Douglas J. Hartman

PUMA-mediated intestinal epithelial apoptosis contributes to ulcerative colitis in humans and mice

Intestinal epithelial cell (IEC) apoptosis contributes to the development of ulcerative colitis (UC), an inflammatory bowel disease (IBD) that affects the colon and rectum. Therapies that target the inflammatory cytokine TNF have been found to inhibit IEC apoptosis in patients with IBD, although the mechanism of IEC apoptosis remains unclear. We therefore investigated the role of p53-upregulated modulator of apoptosis (PUMA), a p53 target and proapoptotic BH3-only protein, in colitis and IEC apoptosis, using patient samples and mouse models of UC. In UC patient samples, PUMA expression was elevated in colitis tissues relative to that in uninvolved tissues, and the degree of elevation of PUMA expression correlated with the severity of colitis and the degree of apoptosis induction. In mice, PUMA was markedly induced in colonic epithelial cells following induction of colitis by either dextran sulfate sodium salt (DSS) or 2,4,6-trinitrobenzene sulfonic acid (TNBS). The induction of PUMA was p53-independent but required NF-κB. Absence of PUMA, but neither absence of p53 nor that of another BH3-only protein (Bid), relieved DSS- and TNBS-induced colitis and inhibited IEC apoptosis. Furthermore, treating mice with infliximab (Remicade), a clinically used TNF-specific antibody, suppressed DSS- and TNBS-induced PUMA expression and colitis. These results indicate that PUMA induction contributes to the pathogenesis of colitis by promoting IEC apoptosis and suggest that PUMA inhibition may be an effective strategy to promote mucosal healing in patients with UC.

Assessing Treatment Effect in Pancreatic Cancer

CONTEXT Pancreatic cancer is one of the most deadly forms of cancer (43,140 new cases per year; 36,800 deaths), and most people with pancreatic cancer do not survive past 5 years. New therapeutic regimens are constantly being evaluated in an attempt to reduce the rapid progression of this disease. Although some patients receive neoadjuvant therapy in an attempt to make a nonresectable or borderline-resectable tumor resectable, more patients with resectable disease are being enrolled in clinical trials that provide neoadjuvant therapy. This means more pancreatic resections must be evaluated for therapy effect. Histologic grading schemes for the assessment of posttherapy response have been described, but difficulties associated with determining the histologic features of treatment effect in pancreatic cancer have not been addressed. OBJECTIVES To critically review the diagnostic criteria for proposed grading schemes for pancreatic cancer treated with neoadjuvant chemoradiation therapy and to provide guidance to surgical pathologists who encounter treated pancreatic cancer resections. DATA SOURCES Published peer-reviewed literature and the personal experience of the authors. CONCLUSIONS Assessment of treatment effect in pancreatic cancer is difficult. Pathologists need to be aware that some histologic features of treatment effect overlap with histologic features seen in untreated pancreatic cancer, such as tumor cell anaplasia, necrosis, and fibrosis. Careful assessment of pancreatic resections, including detailed gross examination and thorough histologic sampling, is important in accurately assessing treatment effect and improving patient outcomes.

Ipilimumab-induced perforating colitis.

Recently, a monoclonal antibody to cytotoxic T-lymphocyte-associated antigen 4, ipilimumab, was approved for the treatment of metastatic melanoma. One of the most common side effects associated with this therapy is diarrhea and colitis. We report 3 cases of perforating colitis induced by ipilimumab requiring colectomy. The histologic findings of mucosal biopsies have been previously described. Herein, we describe novel associated histologic findings (pseudopolyp formation, fissuring ulcers, dilated crypts, and lack of intraepithelial lymphocytosis and epithelial apoptosis) of segmental resections in patients who required subtotal colectomy after perforation due to the severity of their ipilimumab-induced colitis. Although steroid therapy is the standard treatment for ipilimumab-induced colitis, surgery may be necessary. In the setting of progressive or worsening diarrhea after steroid therapy in patients with colitis, bowel perforation should be considered.

Tissue Yield and Diagnostic Efficacy of Fluoroscopic and Cholangioscopic Techniques to Assess Indeterminate Biliary Strictures

BACKGROUND & AIMS Evaluation of indeterminate biliary strictures typically involves collection and analysis of tissue or cells. Single-operator, peroral, cholangioscopic techniques have been developed that allow for a biopsy sample to be obtained from a specific area of the visualized stricture. We investigated whether standard fluoroscopic-guided and cholangioscopic-directed (SpyGlass Direct Visualization System; Boston Scientific, Natick, MA) biopsy collection provide adequate tissue for histologic assessment. METHODS We examined 110 consecutive bile duct specimens collected from 89 patients with indeterminate biliary strictures at a single institution using fluoroscopy or cholangioscopy (from October 2007 to March 2010). Because of the small nature of the intraductal biopsy fragments, special procedures were followed in the pathology laboratory to maximize the amount of tissue for histopathology analysis. RESULTS Only 4 specimens (3.6%) had insufficient material for a diagnosis. More tissue was obtained from standard fluoroscopic-guided than cholangioscopic-directed biopsies (more biopsy fragments, P = .018; larger total biopsy size, P = .001). Fluoroscopy-guided biopsies assessed indeterminate biliary strictures with 76% sensitivity and 88% accuracy; these values were 57% and 78%, respectively, for cholangioscopic-directed biopsies. Each procedure had 100% specificity. CONCLUSIONS Analysis of bile duct biopsies is important in management of patients with indeterminate biliary strictures. Use of a special handling protocol for these small biopsies could reduce the number of cases with insufficient material for diagnosis. Increasing the sample size (either by using larger biopsy forceps or obtaining more biopsy bites) could improve the sensitivity of the SpyGlass technique. As endoscopists and pathologists gain more experience in collecting and handling small biopsies, the diagnostic efficacy of intraductal biopsies will continue to improve.

Quantification of nanoscale nuclear refractive index changes during the cell cycle

Intrigued by our recent finding that the nuclear refractive index is significantly increased in malignant cells and histologically normal cells in clinical histology specimens derived from cancer patients, we sought to identify potential biological mechanisms underlying the observed phenomena. The cell cycle is an ordered series of events that describes the intervals of cell growth, DNA replication, and mitosis that precede cell division. Since abnormal cell cycles and increased proliferation are characteristic of many human cancer cells, we hypothesized that the observed increase in nuclear refractive index could be related to an abundance or accumulation of cells derived from cancer patients at a specific point or phase(s) of the cell cycle. Here we show that changes in nuclear refractive index of fixed cells are seen as synchronized populations of cells that proceed through the cell cycle, and that increased nuclear refractive index is strongly correlated with increased DNA content. We therefore propose that an abundance of cells undergoing DNA replication and mitosis may explain the increase in nuclear refractive index observed in both malignant and histologically normal cells from cancer patients. Our findings suggest that nuclear refractive index may be a novel physical parameter for early cancer detection and risk stratification.

Are Routine Ancillary Stains Required to Diagnose Helicobacter Infection in Gastric Biopsy Specimens? An Institutional Quality Assurance Review

Gastric biopsies are often done to evaluate for Helicobacter gastritis. Given the oncogenic association with Helicobacter gastritis and the relative ease of therapy, it is important for pathology departments to identify all positive cases. We describe an institutional quality assurance study of an institutional method for the diagnosis of Helicobacter gastritis. We reviewed 356 gastric biopsy specimens from a 4-week period at 1 institution. Approximately half were evaluated by 4 methods, H&E stain, Giemsa stain, Warthin-Starry stain, and Helicobacter immunostain, while the remainder were stained only with H&E and Helicobacter immunostains. There were 30 cases of Helicobacter gastritis diagnosed; about 83% of cases were diagnosed on the initial H&E-stained slides. Our study highlights a quality assurance study and a head-to head comparison of 4 methods not previously reported and supports the use of ancillary stains at the discretion of the sign-out pathologist.

Ablation of sensory neurons in a genetic model of pancreatic ductal adenocarcinoma slows initiation and progression of cancer

Significance In humans and genetically engineered mouse models (GEMs), the development of pancreatic ductal adenocarcinoma (PDAC) is accompanied by intimate neural–tumor interactions. Using a PDAC GEM that phenocopies the human disease, we found that many changes in peripheral and central nervous systems, indicative of injury and inflammation, arise at time points prior to overt tumor formation. Ablation of sensory neurons that innervate the pancreas, via neonatal capsaicin treatment, prevented neurogenic inflammation and delayed tumor formation. The slowing of PDAC in capsaicin-treated mice suggests the nervous system is not a bystander with respect to disease progression. Further studies are warranted to examine nervous system–tumor interactions and to identify potential targets for early detection, prevention, and treatment. Pancreatic ductal adenocarcinoma (PDAC) is characterized by an exuberant inflammatory desmoplastic response. The PDAC microenvironment is complex, containing both pro- and antitumorigenic elements, and remains to be fully characterized. Here, we show that sensory neurons, an under-studied cohort of the pancreas tumor stroma, play a significant role in the initiation and progression of the early stages of PDAC. Using a well-established autochthonous model of PDAC (PKC), we show that inflammation and neuronal damage in the peripheral and central nervous system (CNS) occurs as early as the pancreatic intraepithelial neoplasia (PanIN) 2 stage. Also at the PanIN2 stage, pancreas acinar-derived cells frequently invade along sensory neurons into the spinal cord and migrate caudally to the lower thoracic and upper lumbar regions. Sensory neuron ablation by neonatal capsaicin injection prevented perineural invasion (PNI), astrocyte activation, and neuronal damage, suggesting that sensory neurons convey inflammatory signals from Kras-induced pancreatic neoplasia to the CNS. Neuron ablation in PKC mice also significantly delayed PanIN formation and ultimately prolonged survival compared with vehicle-treated controls (median survival, 7.8 vs. 4.5 mo; P = 0.001). These data establish a reciprocal signaling loop between the pancreas and nervous system, including the CNS, that supports inflammation associated with oncogenic Kras-induced neoplasia. Thus, pancreatic sensory neurons comprise an important stromal cell population that supports the initiation and progression of PDAC and may represent a potential target for prevention in high-risk populations.

Neuroplastic Changes Occur Early in the Development of Pancreatic Ductal Adenocarcinoma

Perineural tumor invasion of intrapancreatic nerves, neurogenic inflammation, and tumor metastases along extrapancreatic nerves are key features of pancreatic malignancies. Animal studies show that chronic pancreatic inflammation produces hypertrophy and hypersensitivity of pancreatic afferents and that sensory fibers may themselves drive inflammation via neurogenic mechanisms. Although genetic mutations are required for cancer development, inflammation has been shown to be a precipitating event that can accelerate the transition of precancerous lesions to cancer. These observations led us to hypothesize that inflammation that accompanies early phases of pancreatic ductal adenocarcinoma (PDAC) would produce pathologic changes in pancreatic neurons and innervation. Using a lineage-labeled genetically engineered mouse model of PDAC, we found that pancreatic neurotrophic factor mRNA expression and sensory innervation increased dramatically when only pancreatic intraepithelial neoplasia were apparent. These changes correlated with pain-related decreases in exploratory behavior and increased expression of nociceptive genes in sensory ganglia. At later stages, cells of pancreatic origin could be found in the celiac and sensory ganglia along with metastases to the spinal cord. These results demonstrate that the nervous system participates in all stages of PDAC, including those that precede the appearance of cancer.

Smartphone adapters for digital photomicrography

Background: Photomicrographs in Anatomic Pathology provide a means of quickly sharing information from a glass slide for consultation, education, documentation and publication. While static image acquisition historically involved the use of a permanently mounted camera unit on a microscope, such cameras may be expensive, need to be connected to a computer, and often require proprietary software to acquire and process images. Another novel approach for capturing digital microscopic images is to use smartphones coupled with the eyepiece of a microscope. Recently, several smartphone adapters have emerged that allow users to attach mobile phones to the microscope. The aim of this study was to test the utility of these various smartphone adapters. Materials and Methods: We surveyed the market for adapters to attach smartphones to the ocular lens of a conventional light microscope. Three adapters (Magnifi, Skylight and Snapzoom) were tested. We assessed the designs of these adapters and their effectiveness at acquiring static microscopic digital images. Results: All adapters facilitated the acquisition of digital microscopic images with a smartphone. The optimal adapter was dependent on the type of phone used. The Magnifi adapters for iPhone were incompatible when using a protective case. The Snapzoom adapter was easiest to use with iPhones and other smartphones even with protective cases. Conclusions: Smartphone adapters are inexpensive and easy to use for acquiring digital microscopic images. However, they require some adjustment by the user in order to optimize focus and obtain good quality images. Smartphone microscope adapters provide an economically feasible method of acquiring and sharing digital pathology photomicrographs.

Comparison of Methods for Proliferative Index Analysis for Grading Pancreatic Well-Differentiated Neuroendocrine Tumors

Assessment of proliferative activity is required for grading well-differentiated pancreatic neuroendocrine tumors. However, a standardized method for obtaining the Ki-67 proliferative index is lacking. This study compared proliferative activity obtained by 3 methods: single-field hot spot (Ki-67 HS) and 10 consecutive field average (Ki-67 CFA) using the Ventana image analysis system (Ventana Medical Systems, Tucson, AZ) and mitotic index (MI). These methods resulted in discrepant grades in 30 (67%) of our 45 cases. With the current Ki-67 cutoff of more than 2% for intermediate-grade tumors, MI, CFA, and HS resulted in specificities of 91%, 94%, and 31%, respectively, for detecting metastasis, with positive predictive values (PPVs) of 25%, 67%, and 31%, respectively. At a higher Ki-67 cutoff of 7.5%, HS analysis resulted in a specificity of 94% and PPV of 71% for predicting metastasis. While single-field HS analysis may be practical and reliable at a higher cutoff, this study emphasizes the variability that can exist when different methods of assessment are used.