Andrew M. Bellizzi

Residual Embryonic Cells as Precursors of a Barrett's-like Metaplasia

Barretts esophagus is an intestine-like metaplasia and precursor of esophageal adenocarcinoma. Triggered by gastroesophageal reflux disease, the origin of this metaplasia remains unknown. p63-deficient mice, which lack squamous epithelia, may model acid-reflux damage. We show here that p63 null embryos rapidly develop intestine-like metaplasia with gene expression profiles similar to Barretts metaplasia. We track its source to a unique embryonic epithelium that is normally undermined and replaced by p63-expressing cells. Significantly, we show that a discrete population of these embryonic cells persists in adult mice and humans at the squamocolumnar junction, the source of Barretts metaplasia. We show that upon programmed damage to the squamous epithelium, these embryonic cells migrate toward adjacent, specialized squamous cells in a process that may recapitulate early Barretts. Our findings suggest that certain precancerous lesions, such as Barretts, initiate not from genetic alterations but from competitive interactions between cell lineages driven by opportunity.

Improved detection suggests all Merkel cell carcinomas harbor Merkel polyomavirus

A human polyomavirus was recently discovered in Merkel cell carcinoma (MCC) specimens. The Merkel cell polyomavirus (MCPyV) genome undergoes clonal integration into the host cell chromosomes of MCC tumors and expresses small T antigen and truncated large T antigen. Previous studies have consistently reported that MCPyV can be detected in approximately 80% of all MCC tumors. We sought to increase the sensitivity of detection of MCPyV in MCC by developing antibodies capable of detecting large T antigen by immunohistochemistry. In addition, we expanded the repertoire of quantitative PCR primers specific for MCPyV to improve the detection of viral DNA in MCC. Here we report that a novel monoclonal antibody detected MCPyV large T antigen expression in 56 of 58 (97%) unique MCC tumors. PCR analysis specifically detected viral DNA in all 60 unique MCC tumors tested. We also detected inactivating point substitution mutations of TP53 in the two MCC specimens that lacked large T antigen expression and in only 1 of 56 tumors positive for large T antigen. These results indicate that MCPyV is present in MCC tumors more frequently than previously reported and that mutations in TP53 tend to occur in MCC tumors that fail to express MCPyV large T antigen.

NUT rearrangement in undifferentiated carcinomas of the upper aerodigestive tract.

Background Undifferentiated carcinomas of the upper aerodigestive tract (UCUAT) occur most frequently within the nasopharynx and are most often associated with infection by Epstein-Barr virus (EBV) (WHO undifferentiated nonkeratinizing squamous cell carcinoma). An unusual group of aggressive carcinomas are characterized by translocations that involve Nuclear Protein in Testis (NUT), a novel gene on chromosome 15. In about two-thirds of cases, NUT is fused to BRD4 on chromosome 19. These tumors, here termed NUT midline carcinomas (NMCs), are undifferentiated, may have focal squamous differentiation, and are reported to occur in children and young adults. This study investigates the prevalence of NUT rearrangement and the diagnostic significance of NUT expression in a series of upper aerodigestive tract undifferentiated carcinomas. The histologic features of these tumors are described in detail. Methods All UCUAT not associated with EBV infection seen at the University of Virginia (UVA) over a 16-year period were reviewed. Clinical and histologic features were noted. Additional material was submitted for fluorescent in situ hybridization (FISH) using split-apart probes to the NUT and BRD4 genes. Immunohistochemistry (IHC) was performed on all cases using a polyclonal antibody to NUT, and on select cases with antibody to p63. Results Thirty-one UCUAT were identified. Twenty-five tumors had originally been diagnosed as sinonasal undifferentiated carcinomas. Five of 28 cases (2 males, 3 females; average age 47; range 31 to 78) with interpretable results showed rearrangements of the NUT and BRD4 genes by FISH. Three of these 5 cases showed diffuse (>90%) nuclear staining for NUT by IHC; 22 of 23 other tumors showed at most focal (<50%) nuclear staining. Undifferentiated carcinomas with NUT gene rearrangement had focal abrupt squamous differentiation in 2 cases, and intense and diffuse immunoreactivity with antibody to p63 in 4 cases. Conclusions Approximately, 20% of UCUAT not associated with EBV infection were found to have rearrangements of NUT by FISH. Although previous reports suggest that NMCs afflict only children and young adults, 4 of 5 of the patients described are mature adults older than any heretofore reported, suggesting that previous reports may have been biased in their case selections. Furthermore, because these tumors are indistinguishable from other poorly differentiated carcinomas, IHC using NUT antibody may be a useful method for the identification of these tumors. Despite the lack of overt squamous differentiation in most cases, their p63 immunoreactivity suggests that NMCs may generally be of squamous lineage.

Barbed Suture for Gastrointestinal Closure: A Randomized Control Trial

In an effort to make laparoscopic suturing more efficient, the V-Loc advanced wound closure device (Covidien, Mansfield, MA) has been produced. This device is a self-anchoring barbed suture that obviates the need for knot tying. The goal of this initial feasibility study was to investigate the use of the barbed suture in gastrointestinal enterotomy closure. A randomized study of 12 pigs comparing enterotomy closure with barbed versus a nonbarbed suture of similar tensile strength was performed. To this end, 25 mm enterotomies were made in the stomach (1 control, 1 treatment), jejunum (2 controls, 2 treatments), and descending colon (1 control, 1 treatment). Animals were killed at 3, 7, and 14 days postoperatively (4 each group) and their gastrointestinal tracts harvested; 6 of the 8 enterotomies from each pig underwent burst strength testing. The remaining 2 were fixed in formalin and sent for histological examination. All 12 pigs survived until they were killed without any major complications. Enterotomy closure with barbed suture revealed adhesion scores, burst strength pressures, and histology scores that were similar to those for the control. Jejunal closures resulted in 6 failures at 7 days (3 control, 3 barbed) and 4 failures at 14 days (2 control, 2 barbed). The barbed suture significantly reduced suturing time in the stomach, jejunum, and colon. The V-Loc wound closure device appears to offer comparable gastrointestinal closure to 3-0 Maxon while being significantly faster. Further studies with V-Loc are required to assess its use in laparoscopic surgery.

Colorectal cancer due to deficiency in DNA mismatch repair function: a review.

Lynch syndrome (LS) is an autosomal dominant cancer predisposition syndrome attributable to deleterious germline mutations in mismatch repair (MMR) genes. The syndrome is typified by early-onset, frequently right-sided colorectal cancers (CRCs) with characteristic histologic features and tendency for multiplicity and an increased risk for extracolonic tumors at particular sites; it accounts for 1% to 5% of CRC. Deficient mismatch repair (dMMR) function manifests as immunohistochemically detectable absence of one or more MMR proteins and microsatellite instability (MSI). Approximately 15% of sporadic, noninherited CRC are characterized by high-level MSI, nearly always owing to transcriptional silencing of MLH1; these sporadic and LS cases exhibit considerable phenotypic overlap. Identification of CRC with dMMR is desirable to identify LS and because MSI status is prognostic and potentially predictive. This review will discuss the history of LS, the principles of MMR and MSI, the clinicopathologic features of LS-associated and sporadic high-level MSI CRC, the fundamentals of clinical testing for dMMR CRC, and the results of the Columbus-area Lynch syndrome study. We conclude with our approach to population-based LS screening based on institutional experience with nearly 2000 cases.

POORLY DIFFERENTIATED NEUROENDOCRINE CARCINOMAS OF THE PANCREAS: A CLINICOPATHOLOGIC ANALYSIS OF 44 CASES

Background:In the pancreas, poorly differentiated neuroendocrine carcinomas include small cell carcinoma and large cell neuroendocrine carcinoma and are rare; data regarding their pathologic and clinical features are very limited. Design:A total of 107 pancreatic resections originally diagnosed as poorly differentiated neuroendocrine carcinomas were reassessed using the classification and grading (mitotic rate/Ki67 index) criteria put forth by the World Health Organization in 2010 for the gastroenteropancreatic system. Immunohistochemical labeling for neuroendocrine and acinar differentiation markers was performed. Sixty-three cases were reclassified, mostly as well-differentiated neuroendocrine tumor (NET) or acinar cell carcinoma, and eliminated. The clinicopathologic features and survival of the remaining 44 poorly differentiated neuroendocrine carcinomas were further assessed. Results:The mean patient age was 59 years (range, 21 to 82 y), and the male/female ratio was 1.4. Twenty-seven tumors were located in the head of the pancreas, 3 in the body, and 11 in the tail. The median tumor size was 4 cm (range, 2 to 18 cm). Twenty-seven tumors were large cell neuroendocrine carcinomas, and 17 were small cell carcinomas (mean mitotic rate, 37/10 and 51/10 HPF; mean Ki67 index, 66% and 75%, respectively). Eight tumors had combined components, mostly adenocarcinomas. In addition, 2 tumors had components of well-differentiated NET. Eighty-eight percent of the patients had nodal or distant metastatic disease at presentation, and an additional 7% developed metastases subsequently. Follow-up information was available for 43 patients; 33 died of disease, with a median survival of 11 months (range, 0 to 104 mo); 8 were alive with disease, with a median follow-up of 19.5 months (range, 0 to 71 mo). The 2- and 5-year survival rates were 22.5% and 16.1%, respectively. Conclusions:Poorly differentiated neuroendocrine carcinoma of the pancreas is a highly aggressive neoplasm, with frequent metastases and poor survival. Most patients die within less than a year. Most (61%) are large cell neuroendocrine carcinomas. Well-differentiated NET and acinar cell carcinoma are often misdiagnosed as poorly differentiated neuroendocrine carcinoma, emphasizing that diagnostic criteria need to be clearly followed to ensure accurate diagnosis.

Pancreatic Cytopathology: A Practical Approach and Review

CONTEXT Pancreatic cytopathology plays an important role in the diagnosis and management of patients with solid and cystic lesions of the pancreas. OBJECTIVE To serve as a practical guide to pancreatic cytopathology for the practicing pathologist. Data Sources.-A comprehensive assessment of the medical literature was performed. CONCLUSIONS We review pancreatic cytopathology, with specific discussions of its role in patient management, specimen types and specimen processing, specific diagnostic criteria, and the use of ancillary testing and advanced techniques.

Cord Colitis Syndrome in Cord-Blood Stem-Cell Transplantation

BACKGROUND Diarrhea is a frequent complication of hematopoietic stem-cell transplantation (HSCT). Important causes of diarrhea after HSCT include acute graft-versus-host disease (GVHD), infections, and medications. After the transplantation and engraftment of hematopoietic stem cells from umbilical-cord blood, we observed a new syndrome of culture-negative, antibiotic-responsive diarrhea not attributable to any known cause. METHODS We conducted a retrospective cohort study of all patients undergoing cord-blood HSCT at our center between March 2003 and March 2010. The cord colitis syndrome was defined as a persistent diarrheal illness in such patients that was not due to acute GVHD, viral or bacterial infection, or another identifiable cause. Clinical and histopathological features of patients meeting the case definition were further analyzed. RESULTS Among 104 patients who underwent cord-blood HSCT at our center, the cord colitis syndrome developed in 11 (10.6%). The 1-year Kaplan-Meier cumulative probability of meeting the case definition for the syndrome was 0.16. The median time to onset after transplantation was 131 days (range, 88 to 314). All patients had a response to a 10-to-14-day course of empirical therapy with metronidazole, alone or in combination with a fluoroquinolone. Five of the 11 patients (45%) had recurrent diarrhea shortly after discontinuation of antibiotics, and all patients who had a relapse had a response to reinitiation of antibiotic therapy. On histologic examination, all patients with the cord colitis syndrome had chronic active colitis, with granulomatous inflammation present in 7 of 11 patients (64%). CONCLUSIONS The cord colitis syndrome is clinically and histopathologically distinct from acute GVHD and other causes of diarrhea in patients who have undergone cord-blood HSCT and is relatively common in this patient population. The syndrome should be considered in such patients who have diarrhea that is not attributable to other causes.

Assigning site of origin in metastatic neuroendocrine neoplasms: a clinically significant application of diagnostic immunohistochemistry.

The neuroendocrine epithelial neoplasms (NENs) include well-differentiated neuroendocrine tumors (WDNETs) and poorly differentiated neuroendocrine carcinomas (PDNECs). Whereas PDNECs are highly lethal, with localized Merkel cell carcinoma somewhat of an exception, WDNETs exhibit a range of “indolent” biologic potentials—from benign to widely metastatic and eventually fatal. Within each of these 2 groups there is substantial morphologic overlap. In the metastatic setting, the site of origin of a WDNET has significant prognostic and therapeutic implications. In the skin, Merkel cell carcinoma must be distinguished from spread of a visceral PDNEC. This review intends to prove the thesis that determining the site of origin of a NEN is clinically vital and that diagnostic immunohistochemistry is well suited to the task. It will begin by reviewing current World Health Organization terminology for the NENs, as well as an embryologic and histologic pattern–based classification. It will present population-based data on the relative frequency and biology of WDNETs arising at various anatomic sites, including the frequency of metastases of unknown primary, and comment on limitations of contemporary imaging techniques, as a means of defining the scope of the problem. It will go on to discuss the therapeutic significance of site of origin. The heart of this review is a synthesis of data compiled from >100 manuscripts on the expression of individual markers in WDNETs and PDNECs, as regards site of origin. These include proteins that are considered “key markers” and others that are either useful “secondary markers,” potentially very useful markers that need to be further vetted, or ones that are widely applied despite a lack of efficacy. It will conclude with my approach to the metastatic NEN of unknown origin.

Additive protection against lung ischemia-reperfusion injury by adenosine A2A receptor activation before procurement and during reperfusion

OBJECTIVE Adenosine A2A receptor activation during reperfusion improves lung ischemia-reperfusion injury. In this study we sought to determine whether pretreatment of rabbits with a potent and selective adenosine A2A receptor agonist, ATL-313, before transplantation or whether adding ATL-313 to the preservation solution results in equivalent or additional protection compared with ATL-313 added during reperfusion. METHODS An isolated, ventilated, ex vivo blood-perfused rabbit lung model was used. All groups underwent 2 hours of reperfusion after 18 hours of cold ischemia (4 degrees C). ATL-313 was administered 1 hour before ischemia intravenously, with the preservation solution, and/or during reperfusion. RESULTS Both pretreatment of donor animals with ATL-313 or adding ATL-313 just during reperfusion improved pulmonary function, but significantly greater improvement was observed when pretreatment and treatment during reperfusion were combined (all P < .05). Myeloperoxidase levels, bronchoalveolar lavage tumor necrosis factor alpha levels, and pulmonary edema were all maximally decreased in the combined treatment group. The administration of an equimolar amount of the potent and highly selective adenosine 2A receptor antagonist, ZM 241385, along with ATL-313, resulted in the loss of protection conferred by ATL-313. CONCLUSIONS Adenosine A2A receptor activation with ATL-313 results in the greatest protection against lung ischemia-reperfusion injury when given before ischemia and during reperfusion. Improved pulmonary function observed with adenosine A2A receptor activation was correlated with decreased bronchoalveolar lavage tumor necrosis factor alpha and decreased lung myeloperoxidase. The loss of protection observed with the concurrent administration of the adenosine A2A receptor antagonist, ZM 241385, supports that the mechanism of ATL-313 protection is specifically mediated via adenosine A2A receptor activation.