Eizaburo Sasatomi

Molecular pathogenesis of pulmonary carcinosarcoma as determined by microdissection-based allelotyping.

Pulmonary carcinosarcoma is a rare, biphasic tumor composed of malignant epithelial and mesenchymal elements. Its histogenesis is controversial in light of the presence of divergent cell lineages and the clonal nature of malignancy. To address these issues, we performed an extensive comparative genotypic analysis using microdissection to secure representative mesenchymal and epithelial components from each of six cases of pulmonary carcinosarcoma. Loss of heterozygosity was analyzed with a panel of 12 polymorphic microsatellite markers designed to indicate allelic loss and situated in proximity to known tumor suppressor genes located on 1p, 3p, 5q, 9p, 10q, and 17p. In accordance with the relatively greater biologic aggressiveness of this tumor type, both the epithelial and mesenchymal components showed extensive allelic loss, most notably for 3p, 5q, and 17p. More importantly, we found overall equivalent patterns of acquired allelic loss between the two components on an individual case basis, strongly supporting the monoclonal origin of these neoplasms. Minor differences in the allelic fingerprint between the two cell lineages could be explained by progressive accumulation of allelic loss alterations that appear to occur more frequently in the mesenchymal component of the tumor. The data support the efficacy of microdissection-based allelic fingerprinting to delineate the relationship between different morphologic components of a single neoplasm.

Recurrent disease following liver transplantation for nonalcoholic steatohepatitis cirrhosis

Recurrence of the original disease following liver transplantation is not uncommon and can lead to graft failure. There are limited data on recurrent fatty liver disease following liver transplantation. The aim of this study was to determine the incidence of recurrent fatty liver disease in patients with biopsy‐proven nonalcoholic steatohepatitis, its effect on survival, and whether there are any predictive factors for recurrence. We analyzed patients undergoing liver transplantation for nonalcoholic steatohepatitis cirrhosis from 1997 to 2008 at a single center. Patients undergoing transplantation for cholestatic disease, alcohol, hepatitis C, or cryptogenic cirrhosis were controls. Ninety‐eight patients underwent transplantation for nonalcoholic steatohepatitis cirrhosis. Recurrent fatty liver disease was seen in 70%, 25% had recurrent nonalcoholic steatohepatitis, and 18% had stage II/IV or greater fibrosis at a mean of 18 months. No patients with recurrent nonalcoholic steatohepatitis developed graft failure or required retransplantation at a follow‐up of 3 years. No recipient or donor factors were associated with disease recurrence, although patients with recurrent nonalcoholic steatohepatitis had a higher incidence of diabetes, weight gain, and dyslipidemia at the time of diagnosis of recurrence. One‐third of patients with recurrent nonalcoholic steatohepatitis had normal liver enzymes at the time of diagnosis post‐transplantation. In conclusion, recurrent fatty liver disease is common following liver transplantation for nonalcoholic steatohepatitis cirrhosis but does not lead to early allograft failure. Recurrent nonalcoholic steatohepatitis can occur despite normal liver enzymes, and features of metabolic syndrome are associated with disease recurrence. Liver Transpl 15:1843–1851, 2009.

Pulmonary meningothelial-like nodules: a genotypic comparison with meningiomas.

Background: Minute pulmonary meningothelial-like nodules (MPMNs) are incidental interstitial pulmonary nodules. They share histologic, ultrastructural, and immunohistochemical features with meningiomas (MGs). Design: Sixteen cases yielding 33 separate MPMNs and 10 cases of benign MG were studied. Immunohistochemical studies and mutational analyses were performed on microdissected tissue using 20 polymorphic microsatellite markers targeting 11 genomic regions in an effort to identify genetic similarities of MPMN and MG. Results: A total of 96.6% of MPMNs stained positive for vimentin, 33.3% for epithelial membrane antigen, 3% for S-100, and all were negative for cytokeratin and synaptophysin. Loss of heterozygosity (LOH) was identified in 25% of single MPMN affecting 3 genomic loci. No solitary MPMN had more than 1 LOH event. Multiple LOHs were seen only in MPMN-omatosis syndrome, where 33.3% of MPMNs showed LOH affecting 7 genomic loci. MG showed the highest frequency of LOH with major events seen at 22q (60%), 14q (42.8%), and 1p (44.4%) that were not shared by MPMN. Conclusion: Isolated MPMN lacks mutational damage, consistent with a reactive origin. MPMN-omatosis syndrome might represent the transition between a reactive and neoplastic proliferation. MPMNs are different from MG based on the major molecular genetic events seen in their formation and progression.

A microdissection and molecular genotyping assay to confirm the identity of tissue floaters in paraffin-embedded tissue blocks

CONTEXT A recurring problem in surgical pathology practice is specimen mix-up and floater contamination. While many cases can be resolved histologically, a significant number remain unclear and may have serious clinical and medicolegal implications. OBJECTIVES To design a microdissection and genotyping assay to identify contaminating floater tissues in paraffin-embedded tissues that is optimized for small samples, and to use the assay to resolve a series of clinical cases with floater tissues. MATERIALS AND METHODS Twenty-one cases of possible tissue floater contamination in paraffin-embedded tissue blocks were included. Using 4 unstained, 4-microm-thick histologic sections, multiple sites were microdissected under direct visualization either by hand or by laser capture microdissection. Nonneoplastic and neoplastic tissues were sampled. Polymerase chain reaction was performed for a panel of 10 polymorphic microsatellite markers at 1p34, 3p26, 5q21, 9p21, 10q23, and 17p13. Allele size and content were analyzed semiquantitatively by fluorescent capillary electrophoresis, and the genotypes for the tissues in the paraffin-embedded tissue blocks were compared for identity. RESULTS Tissue identification was successful in all cases, despite small tissue sample size and fixation effects. Comparative analysis of neoplastic tissue floaters and the presumptive source tumor was performed when possible to control for possible allelic loss or microsatellite instability. CONCLUSIONS Microdissection and genotyping are effective and reliable means to objectively resolve problems of possible floater contamination. Even minute tissue samples provide sufficient DNA template for polymerase chain reaction microsatellite analysis. Because of the potential clinical implications of floaters, we recommend that all suspected floaters that would change a diagnosis from benign to malignant be subjected to genotyping assay to confirm the identity of the floater tissue.

Inflammatory fibroid polyps of the gastrointestinal tract: clinical, pathologic, and molecular characteristics.

Inflammatory fibroid polyp (IFP) of the gastrointestinal tract is an uncommon proliferative lesion. When sampled by biopsy, IFP can be mistaken for various lesions, from granulation tissue to high-grade sarcoma. We present an unusual case of IFP and review a large series of IFPs to characterize clinical, histologic, and molecular features of diagnostic value. A total of 42 IFPs were gathered from the pathology archives of the University of Pittsburgh Medical Center over the past 22 years. Clinical, histopathologic, and immunohistochemical features were collected. A random subset of IFPs (n = 12) underwent microdissection genotyping for a broad panel of tumor suppressor gene-associated mutations (loss of heterozygosity). IFPs occurred in both sexes (male, 17; female, 25) over a broad age range (29–85 years). IFPs varied in size from 0.2 to 8 cm. The stomach (n = 19) was the most common location, followed by large bowel (n = 13) and small bowel (n = 10). Most IFPs displayed typical morphologic features (eosinophils admixed with loose, mature fibrous tissue), and in 2 instances, sampled by biopsy, IFP was confused for sarcoma. All IFPs lacked c-kit staining. No mutations were identified in any IFPs examined. IFP is a clinically underrecognized entity with unique morphologic and immunohistochemical features. On biopsy alone, the differential diagnosis may include sarcoma and other malignancies. The absence of mutational change may help to exclude malignant lesions.

Re-examination of the Lymphocytotoxic Crossmatch in Liver Transplantation: Can C4d Stains Help in Monitoring?

C4d‐assisted recognition of antibody‐mediated rejection (AMR) in formalin‐fixed paraffin‐embedded tissues (FFPE) from donor‐specific antibody‐positive (DSA+) renal allograft recipients prompted study of DSA+ liver allograft recipients as measured by lymphocytotoxic crossmatch (XM) and/or Luminex. XM results did not influence patient or allograft survival, or cellular rejection rates, but XM+ recipients received significantly more prophylactic steroids. Endothelial C4d staining strongly correlates with XM+ (<3 weeks posttransplantation) and DSA+ status and cellular rejection, but not with worse Banff grading or treatment response. Diffuse C4d staining, XM+, DSA+ and ABO– incompatibility status, histopathology and clinical–serologic profile helped establish an isolated AMR diagnosis in 5 of 100 (5%) XM+ and one ABO‐incompatible, recipients. C4d staining later after transplantation was associated with rejection and nonrejection‐related causes of allograft dysfunction in DSA– and DSA+ recipients, some of whom had good outcomes without additional therapy. Liver allograft FFPE C4d staining: (a) can help classify liver allograft dysfunction; (b) substantiates antibody contribution to rejection; (c) probably represents nonalloantibody insults and/or complete absorption in DSA– recipients and (d) alone, is an imperfect AMR marker needing correlation with routine histopathology, clinical and serologic profiles. Further study in late biopsies and other tissue markers of liver AMR with simultaneous DSA measurements are needed.

A Critical Appraisal of Methods to Grade Transplant Glomerulitis in Renal Allograft Biopsies

Transplant glomerulitis is an increasingly recognized lesion in renal transplant biopsies. To develop a refined grading system, we defined glomerulitis by the presence of ≥5 leukocytes/glomerulus and evaluated 111 biopsies using three different grading systems: (i) percentage of glomerular involvement, (ii) peak inflammation in the most severely affected glomerulus and (iii) presence/absence of endocapillary occlusion by inflammatory cells. Endocapillary occlusion had no impact on graft survival, but was associated with increased serum creatinine, proteinuria and subsequent transplant glomerulopathy. Grading based on either percent or peak glomerular involvement correlated with graft failure and peritubular capillaritis. However, the percent glomerular involvement method had the additional advantage of displaying associations with: concurrent proteinuria, focal or diffuse immunoperoxidase peritubular capillary C4d staining, 1‐year postbiopsy serum creatinine, subsequent detection of donor‐specific antibody and development of transplant glomerulopathy. Patients with >75% glomerular involvement also revealed persistent high‐grade glomerulitis on follow‐up biopsies despite antirejection treatment. In conclusion, grading of glomerulitis is a meaningful exercise, and a quantification system based on percentage of glomerular involvement shows the most robust associations with clinical parameters and prognosis.

Liver-Specific β-Catenin Knockout Mice Have Bile Canalicular Abnormalities, Bile Secretory Defect, and Intrahepatic Cholestasis

Beta‐catenin plays important roles in liver physiology and hepatocarcinogenesis. While studying the role of β‐catenin in diet‐induced steatohepatitis, we recently found that liver‐specific β‐catenin knockout (KO) mice exhibit intrahepatic cholestasis. This study was undertaken to further characterize the role of β‐catenin in biliary physiology. KO mice and wild‐type (WT) littermates were fed standard chow or a diet supplemented with 0.5% cholic acid for 2 weeks. Chow‐fed KO mice had higher serum and hepatic total bile acid levels and lower bile flow rate than WT mice. Expression levels of bile acid biosynthetic genes were lower and levels of major bile acid exporters were similar, which therefore could not explain the KO phenotype. Despite loss of the tight junction protein claudin‐2, KO mice had preserved functional integrity of tight junctions. KO mice had bile canalicular morphologic abnormalities as evidenced by staining for F‐actin and zona occludens 1. Electron microscopy revealed dilated and tortuous bile canaliculi in KO livers along with decreased canalicular and sinusoidal microvilli. KO mice on a cholic acid diet had higher hepatic and serum bile acid levels, bile ductular reaction, increased pericellular fibrosis, and dilated, misshapen bile canaliculi. Compensatory changes in expression levels of several bile acid transporters and regulatory genes were found in KO livers. Conclusion: Liver‐specific loss of β‐catenin leads to defective bile canalicular morphology, bile secretory defect, and intrahepatic cholestasis. Thus, our results establish a critical role for β‐catenin in biliary physiology. (HEPATOLOGY 2010)

“Plasma Cell Hepatitis” in Liver Allografts: Identification and Characterization of an IgG4‐Rich Cohort

Plasma cell hepatitis (PCH), also known as “de novo autoimmune” hepatitis, is an increasingly recognized, but suboptimally named and poorly understood, category of late allograft dysfunction strongly resembling autoimmune hepatitis (AIH): They share plasma‐cell‐rich necro‐inflammatory activity on biopsy, autoantibodies and steroid responsiveness, but overlap with rejection is problematic. A retrospective study of clinical, serological, histopathological and IgG4 immunohistological features of PCH (n = 20) in liver allograft recipients, native liver AIH (n = 19) and plasma‐cell‐rich renal allograft rejection (n = 20) showed: (1) high frequency (44%) of HLA‐DR15; (2) less female predominance (p = 0.03) and (3) n = 9/20 PCH recipients showed >25 IgG4+ plasma cells/high‐power field (IgG4+ PCH) versus AIH (n = 1/19, p = 0.008) or plasma‐cell‐rich kidney rejection (n = 2/20, p = 0.03). The IgG4+ PCH (n = 9) subgroup showed lower alanine transaminase (ALT) (p < 0.01) and aspartate transaminase (AST) (p < 0.05) at index biopsy but (a) higher plasma cell number/percentage, (b) more aggressive‐appearing portal/periportal and perivenular necro‐inflammatory activity and (c) more severe portal/periportal fibrosis than IgG4− PCH (n = 11). Significant demographic, histopathologic and plasma cell phenotype differences between PCH and AIH suggest distinct pathogenic mechanisms for at least the IgG4+ PCH subgroup likely representing an overlap between allo‐ and auto‐immunity. IgG4+ PCH was associated with fibrosis, but also highly responsive to increased immunosuppression.

Distinguishing de Novo Second Cancer Formation from Tumor Recurrence: Mutational Fingerprinting by Microdissection Genotyping

Patients with one form of cancer are known to possess a higher risk for development of a second tumor, presenting synchronously or metachronously over time. Distinguishing whether the second tumor represents a de novo cancer or a recurrence/metastasis of the first cancer has important implications for treatment and prognosis and specific clinical and pathological evidence is sought to resolve this issue. De novo cancer formation is favored by long latency interval (usually exceeding five years), better differentiation in the subsequent tumor, solitary second tumor formation and occurrence of the later tumor in a site not typical of metastatic spread. Conversely, recurrent metastatic disease is favored by short interval to second cancer formation, similar histology with increased anaplasia in the second tumor and lack of in situ malignancy and multifocal tumor deposits. While these criteria are sufficient in most instances to distinguish between the two, varying degrees of uncertainty can persist with a minority of cases remaining unresolved.