, and immunohistochemical analysis for PHH3 and Ki67 was performed. Mitotic rate was independently assessed by 4 pathologists on hematoxylin and eosin (HE; in 50 high-power fields [HPFs], expressed as mitoses/10 HPF) and PHH3 stains (in 50 HPFs, one 10×, and one 20× hotspot). PHH3 and Ki67 labeling indices were determined on a single 20× hotspot and expressed as the percentage of positive cells to total cells. We found that mitotic counts by various methods significantly correlated with each other and also with PHH3 and Ki67 indices, with the best correlation seen within the 3 different PHH3 counts (in 50 HPFs, one 10× and one 20× hotspot). Moreover, mitotic count on PHH3 was less time consuming than that on HE (1.68 vs. 3.67 min for 50 HPFs, P<0.0001). Histologic grade determined by PHH3 significantly correlated with disease-specific and disease-free survivals, with the best cutoffs of ≥4 mitoses/10 HPF (2 mm2), ≥7 mitoses/10× hotspot, ≥5 mitoses/20× hotspot (log rank test, P<0.0001), and ≥0.16% for PHH3 labeling index (log rank test, P<0.0006). Tumor grades based on PHH3 stain also showed significant correlation with patient survivals in multivariate Cox proportional hazards models (P<0.05). Histologic grades by mitotic counts on PHH3 demonstrated high concordance and &kgr; agreement with grades determined by mitotic count on HE. PHH3 stain also showed improved interobserver agreement in both original mitotic count (intraclass correlation 0.98 vs. 0.79) and final grade assignment (Fleiss &kgr; 0.69 vs. 0.46) as compared with HE. Thus, our data confirmed that histologic grading by PHH3 stain has practical and prognostic values and offers reduced time and improved interobserver reproducibility in mitotic rate assessment and grade assignment. Although larger series are needed for validation, mitotic rate can potentially be determined by counting 1 hotspot, which will greatly facilitate the assessment of histologic grade in pancreatic WDNETs.

Cooperative role of the MHR and the CA dimerization helix in the maturation of the functional retrovirus capsid.

The second helix in the C-terminal domain of retroviral capsid (CA) protein functions as the site of dimerization between subunits in capsid assembly and is believed to participate in a unique interface between Gag molecules in immature particles. This study reports isolation of two substitutions in the dimerization helix of Rous sarcoma virus CA protein that have the ability to suppress lethal defects in core maturation imposed by alterations to the major homology region (MHR) motif just upstream. Together with two previously published suppressors, these define an extended region of the dimerization helix that is unlikely to contribute directly to CA-CA contacts but whose assembly-competence may be strongly affected by conformation. The broad-spectrum suppression and temperature-sensitivity exhibited by some mutants argues that they act through modulation of protein conformation. These findings provide important biological evidence in support of a significant conformational change involving the dimerization helix and the MHR during maturation.

Suppression of a Morphogenic Mutant in Rous Sarcoma Virus Capsid Protein by a Second-Site Mutation: a Cryoelectron Tomography Study

ABSTRACT Retrovirus assembly is driven by polymerization of the Gag polyprotein as nascent virions bud from host cells. Gag is then processed proteolytically, releasing the capsid protein (CA) to assemble de novo inside maturing virions. CA has N-terminal and C-terminal domains (NTDs and CTDs, respectively) whose folds are conserved, although their sequences are divergent except in the 20-residue major homology region (MHR) in the CTD. The MHR is thought to play an important role in assembly, and some mutations affecting it, including the F167Y substitution, are lethal. A temperature-sensitive second-site suppressor mutation in the NTD, A38V, restores infectivity. We have used cryoelectron tomography to investigate the morphotypes of this double mutant. Virions produced at the nonpermissive temperature do not assemble capsids, although Gag is processed normally; moreover, they are more variable in size than the wild type and have fewer glycoprotein spikes. At the permissive temperature, virions are similar in size and spike content as in the wild type and capsid assembly is restored, albeit with altered polymorphisms. The mutation F167Y-A38V (referred to as FY/AV in this paper) produces fewer tubular capsids than wild type and more irregular polyhedra, which tend to be larger than in the wild type, containing ∼30% more CA subunits. It follows that FY/AV CA assembles more efficiently in situ than in the wild type and has a lower critical concentration, reflecting altered nucleation properties. However, its infectivity is lower than that of the wild type, due to a 4-fold-lower budding efficiency. We conclude that the wild-type CA protein sequence represents an evolutionary compromise between competing requirements for optimization of Gag assembly (of the immature virion) and CA assembly (in the maturing virion).

Clinical mutational profiling of bone metastases of lung and colon carcinoma and malignant melanoma using next-generation sequencing

Bone is a common metastatic site for solid tumors and is often the only source for molecular testing. Current routine decalcification protocols for the processing of bone specimens damage nucleic acids, leading to a high failure rate.

Lethal mutations in the major homology region and their suppressors act by modulating the dimerization of the Rous sarcoma virus capsid protein C-terminal domain

An infective retrovirus requires a mature capsid shell around the viral replication complex. This shell is formed by about 1500 capsid protein monomers, organized into hexamer and pentamer rings that are linked to each other by the dimerization of the C‐terminal domain (CTD). The major homology region (MHR), the most highly conserved protein sequence across retroviral genomes, is part of the CTD. Several mutations in the MHR appear to block infectivity by preventing capsid formation. Suppressor mutations have been identified that are distant in sequence and structure from the MHR and restore capsid formation. The effects of two lethal and two suppressor mutations on the stability and function of the CTD were examined. No correlation with infectivity was found for the stability of the lethal mutations (D155Y‐CTD, F167Y‐CTD) and suppressor mutations (R185W‐CTD, I190V‐CTD). The stabilities of three double mutant proteins (D155Y/R185W‐CTD, F167Y/R185W‐CTD, and F167Y/I190V‐CTD) were additive. However, the dimerization affinity of the mutant proteins correlated strongly with biological function. The CTD proteins with lethal mutations did not dimerize, while those with suppressor mutations had greater dimerization affinity than WT‐CTD. The suppressor mutations were able to partially correct the dimerization defect caused by the lethal MHR mutations in double mutant proteins. Despite their dramatic effects on dimerization, none of these residues participate directly in the proposed dimerization interface in a mature capsid. These findings suggest that the conserved sequence of the MHR has critical roles in the conformation(s) of the CTD that are required for dimerization and correct capsid maturation. Proteins 2013.

Therapeutic plasma exchange for hyperviscosity syndrome secondary to high rheumatoid factor

Hyperviscosity syndrome (HVS) is most commonly associated with Waldenstroms macroglobulinemia, where it may be life-threatening. HVS may also occur in autoimmune diseases; data pertaining to efficacy of therapeutic plasma exchange (TPE) in HVS arising in non-malignant gammopathy are limited. We report a case of 71-year-old female with erosive rheumatoid arthritis with profoundly elevated rheumatoid factor (57,400 IU/ml; normal <35) who presented with findings consistent with HVS: profound weakness, headache, epistaxis and plasma viscosity (8.5 centipoise). She was successfully treated with pulsed high-dose steroids and TPE. Her symptoms of HVS have not recurred and the plasma viscosity has remained less than 3 centipoise. Given a slow onset of non-specific symptoms, HVS may be missed, incurring high risk of adverse effect. In symptomatic patients with high RF activity, a high index of suspicion for HVS is necessary to ensure timely identification and treatment with TPE, a safe and effective therapy.

Hemostatic profile and safety of pooled cryoprecipitate up to 120 hours after thawing: THAWED POOLED CRYOPRECIPITATE SHELF LIFE

AABB standards state that cryoprecipitate should be transfused within 4 to 6 hours after thawing. We evaluated coagulation factor levels and sterility of thawed pooled cryoprecipitate to assess whether shelf life can be safely extended.

Assessment of cytotechnologist–cytopathologist interpretative agreement using the Bethesda system for reporting thyroid cytopathology

The Bethesda system for reporting thyroid cytopathology was proposed to provide a clinically relevant framework for interpretations to improve interobserver agreement. Limited data is available regarding the level of interobserver agreement between groups of cytotechnologists (CTs) and cytopathologists (CPs) examining the same thyroid fine needle aspirate (FNA) samples.

Benign Phyllodes Tumor of the Vulva: A Case Report and Literature Review.

Phyllodes tumor is an uncommon breast lesion with characteristic histologic appearance when examined by hematoxylin and eosin staining: leaf-like fronds projecting into cystic spaces on low-power microscopy, and biphasic (epithelial and stromal) components on high-power microscopy. We report a rare primary case of this tumor arising within the vulva. A 34-year old African American female presented with a 3 cm slow-growing vulvar mass initially thought to be an inclusion cyst. The lesion was excised and histologic examination demonstrated this lesion to be a rare case of benign phyllodes tumor with morphologic features similar to those arising from breast tissue. Patient received no further treatment and did not exhibit any recurrence or metastasis. Nearly two years after excision, the patient died due to an unrelated medical cause. This rare tumor should be considered in the differential diagnosis for women presenting with a slow-growing vulvar mass.

Donor-specific antibody to trans-encoded donor HLA-DQ heterodimer.

The majority of de novo donor specific HLA antibodies (DSAs) in transplant patients are directed to HLA-DQ antigens, which consist of a heterodimer of alpha and beta chains. Although a heterodimer can theoretically be cis- or trans-encoded, the sensitizing forms generally appear to be forms. DSA to DQ trans-heterodimer has never been reported. We reviewed 360 post-kidney transplant recipients (transplant: 2002-2013; follow-up: 5.6±3.3years). DQ DSA was detected in 46 of 57 patients who developed DSA. DSA specificity was consistent with donor mismatched DQ trans-heterodimers in three patients: DQ2.5 (DQB1*02, DQA1*05), DQ2.3 (DQB1*02, DQA1*03), and DQ4.3 (DQB1*04, DQA1*03). Two of them eventually lost grafts (2 and 5years later) with allograft nephropathy. In conclusion, post-transplant patients may develop DSA to donor DQ trans-heterodimers. Further studies are warranted to determine the clinical significance of such DSAs.