Alexa J. Siddon

Human herpesvirus 6 positive Reed-Sternberg cells in nodular sclerosis Hodgkin lymphoma.

Classical Hodgkin lymphoma (HL) exhibits a bi‐modal age distribution that suggests an infectious aetiology. However, most cases of nodular sclerosis HL (NSHL) are Epstein–Barr virus (EBV) negative (60–90%). Previous studies regarding human herpesvirus 6 (HHV‐6) positivity of HL have led to conflicting results. In order to clarify this situation, we examined NSHL biopsies for the presence and distribution of HHV‐6 by immunohistochemistry (IHC), polymerase chain reaction (PCR), and fluorescence in situ hybridization (FISH). PCR identified HHV‐6 DNA in 86% of NSHL cases. As HHV‐6 DNA was also identified in most cases of reactive lymphoid hyperplasia, we sought to localize the virus to specific cells by IHC, which detected HHV‐6 in Reed–Sternberg (RS) cells of nearly half (48%) of NSHL cases. Dual CD30/HHV‐6 immunostaining confirmed HHV‐6 immunoreactivity in CD30+ RS cells, and HHV‐6 PCR positivity was confirmed in laser capture microdissection‐isolated CD30+ RS cells. FISH demonstrated multiple copies of HHV‐6 genome in scattered cells. In contrast, EBV+ RS cells were identified in only 24% of the cases. HHV‐6+ cases trended toward a younger age than EBV+ cases. These results conclusively demonstrate that RS cells in many cases of NSHL are HHV‐6 positive, and suggest that HHV‐6 may play a role in NSHL pathogenesis, particularly in younger patients with EBV‐negative disease.

Antigen modulation as a potential mechanism of anti-KEL immunoprophylaxis in mice.

Red blood cell (RBC) alloimmunization is a serious complication of transfusion or pregnancy. Despite the widespread use of Rh immune globulin to prevent pregnancy associated anti-D alloimmunization, its mechanism of action remains elusive. We have previously described a murine model in which immunoprophylaxis with polyclonal anti-KEL sera prevents alloimmunization in wild-type recipients transfused with transgenic murine RBCs expressing the human KEL glycoprotein. To investigate the mechanism of action, we have now evaluated the outcome of immunoprophylaxis treatment in mice lacking Fcγ receptors (FcγRs), complement (C3), both, or none. Whereas polyclonal anti-KEL sera completely prevented alloimmunization in wild-type and single-knockout (KO) mice lacking FcγRs or C3, double-KO mice lacking both FcγRs and C3 became alloimmunized despite immunoprophylaxis. Rapid clearance of essentially all transfused RBCs with detectable KEL glycoprotein antigen occurred within 24 hours in wild-type and single-KO recipients treated with immunoprophylaxis, with the transfused RBCs remaining in circulation having minimal KEL glycoprotein antigen detectable by flow cytometry or western blot. In contrast, transfused RBCs with the KEL glycoprotein antigen fully intact continued to circulate for days in double-KO mice despite treatment with immunoprophylaxis. Further, in vitro phagocytosis assays showed no consumption of opsonized murine RBCs by double-KO splenocytes. Taken in combination, our data suggest that modulation of the KEL antigen (and potentially RBC clearance) by redundant recipient pathways involving both FcγRs and C3 may be critical to the mechanism of action of polyclonal anti-KEL immunoprophylaxis. These findings could have implications for the development of immunoprophylaxis programs in humans.

Use of application containers and workflows for genomic data analysis

Background: The rapid acquisition of biological data and development of computationally intensive analyses has led to a need for novel approaches to software deployment. In particular, the complexity of common analytic tools for genomics makes them difficult to deploy and decreases the reproducibility of computational experiments. Methods: Recent technologies that allow for application virtualization, such as Docker, allow developers and bioinformaticians to isolate these applications and deploy secure, scalable platforms that have the potential to dramatically increase the efficiency of big data processing. Results: While limitations exist, this study demonstrates a successful implementation of a pipeline with several discrete software applications for the analysis of next-generation sequencing (NGS) data. Conclusions: With this approach, we significantly reduced the amount of time needed to perform clonal analysis from NGS data in acute myeloid leukemia.

Pathology Consultation on Evaluating Prognosis in Incidental Monoclonal Lymphocytosis and Chronic Lymphocytic Leukemia

Chronic lymphocytic leukemia (CLL) is a monoclonal B-cell lymphoproliferative disorder generally characterized by an indolent clinical course. However, some patients with CLL will have more aggressive disease progression, and identifying that subgroup may be important for early, or perhaps more aggressive, intervention. In addition, monoclonal B-cell lymphocytosis is often found on routine laboratory evaluation, and it is important to distinguish this entity from overt CLL. Moreover, since many patients with CLL are discovered incidentally and before significant disease progression, prognostic laboratory evaluation may become increasingly efficacious as therapeutic options replace the older strategy of expectant observation. Prognostication may be especially critical if it correctly identifies patients with early stage CLL who are at high risk of clonal evolution and/ or resistance to chemoimmunotherapy. Laboratory studies include surface CD38 and intracellular ZAP-70 expression by flow cytometry, serum β2-microglobulin, and immunoglobulin heavy-chain variable gene mutational status. Cytogenetics for targeted chromosome alterations may similarly aid in predicting outcome and guiding early intervention. This article concisely reviews the utility of commonly performed prognostic markers and addresses the laboratory evaluation in patients with incidentally discovered early stage CLL.

Glial heterotopia of the uterine cervix: DNA genotyping confirmation of its fetal origin.

Uterine glial heterotopia is a rare, yet biologically intriguing lesion, mostly involving the cervix. Although an implantation of the fetal brain tissue is widely accepted as the etiology, there has been no confirming evidence to support this hypothesis. We investigated a case of polypoid glial heterotopia of the uterine cervix in a 42-year-old woman who underwent an elective termination of pregnancy of a Down syndrome fetus. One year earlier, the patient had a pregnancy termination of a fetus with Klinefelters syndrome. Gross and microscopic examination revealed a 2.5 cm polypoid cervical lesion consisting of lobulated mature glial tissue covered by endocervical glandular epithelium. The neural nature of the lesion was confirmed by glial fibrillary acidic protein and S100 immunohistochemistry. DNA genotyping of the cervical polyp, maternal, first and second fetal tissue samples showed an identical genetic profile between the cervical glial tissue and the first aborted fetus. Genotyping also attested the presence of Klinefelters syndrome in the first gestation and Down syndrome in the second gestation. Therefore, this molecular case investigation confirms the fetal origin of uterine glial heterotopia.

Normalized CCND1 expression has prognostic value in mantle cell lymphoma

Mantle cell lymphoma (MCL) is an aggressive B-cell nonHodgkin lymphoma (NHL; Swerdlow et al, 2008). The molecular hallmark for diagnosis of MCL is the t(11;14)(q13; q32) translocation, resulting in constitutive over-expression of CCND1 (Cyclin D1; Campo et al, 1999; Swerdlow et al, 2008). The current median survival in patients with MCL is only 4–5 years (Herrmann et al, 2009), one of the poorest overall survival rates of all NHLs. Yet, some MCL patients have a more indolent course (Martin et al, 2009; Weigert et al, 2009). While several predictors of outcome for MCL have been proposed, their reliability and reproducibility are not well established (Rosenwald et al, 2003; Martin et al, 2009; Weigert et al, 2009). The chronic lymphocytic leukaemia overlap marker CD23 may be associated with a better prognosis in MCL, but this has not been confirmed (Kelemen et al, 2008). High Ki-67 expression and variant blastoid morphology are useful tissue correlates of poor prognosis, but these measures cannot be applied to patients diagnosed in the leukaemic state (Determann et al, 2008). Thus, at this time, there are no laboratory assays that stratify MCL patients into conservative versus aggressive treatment regimens. The primary aim of this study was to evaluate the prognostic utility of normalized quantitative CCND1 mRNA values in the blood or marrow of patients with newly diagnosed MCL. This novel quantitative CCND1 assay has been validated for establishing a diagnosis of MCL in blood/bone marrow (Hui et al, 2003; Howe et al, 2004), but its use as a prognostic marker has not been evaluated. We further compared the normalized CCND1 assay with CD23 expression and circulating levels of malignant B-cells. All cases tested between October 2002 and May 2010 that demonstrated an elevated normalized CCND1 mRNA value were eligible for inclusion (n = 74). Any cases in which the tissue source was not blood or marrow (n = 6) was subsequently excluded, and inclusion in the final study was restricted to only those patients with a new diagnosis of MCL and no prior treatment (n = 40). The institutional review board approved the review and use of patient information in this study. The normalized CCND1 mRNA assay is a relative fold increase (RFI) determined against the background of B-cells (Howe et al, 2004). The RFI method has been shown to strongly correlate with results obtained using fluorescent insitu hybridization (FISH) with CCND1 probes (Hui et al, 2003). The quantitative assay is briefly described here. RNA was extracted using RNeasy reagent (Qiagen, Valencia, CA, USA) according to the manufacturer’s instructions. All primers and probes were synthesized as previously described (Howe et al, 2004) and reaction mixtures contained primers for CCND1 and CD19. Reverse transcription and polymerase chain reaction were performed using an ABI PRISM 7700 Sequence Detector (Applied Biosystems, Carlsbad, CA, USA). Critical threshold (Ct) cycle numbers were obtained for amplification of CCND1 and CD19. The DCt values were then obtained by subtracting the Ct value of CD19 from the Ct value of CCND1. The characteristics of our 40 patients with new a diagnosis of MCL in blood (n = 25) or marrow (n = 15) are presented in Table I. All 40 patients met the molecular criteria for diagnosis of MCL as previously described (Howe et al, 2004).

R634W KIT Mutation in an Adult With Systemic Mastocytosis

Mastocytosis is a clonal neoplasm with the potential to affect various organs within the body. It can range in clinical severity from benign to extremely aggressive. Mastocytosis can be separated into cutaneous, systemic, and leukemic forms, as well as mast-cell sarcoma and extracutaneous mastocytoma. It is most often an acquired condition but can be inherited; the most commonly identified genetic aberrations leading to mastocytosis are activating mutations involving codon 816 of the KIT gene. Herein, we present the case of a 30-year-old Caucasian man with systemic mastocytosis discovered to have a p.Arg634Trp mutation involving KIT. To our knowledge, this mutation has previously only been identified in children with familial urticarial pigmentosa. Ours is the the first case report in the literature of an adult with systemic mastocytosis likely due to a p.Arg634Trp KIT mutation.

Successful Use of Four Factor-Prothrombin Complex Concentrate for Congenital Factor X Deficiency in the Setting of Neurosurgery.

Congenital factor X deficiency is an extremely rare coagulation disorder that can place patients at risk for spontaneous hemorrhage or excessive bleeding in the setting of trauma or invasive procedures. Given the rarity of this disorder, there is little published guidance on how best to prevent or treat bleeding. Herein, we report a case of a 56-year-old white man with congenital factor X deficiency who was scheduled for major neurosurgery and who was treated perioperatively with 4-factor prothrombin complex concentrate (4F-PCC). Doses of 4F-PCC at 15 U per kg, administered immediately preoperatively and once at 24 hours postoperatively, allowed for successful completion of an anterior cervical discectomy and fusion without excessive bleeding. Moreover, no thromboembolic complications were observed. As such, given the wide availability of 4F-PCC, it may be considered as a first-line therapy and an alternative to fresh frozen plasma for factor X deficiencies, particularly in high-risk operative cases.

Catastrophic basilar artery leukostasis in the setting of acute myeloid leukemia

Catastrophic basilar artery leukostasis in the setting of acute myeloid leukemia Rita Abi Raad, Christopher A. Tormey, and Alexa J. Siddon 1 A 43-year-old female with a history of acute myelomonocytic leukemia refractory to treatment was admitted with a new onset of confusion and lethargy. Despite her demonstrated refractoriness a salvage chemotherapy regimen was initiated due to a white blood cell (WBC) count that was noted to be increasing from 50 × 10 to 132 × 10/L with 33% blasts. Her rapidly increasing WBC count, confusion, and lethargy raised concerns for leukostasis. An MRI with angiography of the brain demonstrated absence of flow in the basilar artery and right posterior cerebral artery with multiple areas of associated infarction including the pons, midbrain, thalamus, and cerebellum. Leukapheresis was requested but the patient’s clinical course took a precipitous decline and comfort measures were initiated before the procedure could be performed. The patient expired 4 days later. At autopsy, examination of the brain confirmed basilar artery leukostasis. Histologically, the immature cells were CD34+myeloblasts. Figure A shows a gross image of brain at autopsy. Black arrow indicates basilar artery. Red arrows indicate vertebral arteries. Area of white box is represented by Figure B, which shows a low-power view of the basilar artery with leukostasis. Letter A indicates necrosis with low flow. Area of black box is shown at higher power in Figure C, which shows a high-power image demonstrating clustered myeloblasts in vascular space. Letter A shows the lumen containing numerous blasts. Letter B indicates the artery wall. Leukostasis is a known complication of acute leukemia with a threefold higher early mortality rate in patients with acute myeloid leukemia and elevated WBC counts, which needs to be recognized and treated promptly, often with leukapheresis. Risk for leukostasis-associated complications increases significantly with a WBC count greater than 100 × 10/L in the setting of myeloid leukemia and may be even higher in patients who have monocytic subtypes of acute myeloid leukemia. In addition to chemotherapy and hydration, leukapheresis is recommended to reduce the total circulating blast percentage and is a Category I indication by the American Society for Apheresis. Of note, and of relevance to transfusion medicine, red blood cell transfusions are generally contraindicated in the setting of hyperleukocytosis with leukostasis due to the potential for further increasing blood viscosity. In conclusion, leukapheresis may play an important role in acutely reducing morbidity and mortality in patients with hyperleukocytosis.

Cardiac FDG-PET to assess sarcoidosis in a cardiac allograft.

A 58-year-old man with end-stage heart failure caused by cardiac sarcoidosis underwent an orthotopic heart transplant. His immediate posttransplant course was uncomplicated, but several months later, an endomyocardial biopsy revealed likely recurrent sarcoidosis. A cardiac F-FDG PET study subsequently showed focal anteroseptal uptake that suggested the presence of inflammation. Despite steroid therapy, a study repeated 3 months later showed persistent anteroseptal FDG uptake and new hypokinesis in that region. After escalating his steroid therapy, a final FDG-PET study ultimately demonstrated resolution of the uptake and normalization of anteroseptal wall motion.