Ilyssa O. Gordon

Immune-mediated red cell aplasia after anti-CTLA-4 immunotherapy for metastatic melanoma

A 55-year-old man presented in 2002 with a right lower extremity nodular melanoma, Breslow thickness 1.33 mm, without associated nevus or lymphocytic inWltrate. A sentinel lymph node biopsy was negative, and he received a wide excision and no further therapy. One year later, he presented with a subcutaneous in-transit metastasis which was resected. He began systemic adjuvant therapy on a melanoma vaccine trial containing twelve melanoma peptides in 2004, but 1 year later was found to have progressive disease with liver and subcutaneous metastases. In 2005, he participated in a clinical trial of the Wbroblast activation protein (FAP) inhibitor PT100, but developed progressive disease after 6 months, with new subcutaneous and right inguinal lymph node lesions. He continued to have excellent performance status and in late 2005 participated in a phase III trial of carboplatin and paclitaxel along with Sorafenib or placebo, but developed progressive disease after 7 months. In 2006, he began on Ipilimumab (10 mg/kg once every 3 weeks), an anti-CTLA-4 monoclonal antibody, in a phase II clinical trial approved by the Institutional Review Board at the University of Chicago. He did not have a prior personal or family history of autoimmune disease. He developed vitiligo after 3 months of therapy and had radiographic evidence of response, with decreased size of the right inguinal lymph nodes and stable liver metastases. The following month, he developed hypothyroidism, which was successfully treated with levothyroxine. He had intermittent low grade diarrhea managed with kaopectate, and fatigue, but continued to have an active lifestyle with stable disease. In 2007, after receiving four cycles of induction followed by three cycles of maintenance therapy over a total of 47 weeks, he had radiographic evidence of progressive disease of a subcutaneous mass and underwent re-induction with 10 mg/kg intravenous Ipilimumab, given once every 3 weeks. He continued to have vitiligo, hypothyroidism, and intermittent fatigue. After two cycles of re-induction, he became severely fatigued, pale and tachycardic. His hemoglobin was 7.4 g/dl and hematocrit was 20.4%, compared to a baseline of 14.4 g/dl and 42%, respectively. Ipilimumab dosing was held and he was transfused with packed red blood cells. However, he continued to have severe fatigue and persistent anemia. There was no melena or sign of active bleeding, and a stool Guaiac test was negative. Laboratory evaluation revealed hemoglobin 5.4 g/dl, hematocrit 14.9%, reticulocytes 0.1%, absolute reticulocyte count 1.72 K/ l, reticulocyte production index 0, elevated serum ferritin (839 ng/mL) and serum iron (206 mcg/dl), normal vitamin B12 and folate, and elevated serum erythropoietin (1,284 mIU/ml). Platelets were 173,000 l¡1 and white blood cells were mildly decreased at 2,900 l¡1. Although a direct antiglobulin test was positive, the levels of lactate dehydrogenase, bilirubin, and haptoglobin were normal. He was admitted to the hospital for evaluation and treatment of an apparent underproduction anemia. He was transfused with packed red blood cells and underwent a bone marrow biopsy and aspirate with review of peripheral blood smears. The diVerential diagnosis at this time included red cell aplasia, aplastic anemia, parvovirus, and I. O. Gordon · J. Dickstein (&) · T. F. Gajewski Department of Pathology, University of Chicago, 5841 S. Maryland Ave., TW055, Chicago, IL 60637, USA e-mail: jerome.dickstein@uchospitals.edu

Prostate-specific membrane antigen expression in regeneration and repair

Prostate-specific membrane antigen is a type II transmembrane glycoprotein, expressed in benign and neoplastic prostatic tissue as well as endothelial cells of neovasculature from a variety of tumors. The expression of prostate-specific membrane antigen in nonneoplastic neovasculature has not been well studied. Therefore, we studied nonneoplastic reparative and regenerative human tissues, as well as preneoplastic tissue, to determine the presence of prostate-specific membrane antigen-expressing neovasculature. Formalin-fixed paraffin-embedded tissue from keloids, granulation tissue from heart valves and pleura, proliferative and secretory endometrium, and Barretts mucosa with and without dysplasia were stained for the expression of prostate-specific membrane antigen (3E6). Vessels of proliferative, mid-secretory, and late secretory endometrium were consistently strongly positive for prostate-specific membrane antigen expression in all ten cases of each type (100%). Vessels associated with granulation tissue from pleural peels and heart valves were positive in 10 of 12 cases (83%) and 7 of 10 cases (70%), respectively. Keloids had prostate-specific membrane antigen-expressing endothelial cells in 6 of 15 cases (40%). Prostate-specific membrane antigen was not expressed by vessels associated with Barretts mucosa with low-grade dysplasia (12 foci), high-grade dysplasia (24 foci), or no dysplasia (18 foci). A variety of nonneoplastic neovasculature expresses prostate-specific membrane antigen, including vessels in proliferative endometrium, granulation tissue, and some scars. This is the first study showing that prostate-specific membrane antigen is expressed in neovasculature from physiologic regenerative and reparative conditions. The folate hydrolase activity of prostate-specific membrane antigen may facilitate vasculogenesis and angiogenesis by increasing local availability of folic acid. These findings will enhance our overall understanding of blood vessel development and will enable us to better understand the effects of anti-prostate-specific membrane antigen therapies, which are already being explored in clinical trials.

Effects of Oral Contraceptives or a Gonadotropin-Releasing Hormone Agonist on Ovarian Carcinogenesis in Genetically Engineered Mice

Although epidemiologic evidence for the ability of combined oral contraception (OC) to reduce the risk of ovarian cancer (OvCa) is convincing, the biological mechanisms underlying this effect are largely unknown. We conducted the present study to determine if OC also influences ovarian carcinogenesis in a genetic mouse model and, if so, to investigate the mechanism underlying the protective effect. LSL-K-rasG12D/+PtenloxP/loxP mice were treated with ethinyl estradiol plus norethindrone, contraceptive hormones commonly used in combined OC, or norethindrone alone, or a gonadotropin-releasing hormone agonist. The combined OC had a 29% reduction in mean total tumor weight compared with placebo (epithelial tumor weight, −80%). Norethindrone alone reduced mean total tumor weight by 42% (epithelial tumor weight, −46%), and the gonadotropin-releasing hormone agonist increased mean total tumor weight by 71% (epithelial tumor weight, +150%). Large variations in tumor size affected the P values for these changes, which were not statistically significant. Nonetheless, the OC reductions are consistent with the epidemiologic data indicating a protective effect of OC. Matrix metalloproteinase-2 activity was decreased in association with OC, indicating that OC may affect ovarian carcinogenesis by decreasing proteolytic activity, an important early event in the pathogenesis of OvCa. In contrast, OC increased invasion in a K-ras/Pten OvCa cell line established from the mouse tumors, suggesting that OC hormones, particularly estrogen, may have a detrimental effect after the disease process is under way. Our study results support further investigation of OC effects and mechanisms for OvCa prevention.

Endobronchial Biopsy: A Guide for Asthma Therapy Selection in the Era of Bronchial Thermoplasty

Objective. Bronchial thermoplasty (BT) reduces airway smooth muscle in patients with severe asthma. We developed a novel standardized histologic grading system assessing inflammation and structural remodeling on endobronchial biopsy (EBBx) in severe persistent asthma and evaluated airway structure before and after BT. In addition, we correlated invasive and non-invasive inflammatory markers in severe persistent asthma. Methods. Thirty-three patients with severe persistent asthma underwent bronchoscopy, including bronchoalveolar lavage (BAL) and diagnostic EBBx. The control group (N = 41) underwent EBBx for other clinical indications. Biopsies were graded for airway inflammation and epithelial and submucosal structural features. We also evaluated airway histology in three patients before and after BT. Results. Compared to the control group, patients with severe persistent asthma more often had intraepithelial eosinophils and lymphocytes (67% vs. 17% and 61% vs. 27%; p < .001 and p = .005, respectively) and prominent smooth muscle and goblet cell hyperplasia (88% vs. 29% and 47% vs. 22%, p < .001 and p = .004, respectively). Other features including epithelial denudation and basement membrane thickening were not significantly different. Following BT, airway smooth muscle was no longer prominent due to partial replacement by fibrosis. Increased submucosal eosinophilic inflammation and BAL eosinophilia correlated with exhaled nitric oxide (eNO, p = .05 for both). Conclusions. We developed a clinically applicable standardized histologic grading system which identified structural but not inflammatory changes before and after BT in severe persistent asthmatics. Additionally, we demonstrated that eNO is representative of submucosal eosinophilia in this population. This semi-quantitative assessment will be useful for practicing pathologists assessing EBBx from severe persistent asthma patients for diagnostic and clinical research purposes.

SaLUTaRy: Survey of lung transplant rejection

BACKGROUND The International Society for Heart and Lung Transplantation (ISHLT) guidelines on the interpretation of lung rejection in pulmonary allograft biopsy specimens were revised most recently in 2007. The goal of our study was to determine how these revisions, along with nuances in the interpretation and application of the guidelines, affect patient care. METHODS A Web-based survey was e-mailed to pathologists and pulmonologists identified as being part of the lung transplant team at institutions in the United States with active lung transplant programs as determined from the Organ Procurement and Transplantation Network Web site (http://optn.transplant.hrsa.gov/members/directory.asp). RESULTS Grades B1 and B2 in asymptomatic patients would fall into the same treatment group under the 2007 classification, which combines B1 and B2 into B1R. Also, some pulmonologists would not interpret a pathologic diagnosis of lymphocytic bronchiolitis as grade B rejection, resulting in under-treatment of these patients. Regarding bronchiolitis obliterans, most pulmonologists would treat the patient differently if there were an active mononuclear inflammatory infiltrate, and most pathologists would comment on the presence of such an infiltrate, contrary to the 2007 guidelines, which discourage reporting this infiltrate. We also found discrepancies among pathologists in their interpretation of airway lymphocytic infiltrates, whether eosinophils can be present in bronchial-associated lymphoid tissue, and whether airway inflammation represents rejection or bacterial infection. CONCLUSIONS The issue of grading and treating airway inflammation in pulmonary allograft biopsy specimens continues to be problematic, despite revised ISHLT guidelines. Clarification of guidelines for pathologists and pulmonologists using evidence-based criteria could lead to improved communication and patient care.

Evaluation of microvascular density in Barrett’s associated neoplasia

Angiogenesis has an important role in the carcinogenesis of esophageal adenocarcinoma, however, the diagnostic and prognostic utility of microvascular density counts have not been clinically established. The aim of this study is to assess the correlation between microvascular density and disease progression of non-dysplastic Barrett’s esophagus, low-grade dysplasia, high-grade dysplasia and invasive carcinoma in the superficial aspects of the tissue. Archival histological specimens from two referral centers for Barrett’s esophagus and esophageal cancer were selected for review. A total of 160 regions marked according to histological grade were assessed with digitally interactive software to measure microvascular density. This was quantified in three levels: 0–50, 50–100 and 100–150 μm. In the areas of gastric cardia, Barrett’s esophagus, low-grade dysplasia, high-grade dysplasia and cancer, microvascular density was significantly different (P<0.0001) among the five groups in the most superficial 150 μm of the mucosa. Furthermore, when examining the pairwise difference between the groups, there was a significant difference between cancer and each of the lower grades of histology (P<0.05) and between high-grade dysplasia and each of the lower grades of histology (P<0.05). These statistically significant differences were preserved in examining the depth at the most superficial 50 μm. We have used digital pathology to demonstrate a significant and stepwise increase in microvascular density, which supports the hypothesis that angiogenesis has a key role in Barrett’s carcinogenesis. Furthermore, the differences in the most superficial mucosal layers are consistent with findings of increased vascularity by depth-restricted imaging modalities.

Post-ablation lymphocytic esophagitis in Barrett esophagus with high grade dysplasia or intramucosal carcinoma

In patients who have undergone ablation therapy for treatment of Barretts esophagus with dysplasia, histologic features of eosinophilic esophagitis, but not lymphocytic esophagitis, have been described. We evaluated for histologic evidence of eosinophilic esophagitis and lymphocytic esophagitis and correlated with endoscopic findings in this population. A single-institution Barretts esophagus registry was searched for patients who had received radiofrequency ablation, cryotherapy, or both for treatment of Barretts esophagus with dysplasia. Clinical and endoscopic data were collected and biopsies were reviewed for inflammation and reactive changes at three time points: pre-intervention, first surveillance after ablation therapy, and most recent surveillance. Of the 173 patients initially identified, 102 met the inclusion criteria. Intraepithelial eosinophils were increased at first surveillance (60%, P=0.096) and last surveillance (69%, P=0.048) compared with pre-intervention (50%), although histologic evidence of post-ablation eosinophilic esophagitis was not significant. Prevalence of lymphocytic esophagitis was significantly higher at first surveillance (17%, P=0.02) and at last surveillance (43%, P<0.001), compared with pre-intervention (7%). Smoking, hyperlipidemia, and cryotherapy were identified as independent risk factors for developing histologic lymphocytic esophagitis. This is the first report that histologic evidence of lymphocytic esophagitis increased over time in patients undergoing ablation for Barretts esophagus with dysplasia. Though the pathophysiology of lymphocytic esophagitis remains unknown, patients in our study with a history of smoking, hyperlipidemia, or cryotherapy were more likely to develop post-ablation lymphocytic esophagitis.

The effects of 17β-estradiol and a selective estrogen receptor modulator, bazedoxifene, on ovarian carcinogenesis

OBJECTIVE To test if estrogen promotes carcinogenesis in vitro and in a genetic mouse model of ovarian cancer and whether its effects can be inhibited by a novel selective estrogen receptor modulator (SERM), bazedoxifene. METHODS Bazedoxifene was synthesized and it was confirmed that the drug abrogated the uterine stimulatory effect of 17β-estradiol in mice. To determine if hormones alter tumorigenesis in vivo LSL-K-ras(G12D/+)Pten(loxP/loxP) mice were treated with vehicle control, 17β-estradiol or bazedoxifene. Hormone receptor status of a cell line established from LSL-K-ras(G12D/+)Pten(loxP/loxP) mouse ovarian tumors was characterized using Western blotting and immunohistochemistry. The cell line was treated with hormones and invasion assays were performed using Boyden chambers and proliferation was assessed using MTT assays. RESULTS In vitro 17β-estradiol increased both the invasion and proliferation of ovarian cancer cells and bazedoxifene reversed these effects. However, in the genetic mouse model neither treatment with 17β-estradiol nor bazedoxifene changed mean tumor burden when compared to treatment with placebo. The mice in all treatment groups had similar tumor incidence, metastatic nodules and ascites. CONCLUSION While 17β-estradiol increases the invasion and proliferation of ovarian cancer cells, these effects do not translate into increased tumor burden in a genetic mouse model of endometrioid ovarian cancer. Likewise, while the SERM reversed the detrimental effects of estrogen in vitro, there was no change in tumor burden in mice treated with bazedoxifene. These findings demonstrate the complex interplay between hormones and ovarian carcinogenesis.

Effect of blood collection tubes on the incidence of artifactual hyperkalemia on patient samples from an outreach clinic

BACKGROUND An offsite satellite clinic of the University of Chicago Medical Center (UCMC) requested an investigation by the Clinical Chemistry Laboratory (CCL) into several cases of possible falsely elevated potassium (K⁺) values in their patients. Bloods for K⁺ and chemistry profiles are routinely collected in mint-green, heparinized plasma separator tubes (PST), centrifuged, and transported by courier from satellite clinic to CCL within several hours. Samples from on-site phlebotomy areas are similarly collected but sent uncentrifuged to CCL via a pneumatic tube system within minutes of collection. METHODS Our investigations included extensive QC and QA review of UCMC onsite and offsite outpatient clinics, reference range studies using PST and serum separator tubes (SST), assessment of pre-analytic handling of specimens, including transportation simulation study, and comparison of K⁺ results for samples collected simultaneously using PST and SST tubes at an offsite clinic. RESULTS Our transportation simulation demonstrated elevations in K⁺ concentrations following sample jostling and perturbations. We also observed RBC escape across the gel barrier further contributing to K⁺ elevations. CONCLUSION Serum is preferred sample type for an offsite clinic.

Drug-induced Injury of the Gastrointestinal Tract

The effects of drugs on the gastrointestinal tract are diverse and depend on numerous factors. Diagnosis is centered on histologic findings, with mostly nonspecific patterns of injury that must be interpreted in the correct clinical context. Nonsteroidal antiinflammatory drugs are a common cause of drug-induced gastrointestinal injury, with effects primarily in the gastric mucosa but also throughout the gastrointestinal tract. Another common class of drugs causing a variety of pathologic findings in the gut is chemotherapeutic agents. This article discusses the differential diagnosis of the various patterns of injury, including ischemic damage, and the histologic findings specific for certain drugs.