Human Pathology - Articles in Press

High levels of p53 expression correlate with DNA aneuploidy in (pre)malignancies of the vulva - Corrected Proof

2010-07-26

Summary: The molecular pathogenesis of human papilloma virus–unrelated vulvar squamous cell carcinoma is not well known. Whether malignant progression of lichen sclerosus and differentiated vulvar intraepithelial neoplasia to vulvar squamous cell carcinoma could be accompanied by altered DNA content has not been studied extensively. DNA content in isolated nuclei of microdissected normal vulvar epithelium (n = 2), lichen sclerosus (n = 9), differentiated vulvar intraepithelial neoplasia (n = 13), and squamous cell carcinoma (n = 17) from 22 patients was measured via DNA image cytometry. For additional analysis, 6 differentiated vulvar intraepithelial neoplasia lesions were selected, bringing the number of patients to 28. p53 expression was determined by immunohistochemistry on consecutive tissue sections. Thirty-eight percent (5/13) of differentiated vulvar intraepithelial neoplasia lesions and 65% (11/17) of squamous cell carcinomas were DNA aneuploid or tetraploid. In lesions that contained differentiated vulvar intraepithelial neoplasia and adjacent squamous cell carcinoma, the ploidy status of differentiated vulvar intraepithelial neoplasia did not exceed that of squamous cell carcinoma. We observed a strong correlation between high p53 expression and DNA aneuploidy. This relation was also present at the level of a single nucleus, measured by sequential image cytometry of p53 immunohistochemistry followed by DNA image cytometry on formalin-fixed tissue sections. Similarly, we found p53-positive nonproliferating cells with increased DNA content in the superficial compartment of 6 additional solitary differentiated vulvar intraepithelial neoplasia lesions that were not associated with squamous cell carcinoma, indicating ascending aneuploid cells from the basal compartment. DNA ploidy measurements suggest that differentiated vulvar intraepithelial neoplasia has a higher malignant potential than lichen sclerosus and thus is a more likely precursor of squamous cell carcinoma. Furthermore, high p53 expression correlates with increased DNA content and aneuploidy; but it requires further research to unveil a possible causal relation.

The phosphatidylserine receptors, T cell immunoglobulin mucin proteins 3 and 4, are markers of histiocytic sarcoma and other histiocytic and dendritic cell neoplasms - Corrected Proof

David M. Dorfman, Jason L. Hornick, Aliakbar Shahsafaei, Gordon J. Freeman — 2010-07-26

Summary: The T cell immunoglobulin mucin (TIM) proteins are a family of cell surface phosphatidyserine receptors that are important for the recognition and phagocytosis of apoptotic cells. Because TIM-4 is expressed by macrophages and dendritic cells in human tissue, we examined its expression in a range of histiocytic and dendritic cell neoplasms and found moderate to strong immunohistochemical staining in cases of juvenile xanthogranuloma and histiocytic sarcoma, and lower level staining in interdigitating dendritic cell sarcoma, Langerhans cell histiocytosis, acute monocytic leukemia (leukemia cutis), and blastic plasmacytoid dendritic cell neoplasm (hematodermic tumor). TIM-3 was first described on activated TH1 cells but was recently shown to also be a phosphatidylserine receptor and mediate phagocytosis. We found TIM-3 was expressed by peritoneal macrophages, monocytes and splenic dendritic cells. We found that it, like TIM-4, is expressed in a range of histiocytic and dendritic cell neoplasms, typically with strong immunohistochemical staining. Cases of diffuse large B cell lymphoma, anaplastic large cell lymphoma, metastatic malignant melanoma, and metastatic poorly differentiated carcinoma generally exhibited negative to minimal heterogenous staining for TIM-4 and TIM-3. We conclude that histiocytic and dendritic cell neoplasms consistently express TIM-3 and TIM-4 and that these molecules are new markers of neoplasms derived from histiocytic and dendritic cells.

The presence of isolated tumor cells and micrometastases in the intrathoracic lymph nodes of patients with lung cancer is not associated with decreased survival - Corrected Proof

2010-07-26

Summary: The prognostic role of small intrathoracic nodal metastases in primary patients with lung cancer has been controversial, and it is unclear how their presence should be used for pathologic staging and treatment planning. The intrathoracic lymph nodes from 266 clinical stage I non–small cell carcinoma patients treated at Cedars Sinai Medical Center from 1992 to 2006 were evaluated with immunohistochemistry for keratin AE1/AE3 for the presence of isolated tumor cells and micrometastases, as defined by American Joint Commission on Cancer criteria, correlated with survival using the Kaplan-Meier method and analyzed with power analysis. The English literature from 1995 to 2008 was reviewed to identify best available evidence regarding the prognostic value of isolated tumor cells and micrometastases detected with various immunohistochemistry and molecular methods in non–small cell carcinoma patients. Results were combined with our own data and evaluated with meta-analysis using Comprehensive Meta-analysis 2.0 software (Biostat Inc, Englewood, NJ). Isolated tumor cells and micrometastases were detected in 8 and 67 of 4148 lymph nodes, respectively, and their presence was not significantly associated with survival. Power analysis showed that 3060 cases followed up for 60 months would be needed to achieve 80% power in a study designed to detect survival differences between patients with negative nodes and micrometastases. Meta-analysis of 835 non-small cell carcinoma patients reported in 13 studies showed scanty data to evaluate patients with isolated tumor cells, no significant association between micrometastases and survival and significant data heterogeneity. Current best evidence suggests that non-small cell carcinoma patients should probably not be “upstaged” in the presence of isolated tumor cells and micrometastases. There is no data demonstrating survival benefits for patients treated with adjuvant chemotherapy and/or radiation therapy because of the presence of small nodal metastases.

The chromosome Y-linked testis-specific protein locus TSPY1 is characteristically present in gonadoblastoma - Corrected Proof

Johann D. Hertel, Phyllis C. Huettner, Louis P. Dehner, John D. Pfeifer — 2010-07-26

Summary: Gonadoblastoma is a rare gonadal neoplasm that occurs almost exclusively in individuals who are phenotypically females. Most cases develop in women who have an abnormal karyotype in which at least a portion of the centromeric region of the short arm of chromosome Y is present, a region often referred to as the GBY locus. Of the several genes present in the GBY locus, the TSPY1 gene (which encodes testis-specific protein, a protein thought to have a role in cell cycle regulation) appears to be the most likely to have a critical role in the pathogenesis of gonadoblastoma. To evaluate the association of TSPY1 with the tumor, we developed an interphase fluorescent in situ hybridization assay that uses probes that target the region of the GBY locus that contains TSPY1 and a commercially available chromosome X CEP probe. Using this set of probes in a dual-color approach, we evaluated 6 cases of gonadoblastoma identified from our files and found that both TSPY1 and chromosome X were present in 5 (84%) of 6 cases; in these 5 cases, the adjacent nonneoplastic gonadal parenchyma showed the same genotype as the tumor. Of 6 cases, 1 (16%) showed no evidence of TSPY1; in this case, which occurred in a gravida 2 para 2 woman, 2 X chromosomes were present in the nonneoplastic ovary, the gonadoblastoma, and associated dysgerminoma and granulosa cell tumors. From a basic science perspective, our data demonstrate that the TSPY1 gene is present in most gonadoblastomas, supporting the hypothesized role for TSPY1 in gonadoblastoma tumorigenesis; the lack of TSPY1 in a fertile woman suggests that other loci can, however, substitute for TSPY1 in the development of the tumor. From a clinical perspective, our data show that interphase fluorescence in situ hybridization targeting TSPY1 is a straightforward approach that can be used in the evaluation of Y-associated intersex disorders in women who develop gonadoblastoma.

Anaplastic sarcoma of the kidney with chromosomal abnormality: first report on cytogenetic findings - Corrected Proof

2010-07-26

Summary: We report a case of anaplastic sarcoma of the kidney (ASK) with cytogenetic findings. A 12-year-old Japanese girl presented with buttock pain and urinary incontinence. Radiological investigations revealed a right renal tumor with multiple distant metastases and multicystic thyroid tumor. She underwent radical right nephrectomy and subsequently received chemotherapy and radiation therapy. Histologically, the renal tumor demonstrated admixture of various types of mesenchymal elements: cellular spindle cells with anaplastic features, cartilage, and rhabdomyoblastic cells consistent with ASK. Chromosomal analysis revealed the karyotype of the tumor cells to be 46, XX, +8, –10, der (18) t (10; 18) (q21; p11.2). The thyroid tumor was removed later and diagnosed as adenomatous goiter. To our knowledge, this is the first case of ASK with chromosomal abnormality and may provide new insight into the molecular biologic basis of this rare renal tumor.

Posttransplantation lymphoproliferative disease involving the pituitary gland - Corrected Proof

2010-07-26

Summary: Posttransplantation lymphoproliferative disorders (PTLD) are heterogeneous lesions with variable morphology, immunophenotype, and molecular characteristics. Multiple distinct primary lesions can occur in PTLD, rarely with both B-cell and T-cell characteristics. Lesions can involve both grafted organs and other sites; however, PTLD involving the pituitary gland has not been previously reported. We describe a patient who developed Epstein-Barr virus–negative PTLD 13 years posttransplantation involving the terminal ileum and pituitary, which was simultaneously involved by a pituitary adenoma. Immunohistochemistry of the pituitary lesion showed expression of CD79a, CD3, and CD7 with clonal rearrangements of both T-cell receptor gamma chain (TRG@) and immunoglobulin heavy chain (IGH@) genes. The terminal ileal lesion was immunophenotypically and molecularly distinct. This is the first report of pituitary PTLD and illustrates the potentially complex nature of PTLD.

Aurora kinase A in Barrett’s carcinogenesis - Corrected Proof

2010-07-26

Summary: In Barrett's mucosa, both aneuploidy and TP53 mutations are consistently recognized as markers of an increased risk of Barrett's adenocarcinoma. Overexpression of the mitotic kinase encoding gene (AURKA) results in chromosome instability (assessed from the micronuclei count) and ultimately in aneuploidy. Eighty-seven esophageal biopsy samples representative of all the phenotypic lesions occurring in the multistep process of Barrett's carcinogenesis (gastric metaplasia in 25, intestinal metaplasia in 25, low-grade intraepithelial neoplasia in 16, high-grade intraepithelial neoplasia in 11, and Barrett's adenocarcinoma in 10) were obtained from long segments of Barrett's mucosa. Twenty-five additional biopsy samples of native esophageal mucosa were used for control purposes. In all tissue samples, the immunohistochemical expression of both AURKA and TP53 gene products was scored; and the micronuclei index was calculated. AURKA immunostaining increased progressively and significantly along with dedifferentiation of the histologic phenotype (P < .001). Nine of 10 Barrett's adenocarcinomas showed AURKA immunostaining. AURKA expression correlated significantly with p53 expression and the micronuclei index (both Ps < .001). AURKA overexpression is significantly associated with Barrett's mucosa progressing to Barrett's adenocarcinoma and contributes to esophageal carcinogenesis via chromosome instability. The identification of AURKA as a novel molecular target of cancer progression in Barrett's mucosa provides a lead for the development of new therapeutic approaches in Barrett's mucosa patients.

Heterogenous high-level HER-2 amplification in a small subset of colorectal cancers - Corrected Proof

2010-07-26

Summary: HER-2 is the molecular target for antibody-based treatment of breast cancer (trastuzumab). The potential benefit of anti–HER-2 therapy is currently investigated in several other HER-2 amplified cancers. For example, trastuzumab was recently shown to be effective in HER-2 positive gastric cancer. To address the potential applicability of anti–HER-2 therapy in colorectal cancer, tissue microarray sections and colorectal resection specimens of 1851 colorectal cancers were analyzed for HER-2 overexpression and amplification using FDA approved reagents for immunohistochemistry and fluorescence in situ hybridization. HER-2 amplification was seen in 2.5% and HER-2 overexpression in 2.7% of 1439 interpretable colorectal cancers. Amplification was often high level with HER-2 copies ranging from 4 to 60 per tumor cell and was strongly related to protein overexpression. HER-2 amplification and overexpression were unrelated to histological tumor type, tumor localization, grading, pT, pN, pM or survival. As heterogeneity of drug target expression could represent a major drawback for targeted cancer therapy we next studied HER-2 heterogeneity in selected cases. Extensive evaluation of all available large sections from patients with HER-2 positive colorectal cancer revealed heterogenous findings in 3 of 4 cases. In summary, high-level HER-2 amplification occurs in a small fraction of colorectal cancers. Heterogeneity of amplification may limit the utility of anti- HER-2 therapy in some of these tumors and therefore, adequate clinical trials are needed to further evaluate this approach.

Pdcd4 expression in intraductal papillary mucinous neoplasm of the pancreas: its association with tumor progression and proliferation - Corrected Proof

2010-07-26

Summary: Intraductal papillary mucinous neoplasm is characterized by cystically dilated main and/or branch pancreatic duct with mucus. According to the degree of atypia, intraductal papillary mucinous neoplasm is classified into 3 groups: adenoma, borderline, and carcinoma. Furthermore, intraductal papillary mucinous neoplasm is considered to progress through an adenoma-carcinoma sequence like colorectal carcinoma. Programmed cell death 4 is a recently identified tumor suppressor that was found to inhibit translation. Programmed cell death 4 has been reported to inhibit tumorigenesis, tumor progression, proliferation, invasion, and metastasis in several human malignancies. We examined 108 cases of intraductal papillary mucinous neoplasm by immunohistochemistry and revealed that programmed cell death 4 expression was recognized in both the nucleus and cytoplasm in intraductal papillary mucinous neoplasm. The positive rate of programmed cell death 4 was 79%, 43%, and 10% in adenoma, borderline, and carcinoma, respectively. The positive rate of programmed cell death 4 decreased from adenoma to carcinoma (P < .0001, both adenoma versus borderline and borderline versus carcinoma), indicating that programmed cell death 4 might inhibit tumor progression in intraductal papillary mucinous neoplasm. Programmed cell death 4 expression had a strong relationship with p21 expression (P < .0001) and an inverse correlation with Ki-67 labeling index (r = −0.6255, P < .0001). Thus, programmed cell death 4 might inhibit the proliferation of intraductal papillary mucinous neoplasm; and its inhibition might partly result from cell cycle arrest caused by the up-regulation of p21. In conclusion, programmed cell death 4 may inhibit tumor progression in intraductal papillary mucinous neoplasm; and the loss of programmed cell death 4 expression is representative of the malignant potential of intraductal papillary mucinous neoplasm including the proliferative activity. Therefore, programmed cell death 4 can be an important biomarker for intraductal papillary mucinous neoplasm.

Correlation of the detection of Clostridium difficile toxins in stools and presence of the clostridia in tissues of children - Corrected Proof

2010-07-26

Summary: Clostridium difficile toxin is frequently found in the stool of children; however, pseudomembranous colitis is rare. Studying the usefulness of Clostridium difficile toxin assays in pediatrics is required. We performed a correlation between presence of Clostridium difficile toxin in stool and evidence of Clostridium difficile in gastrointestinal pediatric tissue samples using immunohistochemistry (with a pan-clostridial antibody) and polymerase chain reaction (with primers for toxin genes). We studied 11 patients with a median age of 8 years (range, 4 weeks to 17 years); 4 (36%) were female. The median time between detection of Clostridium difficile toxin in stool and obtaining tissue was 3 days. Ten patients survived. Endoscopy was performed in 8 survivors and showed normal mucosa in 2, pseudomembranes in 2, erythema and friability in 1, aphthae in 1, increased mucous production in 1, and colitis in 1. Two survivors underwent laparotomy for either obstruction or resection of necrotic bowel. Histopathologic studies in these 10 surviving patients showed necrosis in 2 samples, granulomatous inflammation in 1, moderate colitis in 1, and mild to minimal pathology in 7. There was no antigenic or molecular evidence of clostridia in the tissue of these patients. Histopathologic evidence of pseudomembranes and immunohistochemical evidence of clostridia were present in postmortem intestinal tissues of the only patient that died. Our findings indicate that Clostridium difficile toxin in stool does not correlate with the presence of clostridia and may not contribute to pathology in intestinal tissues of children. Clostridial antigens were only observed with histopathologic evidence of pseudomembranes.

Gastric foveolar metaplasia and gastric heterotopia in the duodenum: no evidence of an etiologic role for Helicobacter pylori - Corrected Proof

Robert M. Genta, R. Shawn Kinsey, Anuradha Singhal, Smita Suterwala — 2010-07-26

Summary: Gastric-type epithelium and islands of oxyntic mucosa in duodenal biopsies are considered by some to be part of a spectrum of metaplastic change related to peptic disorders. This study was designed to assess prevalence and associations of metaplastic-heterotopic gastric mucosa in the duodenum. Demographic, clinical, and histopathologic data from patients who had duodenal biopsy specimens for a 12-month period were collected from a national database. The duodenal findings of patients with duodenitis, gastric metaplasia, and gastric heterotopia were correlated with gastric pathology, Helicobacter pylori status, and clinical information. Of 28 210 patients with duodenal biopsy specimens, 80.9% were healthy, 2.1% had active duodenitis, 2.2% gastric foveolar metaplasia without active inflammation (“peptic duodenopathy”), 4.8% gastric foveolar metaplasia with active inflammation (“peptic duodenitis”), and 1.9% gastric heterotopia. Helicobacter pylori was documented in 9.8% of patients with normal duodenum, 6.9% of those with gastric metaplasia without active inflammation, 15.8% of those with active duodenitis, and 29.1% of those with gastric foveolar metaplasia with active inflammation; 2.2% of 543 patients with gastric heterotopia had H pylori gastritis. Helicobacter pylori was detected in the metaplastic epithelium of 67.6% of patients with active inflammation and in 16.4% of those with metaplasia without inflammation. Gastric heterotopia was strongly associated with concurrent fundic gland polyps. In conclusion, active duodenitis was more common in patients with H pylori infection, but gastric metaplasia was not. We suggest that there is insufficient evidence to ascribe duodenitis with foveolar metaplasia to a “peptic” disorder, as “peptic duodenopathy” and “peptic duodenitis” seem to imply. Gastric heterotopia is likely a congenital lesion; its association with fundic gland polyps suggests that use of proton pump inhibitors may enhance its endoscopic detection.

Merkel cell polyomavirus is present in common warts and carcinoma in situ of the skin - Corrected Proof

2010-07-23

Summary: Polyomaviruses have been linked to diseases of immunosuppressed patients. We sought to determine the prevalence of Merkel cell polyomavirus in benign epithelial skin neoplasms and nonmelanoma skin cancer of immunosuppressed renal transplant recipients and long-term dialysis patients. Merkel cell polyomavirus DNA was detected by polymerase chain reaction (PCR) in 2 (10%) of 20 patients, in carcinomas in situ (Bowen's disease). In one of our patients with Merkel cell polyomavirus-positive carcinoma in situ, 9 (39.1%) of 23 skin lesions at various anatomical locations tested positive for Merkel cell polyomavirus sequences by PCR, including all of his common warts (4/4), half of his carcinoma in situ lesions (3/6), and 2 of his 3 seborrheic keratoses. In a second cohort of immunosuppressed renal transplant recipients, Merkel cell polyomavirus DNA was found in 1 (6.3%) of 16 common warts and in 2 (9.5%) of 21 carcinomas in situ. In immunocompetent individuals, Merkel cell polyomavirus DNA was found in 2 (6.7%) of 30 common warts and in 2 (8.3%) of 24 carcinomas in situ. DNA of other human polyomaviruses was not detected in any of the investigated skin neoplasms. We conclude that common warts and carcinomas in situ can be positive for Merkel cell polyomavirus in immunosuppressed as well as immunocompetent individuals. Remarkably, some of the Merkel cell polyomavirus-positive common warts did not contain human papillomavirus. Furthermore, Merkel cell polyomavirus can be found in various skin neoplasms of the same individual.

Myopericytoma of the kidney - Corrected Proof

Sean K. Lau, Roman Klein, Zhong Jiang, Lawrence M. Weiss, Peiguo G. Chu — 2010-07-23

Summary: Myopericytoma is a rare, histologically distinctive tumor that shows evidence of differentiation toward perivascular myoid cells. Myopericytoma is largely considered a neoplasm of skin and soft tissues, with examples of this lesion involving visceral sites being extremely limited. We present the clinical and pathologic details of an unusual case of myopericytoma occurring in the kidney. Histologically, the tumor was richly vascularized and composed of a perivascular proliferation of oval to spindle-shaped cells with bland cytologic features. The neoplastic cells were arranged in a concentric fashion around vascular lumina and also surrounded dilated, branching vessels, with a glomangiopericytomatous appearance. Mitotic figures were inconspicuous, and necrosis was absent. Perivascular myoid differentiation was supported by positive immunoreactivity for muscle-specific and smooth muscle actins, and absence of reactivity for desmin. The present case serves to expand the anatomical distribution of myopericytoma and also broadens the spectrum of primary mesenchymal neoplasms that may be encountered in the kidney.

Disseminated Mycobacterium genavense infection in a healthy boy - Corrected Proof

2010-07-23

Summary: Mycobacterium genavense (M genavense) has been recognized as a life-threatening pathogen in severely immunocompromised patients. To our knowledge, disseminated M genavense infection has never been described in immunocompetent individuals. Here, we report a case of disseminated M genavense infection in a healthy Japanese boy. A 15-year-old boy who had never been diagnosed with an immunodeficiency disorder was hospitalized because of ileus. Tumorous lesions were identified in the ileum, cecum, and ascending colon, resulting in stenosis of ileocecal valve. There was diffuse proliferation of histiocytes throughout the intestinal wall, along with lymphocytic infiltration. No nuclear or cellular atypia was present in these cells. Ziehl-Neelsen staining revealed numerous acid-fast bacteria in histiocytes. After surgery, systemic lymph node swelling was noticed by generalized examination, including the mesenteric and cervical lymph nodes. M genavense DNA was identified by direct sequencing of 16S ribosomal DNA that had been amplified by polymerase chain reaction.

Reduced membranous and ectopic cytoplasmic expression of DSC2 in esophageal squamous cell carcinoma: an independent prognostic factor - Corrected Proof

2010-07-12

Summary: Desmocollin 2, a desmosomal component, is a key membrane glycoprotein critically involved in cell-cell adhesion and the maintenance of normal tissue architectures in epithelia. Reports exploring the link of desmocollin expression to cancers are limited. The aim of this study was to investigate the expression of desmocollin 2 in esophageal squamous cell carcinoma and, in particular, to determine the extent to which the patterns of desmocollin 2 expression correlated with the clinical parameters. Desmocollin 2 expression was evaluated in 308 cases of esophageal squamous cell carcinoma using immunohistochemistry. Western blotting and reverse transcriptase polymerase chain reaction were performed to characterize the relative expression levels of desmocollin 2 isoforms. The results indicated that desmocollin 2 expression was reduced significantly in esophageal cancer in both protein and messenger RNA levels and that this reduction was associated with poor survival (P = .011). The expression of desmocollin 2 was prominent in normal esophageal epithelia and highly differentiated esophageal tumors, but was reduced or absent in poorly differentiated tumor specimens. Furthermore, in 74.7% of tumor tissues, desmocollin 2 immunoreactivity displayed an abnormal cytoplasmic localization that was correlated with poor tumor differentiation (P < .001), regional lymph node metastasis (P < .001), pathologic tumor-node-metastasis stages (P < .001), and poor prognosis (P = .048). Multivariate analysis showed that desmocollin 2 expression level was an independent prognostic factor for esophageal squamous cell carcinoma. These data suggest that desmocollin 2 is involved in the transformation and development of esophageal tumors and that desmocollin 2 expression level and intracellular localization may serve as a predictor for patient outcomes.

Characterization of CD24 expression in intraductal papillary mucinous neoplasms and ductal carcinoma of the pancreas - Corrected Proof

2010-07-12

Summary: CD24 is a molecule involved in cell adhesion and tumor metastasis. The aims of this study were (1) to evaluate the association between CD24 expression and the progression of intraductal papillary mucinous neoplasms of the pancreas and (2) to investigate the association between CD24 expression in pancreatic cancer and the prognosis of patients who underwent curative pancreatectomy. Immunohistochemical analysis of CD24 was performed for 95 intraductal papillary mucinous neoplasms of the pancreas and 83 pancreatic cancers. We investigated the association between CD24 expression and the histologic grade of intraductal papillary mucinous neoplasms of the pancreas, the clinicopathologic parameters of pancreatic cancers, and the survival time of pancreatic cancer patients who underwent pancreatectomy. The positive rates of CD24 expression in intraductal papillary mucinous adenoma, borderline intraductal papillary mucinous neoplasm, noninvasive intraductal papillary mucinous carcinoma, and invasive intraductal papillary mucinous carcinoma were 5 (20%) of 24, 12 (48%) of 25, 10 (43%) of 23, and 15 (65%) of 23, respectively. The CD24-positive rates were significantly higher in borderline intraductal papillary mucinous neoplasm and intraductal papillary mucinous carcinoma compared with intraductal papillary mucinous adenoma (P = .046 and P = .007, respectively). The staining scores, which were determined from the percentage of stained cells and the staining intensity, were significantly higher in invasive intraductal papillary mucinous carcinoma than in noninvasive intraductal papillary mucinous carcinoma (P = .043). In the pancreatic cancers, higher tumor stage (P = .007), nodal metastasis (P = .021), and higher-grade tumors (P < .001) were more frequent in the CD24-positive group compared with the CD24-negative group. CD24 expression was associated with shorter survival in univariate analysis (P = .028) However, based on the multivariate analysis, the CD24 expression was not associated with survival. In conclusion, CD24 is involved in the progression of intraductal papillary mucinous neoplasms of the pancreas and in the malignant behavior of pancreatic cancers.

Hypoxia-inducible adenosine A2B receptor modulates proliferation of colon carcinoma cells - Corrected Proof

2010-07-12

Summary: Extracellular adenosine regulates a wide variety of physiological processes by interacting with 4 adenosine receptor subtypes: A1, A2A, A2B, and A3. However, little is known of their pathophysiological roles in human cancers. In this study, we examined the expression pattern of adenosine receptors in various colorectal tissues and human colon carcinoma cell lines and investigated the biologic functions regarding colon carcinogenesis. Using reverse transcriptase polymerase chain reaction and Western blotting, we found that adenosine receptor A2B (ADORA2B) was consistently up-regulated in colorectal carcinoma tissues and colon cancer cell lines compared with normal colorectal mucosa. In immunohistochemistry, we observed diffuse immunopositivity of ADORA2B in 67% of colorectal adenocarcinomas (39/58), 17% of tubular adenomas (5/30), and 0% of normal colon glands (0/62). During a hypoxic state, there was also a significant induction of ADORA2B expression in the messenger RNA level at 8 hours of incubation and in the protein level at 24 hours of incubation in colon carcinoma cell lines. To examine the function of ADORA2B, we applied an ADORA2B-selective antagonist (MRS1754) to the colon carcinoma cells, which significantly inhibited cell growth in a dose-dependent manner as demonstrated with a 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide cell proliferation assay. In conclusions, ADORA2B was overexpressed in colorectal carcinomas grown under a hypoxic state, presumably promoting cancer cell growth. Our data suggest that this adenosine receptor is a potential therapeutic target for colorectal cancer.

Cell adhesion molecules P-cadherin and CD24 are markers for carcinoma and dysplasia in the biliary tract - Corrected Proof

2010-07-12

Summary: P-cadherin (CDH3) and CD24 are cell adhesion molecules that control morphogenic processes, cell motility, and invasive growth of tumor cells. The aim of our study was to investigate P-cadherin and CD24 expression in carcinomas and dysplastic lesions of the biliary tract and to evaluate the potential diagnostic usefulness of these cell adhesion molecules. Using immunohistochemistry on tissue microarrays, we analyzed P-cadherin, CD24, and p53 expression in 117 carcinomas of the biliary tract (19 intrahepatic cholangiocarcinomas, 59 extrahepatic cholangiocarcinomas, and 39 gallbladder carcinomas) and correlated our findings with clinicopathologic parameters. We found P-cadherin positivity in 37% of intrahepatic cholangiocarcinomas, 73% of extrahepatic cholangiocarcinomas, and 64% of gallbladder carcinomas, respectively. CD24 reactivity was observed in 21% of intrahepatic cholangiocarcinomas, 58% of extrahepatic cholangiocarcinomas, and 42% of gallbladder carcinomas. Nuclear p53 expression was found in 37% of intrahepatic cholangiocarcinomas, 46% of extrahepatic cholangiocarcinomas, and 45% of gallbladder carcinomas. We also studied P-cadherin, CD24, and p53 expression in normal (n = 30), inflamed (n = 22), and dysplastic (n = 21) biliary epithelium of extrahepatic bile ducts. Dysplastic biliary epithelium was positive for P-cadherin in 91%, for CD24 in 71%, and for p53 in 24% of lesions, respectively. In contrast, normal and inflamed epithelia were negative for all 3 proteins. We conclude that P-cadherin and CD24 are expressed in carcinomas of the biliary tract with high frequency and at an early stage of carcinogenesis. Therefore, they may be useful markers for early detection and as targets for therapy of cholangiocarcinoma.

Bone-marrow–derived CXCR4-positive tissue-committed stem cell recruitment in human right ventricular remodeling - Corrected Proof

2010-07-12

Summary: The epicardium contributes to cardiac formation, particularly during embryogenesis. It remains to be seen if it is also involved in postnatal myocardial homeostasis. This study evaluates the topographic distribution of stem cells (c-Kit) and extracardiac progenitor cells (CXCR4+) and their contribution to ventricular remodeling in a model of pressure volume overload leading to right ventricle hypertrophy. Eleven specimens with hypoplastic left heart syndrome were evaluated and compared with 6 normal hearts from subjects matched for age and weight. All underwent Norwood procedure with the right ventricle becoming a systemic one, with pressure and volume overload leading to right ventricle remodeling. Transmural cardiac tissue samples from the right ventricle were analyzed by immunohistochemistry and morphometry. This is the first study to demonstrate that c-Kit–positive progenitor cells and tissue-committed stem cells (CXCR4+/CD45−) are higher in children with systemic right ventricle remodeling. We also show that the localization of cardiac progenitor and recruited CXCR4+ stem cells in the myocardium is site specific in hearts with right ventricle hypertrophy. These cells are mainly scattered in the interstitium of the epicardial layer. In contrast, myocyte proliferation is not a key process in right ventricular hypertrophy. Induced by the overexpression of SDF-1α by the myocardium, CXCR4 cell mobilization resembles SDF-1 homing factor distribution, showing transmural enhanced expression from the endocardium toward the epicardium. The study provides evidences of the site-specific epicardial localization of stem cells in a model of pressure/volume overload and suggests that the epicardium acts as a permissive niche in normal and pathologic conditions.

Diagnostic utility of dual-color break-apart chromogenic in situ hybridization for the detection of rearranged SS18 in formalin-fixed, paraffin-embedded synovial sarcoma - Corrected Proof

Toru Motoi, Arisa Kumagai, Kaori Tsuji, Tetsuo Imamura, Toshio Fukusato — 2010-07-02

Summary: Pathological diagnosis of synovial sarcoma is often problematic due to its broad spectrum of histology. Because synovial sarcoma consistently carries a specific chromosomal translocation, t(X;18), and its derivative chimeric gene, either SS18-SSX1 or SS18-SSX2, detecting these abnormalities by reverse transcription polymerase chain reaction or fluorescence in situ hybridization has been recognized as a powerful aid for diagnosis. Recently, chromogenic in situ hybridization, which enables simultaneous visualization of both genomic abnormality and the morphology of tumor cells, has gained attention. This study investigated the diagnostic utility of dual-color break-apart chromogenic in situ hybridization as a novel method for detecting SS18 rearrangement in synovial sarcoma. Formalin-fixed, paraffin-embedded tissue samples from 16 cases of synovial sarcoma and 10 cases of 5 other types of soft tissue sarcoma were collected. Dual-color break-apart probes were designed against the genomic region adjacent to SS18. Fluorescence and chromogenic in situ hybridization studies were performed using the same sections. In both assays, the number of signals was counted for sixty nuclei per sample. Scoring ratios (unpaired signals/paired signals) were calculated. Subsequently, SS18-SSX1 and SS18-SSX2 were examined by reverse transcription polymerase chain reaction. The results of chromogenic in situ hybridization, fluorescence in situ hybridization, and reverse transcription polymerase chain reaction were correlated. Unpaired signals were clearly observed in all the synovial sarcoma samples, which mostly indicated rearranged SS18. Synovial sarcoma and non-synovial sarcoma samples were clearly distinguished from each other by the scoring ratios. Reverse transcription polymerase chain reaction demonstrated SS18 chimeric gene transcripts in all the synovial sarcoma cases, while no fusion genes were detected in the non-synovial sarcoma cases. Taken together, unpaired signals in synovial sarcoma reflected rearranged SS18. The present chromogenic in situ hybridization-based SS18 rearrangement detection system provides a highly sensitive and specific method for the diagnosis of synovial sarcoma. Chromogenic in situ hybridization-based methods have great potential for routine use in the diagnosis of synovial sarcoma.

Merkel cell carcinoma: correlation of KIT expression with survival and evaluation of KIT gene mutational status - Corrected Proof

2010-07-02

Summary: Merkel cell carcinoma is one of the most aggressive primary cutaneous malignancies. Because some Merkel cell carcinomas express the receptor tyrosine kinase KIT, we aimed to evaluate the correlation of KIT expression with the outcome and the presence of activating mutations in the KIT gene in Merkel cell carcinoma. A total of 49 tumors from 40 patients with a diagnosis of Merkel cell carcinoma were identified, of which 30 cases from 21 patients were used in the study. KIT expression was assessed by immunohistochemistry on formalin-fixed, paraffin-embedded material. Cases were divided into low expressors (0-1+ staining intensity) and high expressors (2-3+ staining intensity). Direct sequencing of exons 9, 11, 13, 17, and 18 of the KIT gene spanning the extracellular, juxtamembrane, and tyrosine kinase domains was performed for cases with high KIT expression. Thirty tumors from 21 patients were analyzed for KIT expression. High KIT expression was seen in 67% of the patients. Five-year survival rates in tumors expressing high versus low levels of KIT were 0% versus 57.8%, respectively; however, this dramatic difference did not reach statistical significance (P = .07). A total of 4 point mutations were identified in 18 tumors analyzed. Two of these were silent mutations involving exons 17 and 18, and 2 involved intron 16-17. Two of the identified mutations may represent novel polymorphisms. Our work suggests a correlation between KIT expression and a worse prognosis in Merkel cell carcinoma patients, raising the possibility of an active role of this receptor in tumor progression and metastasis. However, we did not identify KIT activating mutations in any of the tumors analyzed.

Clinicopathologic study of 85 colorectal serrated adenocarcinomas: further insights into the full recognition of a new subset of colorectal carcinoma - Corrected Proof

2010-07-02

Summary: Colorectal serrated adenocarcinoma represents a subtype of colorectal carcinoma that originates from serrated adenomas. Previous studies have suggested a more aggressive course, but this has not been verified. The aim of this work was to test the diagnostic reproducibility of previously proposed histologic criteria for serrated adenocarcinoma and to analyze the clinicopathologic features and outcome that would warrant its recognition as a new subtype of colorectal cancer. Nine hundred twenty-seven consecutive colorectal cancer specimens were used to search for cases fulfilling the criteria of serrated adenocarcinoma and matched controls. Clinicopathologic findings of 85 serrated adenocarcinomas were compared with a matched control group of conventional cancers. Serrated adenocarcinomas were encountered in 9.1% (n = 85) of cases. Residual serrated adenoma was present in 44 (51.7%). Absence of residual adenoma did not have any influence on the parameters studied. Interobserver variation between 2 Spanish and a Finnish pathologist showed moderate agreement (κ = .5873). Compared with their matched controls, serrated adenocarcinomas were more often accompanied by synchronous residual serrated adenomas (P < .0001), remote serrated adenomas (P = .0035), and serrated adenocarcinomas or cancers representing partial features of these tumors (P = .002). They had a less favorable 5-year survival than conventional cancers (P = .048 Breslow, Kaplan-Meier), and left-sided ones had the worst prognosis (P = .001). Serrated adenocarcinoma is an identifiable subset of colorectal cancer; and the histopathologic differences, in addition to its less favorable prognosis, may justify its recognition as a distinct subset of colorectal cancer warranting the search for specific clinical management strategies.

Glioblastoma with signet-ring morphology—reply - Corrected Proof

Sarah E. Martin, Eyas M. Hattab — 2010-07-02

Pusztaszeri and Lobrinus raise an important point in their letter regarding our case report of a glioblastoma with signet-ring morphology. We agree that there is significant overlap between the designation of rhabdoid and signet-ring cell features, and a glioblastoma with rhabdoid features should be considered in the differential diagnosis for this case. However, we feel that the cells highlighted in this case appear strikingly epithelioid, more closely resembling a carcinoma than a rhabdoid tumor. Although the original “signet-ring” designation did refer to adenocarcinoma cells containing a droplet of mucin in the cytoplasm causing the nucleus to be displaced peripherally, the term was later expanded to encompass any cell with the light microscopic finding of a displaced nucleus and ballooning of the cell body, whether due to excessive accumulation of secretory products, metabolites, or hyperplastic organelles . The electron microscopy finding of bundles of intermediate filaments is nonspecific and has been identified in reported cases of signet-ring oligodendrogliomas . By using the terminology signet-ring morphology, we aimed to emphasize the importance of distinguishing such an entity from metastatic carcinoma.

Evidence for the role of matrix metalloproteinase-13 in bone resorption by giant cell tumor of bone - Corrected Proof

2010-06-23

Summary: Giant cell tumor of bone (GCT) is an aggressively osteolytic primary bone tumor that is characterized by the presence of abundant multinucleated osteoclast-like giant cells, hematopoietic monocytes, and a distinct mesenchymal stromal cell component. Previous work in our laboratory has shown that matrix metalloproteinase (MMP)-13 is the principal proteinase expressed by the stromal cells of GCT. The release of cytokines, particularly interleukin-1β, by the giant cells of GCT acts on stromal cells to stimulate a surge in MMP-13 secretion. The purpose of this study was to determine the bone resorption capabilities of the cellular elements of GCT and the significance of the MMP-13 expression involved in GCT bone resorption. We present a 3-dimensional histomorphometric technique developed to analyze resorption pit depth and yield an accurate measurement of bone resorption with a direct physical view of lacunae on bone slices. In this study, we demonstrate that the mesenchymal stromal cells and the multinucleated giant cells of GCT are independently capable of bone resorption. However, coculture of these 2 cell fractions shows a synergistic increase in bone resorption. In addition, inhibition of MMP-13 reduces resorptive activity of the cells indicating that MMP-13 likely plays an important role in this tumor. This cell-cell cooperation involves giant cell-derived cytokine up-regulation of MMP-13 in the stromal cells, which in turn assists the giant cells in bone resorption. Future research will involve elucidation of the role of cell-cell/matrix communication pathways in bone resorption and tumorigenesis in GCT.

Mesothelin (MSLN) promoter is hypomethylated in malignant mesothelioma, but its expression is not associated with methylation status of the promoter - Corrected Proof

2010-06-23

Summary: Gene methylation leads to malignant progression in some tumors. The mechanism by which mesothelin is expressed in malignant mesothelioma (MM) is not well understood. MM is histologically divided into 3 subtypes, that is, the epithelioid, sarcomatoid, and biphasic types, and it was shown that mesothelin expression was restricted to the epithelioid type and the epithelioid component of the biphasic type of MM. However, its regulatory mechanism of expression has not been clarified. Here, we studied the expression of mesothelin by immunohistochemistry along with the methylation status of 20 CpG sites in the promoter of the mesothelin gene (MSLN) in 118 lung specimens, including 39 MM, 41 lung carcinoma, 26 nonneoplastic pulmonary lesions, and 12 normal lung tissue samples by the methylation-sensitive single nucleotide primer extension technique. We confirmed that mesothelin was expressed in the epithelioid type and epithelioid component of the biphasic type of MM but neither in the sarcomatoid type nor sarcomatous component of the biphasic type. Surprisingly, the MSLN promoter was significantly hypomethylated in the MM cases regardless of its subtype, compared with the other pulmonary lesions and normal lung tissue samples. These findings suggested that hypomethylation of the MSLN promoter may be specifically associated with the formation of MM, regardless of its expression status, and that the expression of mesothelin protein was lost in the sarcomatoid type by some unknown posttranscriptional regulatory mechanism. We also identified 4 CpG sites, among the 20 sites studied, to be more specifically hypomethylated in MM cases.

Interobserver variability in the evaluation of mismatch repair protein immunostaining - Corrected Proof

2010-06-23

Summary: Immunohistochemical staining for mismatch repair proteins has during recent years been established as a routine analysis in many pathology laboratories with the aim to identify tumors linked to the hereditary nonpolyposis colorectal cancer syndrome. Despite widespread application, data on reliability are lacking. We therefore evaluated interobserver variability among 6 pathologists, 3 experienced gastrointestinal pathologists and 3 residents. In total, 225 immunohistochemically stained colorectal cancers were evaluated as having normal, weak, loss of, or nonevaluable mismatch repair protein staining. Full consensus was achieved in 51% of the stainings for MLH1, 61% for PMS2, 83% for MSH2, and 45% for MSH6. Weak stainings were the main cause of reduced consensus, whereas contradictory evaluations with normal as well as loss of staining were reported in 2% to 6% of the tumors. Interobserver variability was considerable, though experienced pathologists and residents reached the same level of consensus. Because results from immunohistochemical mismatch repair protein stainings are used for decisions on mutation analysis and as an aid in the interpretation of gene variants of unknown significance in hereditary nonpolyposis colorectal cancer, the interobserver variability identified highlights the need for quality assessment programs, including guidelines for classification of different expression patterns.

Implications of enhancer of zeste homologue 2 expression in pancreatic ductal adenocarcinoma - Corrected Proof

2010-06-23

Summary: Pancreatic ductal adenocarcinoma is the fourth leading cause of cancer deaths in the United States. Single-agent gemcitabine remains the standard treatment of advanced pancreatic adenocarcinoma. A recently discovered histone methyltransferase termed enhancer of zeste homologue 2 (EZH2) was found to be overexpressed in a variety of carcinomas including pancreatic adenocarcinoma. Silencing of E-cadherin was proposed as a mechanism by which enhancer of zeste homologue 2 mediates tumor aggressiveness, and enhancer of zeste homologue 2 depletion has been found to sensitize pancreatic cancer cells to gemcitabine. In this study, we correlated enhancer of zeste homologue 2 with E-cadherin expression in pancreatic adenocarcinoma and evaluated response to gemcitabine in relation to enhancer of zeste homologue 2 expression in tumor cells. Fifty-four pancreatic adenocarcinomas, 13 intraductal papillary mucinous neoplasms, and 6 chronic pancreatitis cases were stained with antibodies against enhancer of zeste homologue 2 and E-cadherin. Enhancer of zeste homologue 2 staining was scored from 1 to 4+ and classified as either low (1-2+ in <25% of tumor nuclei) or high (3-4+ in >25% of tumor nuclei). E-cadherin expression was scored on membrane positivity as follows: 0 (0%-10%), 1 (10%-25%), 2 (25%-75%), and 3 (>75%). High enhancer of zeste homologue 2 expression in pancreatic adenocarcinoma was significantly associated with decreased E-cadherin expression and more aggressive disease. There was significantly longer survival in gemcitabine-treated patients with low versus high enhancer of zeste homologue 2 expression. High enhancer of zeste homologue 2 expression was detected in intraductal papillary mucinous neoplasms with moderate to severe dysplasia, but not in chronic pancreatitis. Our study suggests that E-cadherin down-regulation may lead to enhancer of zeste homologue 2–mediated invasion and metastasis.

World Health Organization classification of thymomas provides significant prognostic information for selected stage III patients: evidence from an international thymoma study group - Corrected Proof

2010-06-23

Summary: Information regarding prognosis of thymoma patients stratified by both World Health Organization classification and Masaoka staging system is very limited. Analyze 5-year survival data from a large number of thymoma patients stratified by both World Health Organization histologic type and Masaoka stage using meta-analysis. Perform power analysis to estimate the number of cases that would be needed to test the null hypothesis to a power of 80%. Five-year survival data from 905 thymoma patients treated with thymectomy at seven hospitals in America, Japan, Korea, India, Italy, and Germany. Survival data was reported as “dead” or “alive” to facilitate meta-analysis. Significant differences were detected only when comparing survival rates of thymoma patients in stages I to III with those of stage IV disease. Analysis by World Health Organization histologic type and stage yielded significant differences only in patients with thymomas A vs. B2 and A vs. B3 in stage III disease. No significant data heterogeneity was detected with funnel plots and Egger's regression test. Power analysis estimated that a study with 7077 patients is needed to evaluate the prognostic significance of all thymomas stratified by both World Health Organization histologic type and stage to a power of 80%. Selected World Health Organization histologic types are significantly associated with prognosis in stage III thymoma patients and may help select individuals benefiting from neoadjuvant therapy. Power analysis shows that studies with much larger number of patients are needed to exclude the possibility that histologic type may provide significant prognostic information in other stages of the disease.

Prognostic impact of blood vessel invasion in gastrointestinal stromal tumor of the stomach - Corrected Proof

2010-06-23

Summary: Gastrointestinal stromal tumors have a wide spectrum of biologic behavior, and occasional cases show liver metastases. The modified risk grade based on tumor size and mitotic counts has been proposed to predict the biologic behavior in gastric gastrointestinal stromal tumors. Blood vessel invasion (BVI) is important in the development of metastasis of various kinds of cancer. The aim of this study was to elucidate the potential role of blood vessel invasion in gastric gastrointestinal stromal tumors. Blood vessel invasion was found in 17 of 122 cases (13.9%) of gastrointestinal stromal tumors, and was significantly correlated with larger tumor size, higher mitotic count and higher modified risk grade. Among 83 cases of primary, localized gastric gastrointestinal stromal tumors available for follow-up information, liver metastasis was observed in 14 cases (16.9%). When blood vessel invasion was positive in the primary tumor, liver metastasis occurred in 80% of cases after the initial surgery, indicating that blood vessel invasion was a significant risk factor of liver metastasis (P < .0001). In univariate and multivariate analyses, tumor size (>5 cm), mitotic count (>5/50 high-power fields) and blood vessel invasion (positive) were significantly associated with a shorter period of disease-free survival. Our results suggest that the evaluation of blood vessel invasion may be useful for predicting the risk of liver metastasis and aggressive biologic behavior of gastrointestinal stromal tumors, and may serve as important information for determining the therapeutic strategies including adjuvant molecular target therapy.

Rete testis invasion by malignant germ cell tumor and/or intratubular germ cell neoplasia: what is the significance of this finding? - Corrected Proof

Adam P. Vogt, Zhengjia Chen, Adeboye O. Osunkoya — 2010-06-23

Summary: Pathologic stage and postsurgical treatment guidelines of malignant germ cell tumors, currently take into account angiolymphatic invasion, degree of extra testicular invasion, and serum tumor marker levels. The significance of rete testis invasion by malignant germ cell tumors or intratubular germ cell neoplasia however remains controversial. A search through the surgical pathology and expert consultation files at our institution from 2002 to 2009 was made for malignant germ cell tumors and intratubular germ cell neoplasia in orchiectomy specimens. Clinicopathologic data including rete testis status were obtained. Two hundred ninety-two orchiectomy specimens were identified. One hundred thirty-six were associated with malignant germ cell tumors. Mean patient age was 33 years (range, 14-67 years). The mean greatest tumor dimension was 4.1 cm (range, 0.8-18 cm). Fifty-six were pure seminoma (40%), 50 were nonseminomatous malignant germ cell tumors (35%), and 35 were mixed malignant germ cell tumors including a seminoma component (25%). Intratubular germ cell neoplasia was identified in 99 cases (70%). Pathologic stage at presentation was as follows: stage 1, 71 patients (50%); stage 2, 62 patients (45%); stage 3, 2 patients (1%); and indeterminate, 6 patients (4%). Seventy-eight patients had documented rete testis status: rete testis invasion, 41 (53%); no rete testis invasion, 37 (47%). Angiolymphatic invasion was present in 62 cases (44%). Follow-up information was available in 43 patients with known rete testis status. Mean follow-up duration was 43 months (range, 3-65 months). Twenty patients had rete testis invasion, and 23 patients had no rete testis invasion. Intratubular germ cell neoplasia was present in patients with rete testis invasion in 18 cases (90%), compared to only 13 cases (57%) in patients without rete testis invasion, P = .02. Serum markers were elevated in 10 patients (50%) with rete testis invasion compared to only 6 patients (26%) without rete testis invasion, P = .05. The combination of rete testis invasion and angiolymphatic invasion were present in 8 cases and were found to be associated with elevated serum tumor markers in 7 (88%) of the 8 cases, compared to the combination of no invasion of the rete testis and angiolymphatic invasion showing elevated serum tumor markers in 3 (38%) of 8 cases. However, 7 patients (35%) with rete testis invasion developed metastatic disease, and 11 patients (48%) without rete testis invasion developed metastatic disease. Rete testis status should be documented in orchiectomy specimens with malignant germ cell tumors. Intratubular germ cell neoplasia may be the only component of a malignant germ cell tumor involving the rete testis. In this series, elevated tumor markers were more likely associated with angiolymphatic invasion and positive rete testis status. Positive rete testis status does not appear to be an independent predictor of patient outcome.

Mutational and expressional analysis of RFC3, a clamp loader in DNA replication, in gastric and colorectal cancers - Corrected Proof

2010-06-23

Summary: Parts of gastric (GC) and colorectal cancers (CRC) exhibit microsatellite instability (MSI) that causes frameshift mutations and contributes to cancer development. DNA replication and repair play crucial roles in maintenance of genome stability, and their alterations contribute to cancer development. In this study, we analyzed mutation of RFC1 and RFC3, clamp loaders in DNA replication, in GC and CRC with MSI. We analyzed mononucleotide repeats in RFC1 and RFC3 in 29 GC with high MSI (MSI-H), 20 GC with low MSI (MSI-L), 45 GC with stable MSI (MSS), 35 CRC with MSI-H, 20 CRC with MSI-L, and 45 CRC with MSS by single-strand conformation polymorphism. We also analyzed RFC3 expression in the GC and CRC. We found RFC3 frameshift mutations in 7 GC (24.1%) and 9 CRC with MSI-H (25.7%) but not in cancers with MSI-L or MSS. The mutations consisted of 14 c.244delA, one 243_244delAA, and one c.244dupA, which would result in premature stops of RFC3 amino acid synthesis. Loss of RFC3 expression was observed in 51% of the GC and 65% of the CRC, but all of the cancers with RFC3 frameshift mutations were weak or negative. Our data indicate RFC3 mutation and loss of RFC3 expression occur in large fractions of GC and CRC and suggest that these alterations may contribute to the cancer pathogenesis by deregulating DNA repair and replication.

Oligoarray comparative genomic hybridization of renal cell tumors that developed in patients with acquired cystic renal disease - Corrected Proof

Eva Kuntz, Maria V. Yusenko, Anetta Nagy, Gyula Kovacs — 2010-06-21

Summary: Renal cell carcinoma occurs at higher frequency in acquired cystic renal disease than in the general population. We have analyzed 4 tumors obtained from the kidneys of 2 patients with acquired cystic renal disease, including 2 conventional renal cell carcinomas and 2 acquired cystic renal disease–associated tumors, for genetic alterations. DNA changes were established by applying the 44K Agilent Oligonucleotide Array-Based CGH (Agilent Technologies, Waldbronn, Germany), and mutation of VHL gene was detected by direct sequencing of the tumor genome. DNA losses and mutation of the VHL gene, which are characteristic for conventional renal cell carcinomas, were seen in 2 of the tumors. The acquired cystic renal disease–associated eosinophilic-vacuolated cell tumor showed gain of chromosomes 3 and 16. No DNA alterations occurred in the papillary clear cell tumor. We suggest that not only the morphology but also the genetics of renal cell tumors associated with acquired cystic renal disease may differ from those occurring in the general population.

Expression analysis of Ubc9, the single small ubiquitin-like modifier (SUMO) E2 conjugating enzyme, in normal and malignant tissues - Corrected Proof

2010-06-21

Summary: Unlike ubiquitination, which targets proteins for degradation, sumoylation modulates protein-protein interactions of target proteins. Although there are multiple E2 enzymes required for ubiquitination, there is only one E2-conjugating enzyme for sumoylation, which is Ubc9. In line with increasing evidence that sumoylation plays an important role in tumorigenesis, we recently demonstrated that Ubc9 is expressed at high levels in advanced melanomas and that blocking expression of Ubc9 sensitizes melanomas to the cytotoxic effects of chemotherapeutic drugs. To determine whether and to what extent Ubc9 is expressed in other malignancies and their normal tissue counterparts, we undertook a detailed analysis of colon, lung, prostate, and breast cancer tissue microarrays. The findings, presented here, document that in primary colon and prostate cancer, Ubc9 expression is increased compared with their normal tissue counterparts, whereas in metastatic breast, prostate, and lung cancer, it is decreased in comparison with their corresponding normal and primary adenocarcinoma tissues. We also provide evidence that Ubc9 expression correlates positively with Dukes' stage and negatively with the Gleason score as well as breast cancer grade and that Ubc9 expression is substantially higher in the luminal than in the nonluminal type of breast cancer.

Localization of nephritis-associated plasmin receptor in acute poststreptococcal glomerulonephritis - Corrected Proof

2010-06-18

Summary: The nephritis-associated plasmin receptor is a recently identified nephritogenic antigen associated with acute poststreptococcal glomerulonephritis and proposed to play a pathogenic role, but its precise glomerular localization in acute poststreptococcal glomerulonephritis has not been elucidated. We therefore analyzed renal biopsy sections from 10 acute poststreptococcal glomerulonephritis patients by using immunofluorescence staining with anti–nephritis-associated plasmin receptor antibody and various markers of glomerular components. Nephritis-associated plasmin receptor was detected in the glomeruli of all patients, and double staining for nephritis-associated plasmin receptor and collagen IV showed nephritis-associated plasmin receptor to be predominantly on the inner side of the glomerular tufts. Nephritis-associated plasmin receptor–positive areas within glomerular tufts were further characterized with markers for neutrophils, mesangial cells, endothelial cells, and macrophages. In 6 of the patients, nephritis-associated plasmin receptor staining was seen mainly in neutrophils and to a lesser degree in mesangial and endothelial cells. In the other 4 patients, nephritis-associated plasmin receptor staining was seen mainly in mesangial cells and to a lesser degree in neutrophils and endothelial cells. In all patients, macrophages showed little staining. Elevated plasmin activity in glomerular neutrophils was identified by combining in situ zymography staining for plasmin activity and immunofluorescence staining for neutrophils. The glomerular localizations of nephritis-associated plasmin receptor and another nephritogenic antigen, streptococcal pyrogenic exotoxin B, were compared by double immunofluorescence staining and found to be similar. These findings indicate the nephritogenic potential of nephritis-associated plasmin receptor and offer valuable information with respect to the pathogenic mechanism of acute poststreptococcal glomerulonephritis.

Squamous cell carcinoma arising in a communicating bronchopulmonary-foregut malformation - Corrected Proof

2010-05-31

Summary: Communicating bronchopulmonary-foregut malformation, a variant of bronchopulmonary sequestration, is a rare anomaly characterized by communication between an isolated portion of the respiratory tree and the gastrointestinal tract. We report herein a unique case involving a 43-year-old man with squamous cell carcinoma arising in communicating bronchopulmonary-foregut malformation. This patient had a workup for a chief complaint of exacerbation of constitutional dysphagia, resulting in detection of squamous cell carcinoma involving the lower esophagus. Under the clinical diagnosis of esophageal carcinoma, esophagectomy was performed after neoadjuvant chemoradiotherapy. Pathologic findings showed that squamous cell carcinoma had arisen in malformed bronchopulmonary tissue constituting part of the distal esophagus segmentally. This case was unique in that squamous cell carcinoma developed in an extremely rare type of congenital abnormality that had functioned as a passageway for food from birth, as a result of chronic irritation for more than 4 decades.

Clonal analysis of bilateral, recurrent, and metastatic papillary thyroid carcinomas - Corrected Proof

2010-05-17

Summary: Papillary thyroid carcinoma usually presents as a multifocal disease; and tumors often recur in the contralateral thyroid lobe, raising questions concerning their clonal origins. The clonality of tumors appearing simultaneously in both lobes or recurring in the contralateral lobe remains unknown. Accordingly, we examined 25 pairs of bilateral papillary thyroid carcinomas (synchronous or metachronous) and 15 matched metastatic lymph nodes. BRAF gene mutation analysis combined with X-chromosome inactivation was used to evaluate these tumors' clonal origins. Genomic DNA was prepared from paraffin-embedded tissues after microdissection. In total, 62 tumors yielded DNA of adequate quality. Eighteen (18/21, 85.7%) of 21 informative cases showed concordant BRAF status in tumors from both thyroid lobes, being either BRAF mutation positive (12 patients) or BRAF mutation negative (6 patients). Metastatic lymph nodes in 12 patients (12/15, 80%) had a complete concordance of BRAF state with their primaries. Of the 18 studied female patients, 11 were suitable for X-chromosome inactivation assay. Nine cases (9/11, 81.1%) showed the same pattern of inactivation in bilateral tumors. A close correlation was found between BRAF mutation and X-chromosome inactivation analysis. In conclusion, our data provide evidence that bilateral, recurrent, and metastatic papillary thyroid carcinomas often arise from a single clone and that intrathyroidal metastasis may play an important role in the development of bilateral tumors, as well as in the recurrence of this malignancy.

Agrin immunohistochemistry facilitates the determination of primary versus metastatic origin of liver carcinomas - Corrected Proof

2010-05-17

Summary: In our earlier work, we demonstrated that agrin, a multifunctional heparan sulfate proteoglycan, accumulates in hepatocellular carcinoma (HCC) and cholangiocellular carcinoma (CCC). In addition, we proved the utility of agrin immumohistochemistry in discriminating between HCCs and benign parenchymal lesions. Here, we have examined the expression of agrin in metastatic liver carcinomas in comparison with primary liver tumors. Immunohistochemistry for agrin was performed on 25 HCC, 16 intrahepatic CCC, 20 colorectal cancer metastasis (CRCm), and 18 pancreatic ductal carcinoma metastasis (PDCm) samples and evaluated with both quantitative and qualitative methods. Agrin/CD34 double immunofluorescent staining was carried out on snap-frozen sections. Agrin mRNA expression was measured in 11 HCC, 7 CCC, 11 CRCm, and 12 normal liver tissues. Regardless of tumor grade, agrin immunostaining was strong in the microvessels of HCCs. As opposed to HCC, agrin immunostaining was faint or nearly absent from the CD34-positive microvessels of CCC, CRCm, and PDCm; rather, it was detected in the basement membranes surrounding tumor cell pseudoglandules. While agrin was preserved in the basement membranes of Grade III CCCs, it was nearly absent from poorly differentiated metastatic adenocarcinomas. Agrin mRNA levels were the highest in CCC and lower, but still elevated in HCC and CRCm. By qualitative evaluation of agrin immunoreactions, CCC was differentiated from CRCm and PDCm with a sensitivity of 0.81 and a specificity of 0.82. HCCs were unequivocally identified on the basis of microvascular agrin labeling. Thus, agrin immunohistochemistry may facilitate determination of primary versus metastatic origin in problematic liver cancer cases.

Involvement of inactive GSK3β overexpression in tumorigenesis and progression of gastric carcinomas - Corrected Proof

Hua-chuan Zheng, Xiao-yan Xu, Pu Xia, Miao Yu, Hiroyuki Takahashi, Yasuo Takano — 2010-05-06

Summary: Glycogen synthase kinase 3β is reported to repress Wnt/β-catenin pathway and regulate the balance between cellular proliferation and apoptosis. Its inactivation by ser9 phosphorylation might play a critical role in the tumorigenesis and development of malignancies. Here, the expression of phosphorylated glycogen synthase kinase 3β at ser9 was examined in gastric carcinoma and adjacent non-neoplastic mucosa by immunohistochemistry and Western blot, and compared with the clinicopathological parameters of carcinomas, including the expression of vascular endothelial growth factor, extracellular matrix metalloproteinase inducer and β-catenin, and microvessel density labeled by CD34, as well as survival data. Gastric carcinoma cell lines (MKN28, MKN45, AGS, GT-3 TKB, KATO-III and HGC-27) were studied for the phosphorylated kinase by Western blot and for its encoding mRNA by RT-PCR, followed by sequencing. All carcinoma cell lines showed strong expression of the phosphorylated kinase and its encoding mRNA.There were two isoforms of glycogen synthase kinase 3β in all carcinoma cell lines and a synonymous mutation in HGC-27 carcinoma cell line at codon 65(GGA→GGT: Gly). The phosphorylated kinase was localized in the cytoplasm of gastric carcinoma cell lines or carcinomas. It was more expressed in gastric carcinomas than that in non-neoplastic mucosa (P < .05) in line with the data of Western blot. There was a higher expression of the phosphorylated kinase in men carcinoma patients than that in women (P < .05). Its expression was positively correlated with depth of invasion, lymphatic and venous invasion, lymph node metastasis, Union Internationale le Contre Cancer staging, expression of vascular endothelial growth factor and extracellular matrix metalloproteinase inducer in gastric carcinoma (P < .05). Survival analysis indicated the phosphorylated kinase expression to be positively linked to poor prognosis (P< 05), but not independent (P>.05). Three independent prognostic factors, depth of invasion, lymphatic and venous invasion, concordantly influenced its relationship with prognosis (P < .05). Our study indicated that up-regulated expression of phosphorylated glycogen synthase kinase 3β at ser9 was closely linked to gastric carcinogenesis and subsequent progression, and could be employed as a good indicator of aggressive behaviors and prognosis of gastric carcinoma.

Podocyte membrane vesicles in urine originate from tip vesiculation of podocyte microvilli - Corrected Proof

2010-05-06

Summary: Podocyte injury is involved in both the onset and progression of glomerular diseases. Our previous studies revealed that apical cell membranes of podocyte are shed into urine sediment and that urinary podocalyxin is a useful biomarker of podocyte injury. In this study, we examined the origin of urinary podocalyxin. Urine samples and kidney specimens from healthy children (n = 126) and patients with glomerular diseases (n = 77) were analyzed by immunohistologic methods. Immunofluorescence studies demonstrated that urinary podocalyxin was shed as granular structures into both the urine sediment and supernatant. Large amounts of podocalyxin were shed into both the urine sediment (17.2 ± 3.2 ng/mg creatinine) and the supernatant (172.6 ± 24.6 ng/mg creatinine) of patients, compared with the small amounts of urinary podocalyxin in healthy controls (sediment, 0.5 ± 0.1 ng/mg creatinine; supernatant, 24.3 ± 3.5 ng/mg creatinine). Electron and immunoelectron microscopic examinations showed that podocalyxin-positive vesicles in the sediment (125.6 ± 8.8 nm) and the supernatant (121.2 ± 6.4 nm) were similar in size to podocyte microvilli in biopsy specimens (123.6 ± 8.9 nm), differentiating them from the much smaller urine exosomes (30-80 nm in diameter). Urine podocalyxin-positive vesicles tested negative in immunofluorescence microscopy on both exosomal markers CD24 and CD63. Podocalyxin-positive vesicles also tested negative for cytoskeletal markers, and electron microscopic examination revealed tip vesiculation of microvilli. We conclude that human urinary apical cell membrane vesicles appear to originate not from podocyte exosomes but from tip vesiculation of glomerular podocyte microvilli.

The cutaneous lesions of Kikuchi's disease: a comprehensive analysis of 16 cases based on the clinicopathologic, immunohistochemical, and immunofluorescence studies with an emphasis on the differential diagnosis - Corrected Proof

Jang Hee Kim, Young Bae Kim, Sung Il In, You Chan Kim, Jae Ho Han — 2010-04-30

Summary: Kikuchi's disease is a self-limited necrotizing lymphadenitis that is characterized by cervical lymphadenopathy and fever. Although it has been reported that some patients with Kikuchi's disease have cutaneous manifestations, the specific skin changes of patients with Kikuchi's disease have not been fully described. We report here on 16 patients of Kikuchi's disease with cutaneous manifestations. We reviewed the clinical histories of the patients who underwent lymph node and skin biopsies. Immunohistochemistry, immunofluorescence, and Epstein-Barr virus-encoded RNA (EBER) in situ hybridization were performed. The patients ranged in age from 7 to 39 years and included 4 males and 12 females. All the patients had histiocytic necrotizing lymphadenitis. The clinical impression was variable according to the various cutaneous manifestations. The skin biopsies showed vacuolar interface changes (12/16; 75.0%), necrotic keratinocytes (11/16; 68.8%), superficial (16/16; 100.0%) and deep (9/16; 56.3%) lymphohistiocytic infiltration, karyorrhexis (16/16; 100.0%), deposition of mucin (5/16; 31.3%), and panniculitis (9/15; 60.0%). Based on immunohistochemistry, the infiltrating cells were predominantly CD68 and CD163-positve histiocytes and CD3-positive T lymphocytes. Of the 16 patients, 13 (81.3%) had a slight predominance of CD8-positive lymphocytes. Direct immunofluorescence staining and EBER in situ hybridization were all negative. Although the clinical and histopathologic findings are very heterogenous, the presence of a lymphohistiocytic infiltration with nonneutrophilic karyorrhexis helps to make the diagnosis of Kikuchi's disease with skin involvement.

Enteropathy-associated T-cell lymphoma—a clinicopathologic and array comparative genomic hybridization study - Corrected Proof

2010-04-19

Summary: According to the new World Health Organization classification system, there are 2 types of enteropathy-associated T-cell lymphoma. Type 1 is associated with celiac disease and accounts for the majority of cases in Western countries, whereas type 2 is not associated with celiac disease. To characterize enteropathy-associated T-cell lymphoma types in Korea, we carried out clinicopathologic and immunophenotypic analyses of 8 Koreans with enteropathy-associated T-cell lymphoma and investigated genomic profile using array comparative genomic hybridization. The tumors involved the small intestine in 5 patients and the colorectum in 3 patients. Two patients carried an HLA DQB1⁎0302 allele that corresponds to HLA DQ8. None of the patients had gluten-sensitive malabsorption syndrome. Intraepithelial lymphocytosis was observed in all patients. The sizes of the tumor cells were small or small-to-medium in 7 cases and medium-to-large in 1 case. The immunophenotypes of the tumor cells were CD4−CD8+CD56+ in 4 cases, CD4−CD8+CD56− in 1 case, CD4−CD8−CD56+ in 2 cases, and CD4−CD8−CD56− in 1 case. Array comparative genomic hybridization analysis showed that chromosome 9q33-q34.1 gain was present in 4 (80%) of the 5 cases examined. Other recurrent genomic alterations were gain of 6p21.1-21.31 (3/5, 60%), gain of 19q (2/5), and the loss of 3p12.1-p12.2 (2/5) and 3q26.31 (2/5). These results suggest that the most prevalent type of enteropathy-associated T-cell lymphoma in this geographic region is type 2, and the genetic changes associated with it are similar to those in Western countries.

Galectin-1 is a powerful marker to distinguish chondroblastic osteosarcoma and conventional chondrosarcoma - Corrected Proof

2010-04-19

Summary: The clinical management of osteosarcoma differs significantly from that of chondrosarcoma; therefore, it is extremely important to diagnose these 2 types of bone tumor accurately. In the absence of a specific marker, differential diagnosis by histochemistry is sometimes impossible, especially between chondroblastic osteosarcoma and conventional chondrosarcoma. We analyzed 165 bone sarcomas by immunohistochemical staining of tissue microarrays for expression of the galectin-1 (GAL1) lectin and by Western blot experiments. We found that GAL1 was abundant in normal human osteoblasts from benign proliferations and in osteosarcomas, including chondroblastic osteosarcomas, but not in chondrosarcomas. There was a highly significant statistical difference in the percentage of stained cells (P < 10−4) and in the staining intensity (P < 10−3) of chondroblastic osteosarcomas compared to conventional chondrosarcomas. This discriminatory potential of GAL1 staining for osteosarcoma-derived tumors was confirmed by Western blotting. We propose a diagnostic test for bone tumors that takes into account the optimal discriminative values for the percentage of cells stained and the intensity of staining. The positive and negative predictive values were 85.7% (trust interval of 63.7%-97%) and 90% (trust interval of 80%-95.9%), respectively, demonstrating the pertinence of the test. Altogether, our data indicate that GAL1 is a powerful diagnostic marker that distinguishes chondroblastic osteosarcomas from conventional chondrosarcomas.

S100P, von Hippel-Lindau gene product, and IMP3 serve as a useful immunohistochemical panel in the diagnosis of adenocarcinoma on endoscopic bile duct biopsy - Corrected Proof

Mary Levy, Fan Lin, Haodong Xu, Deepti Dhall, Betsy O. Spaulding, Hanlin L. Wang — 2010-04-12

Summary: Histopathologic distinction between benign and malignant bile duct epithelial lesions on endoscopic biopsies can be extremely challenging because of limited material, crush artifact, and compounding inflammatory and/or reactive changes particularly after stent placement. In this study, a total of 72 endoscopic bile duct biopsies, including 40 adenocarcinomas and 32 benign cases, were immunohistochemically examined for the expression of S100P, von Hippel-Lindau gene product (pVHL), and IMP3 to evaluate their diagnostic value. The results showed that 36 adenocarcinomas (90%) exhibited strong nuclear and cytoplasmic staining for S100P, of which 30 (83.3%) showed diffuse immunoreactivity. Intermediate to strong cytoplasmic staining for IMP3 was demonstrated in 31 tumors (77.5%) (15 diffuse, 16 focal). Completely negative staining for pVHL was observed in 37 adenocarcinomas. In the remaining 3 tumors, focal (1) or diffuse (2) membranous and cytoplasmic pVHL immunoreactivity was detected. Twenty-eight tumors (70%) showed a S100P+/IMP3+/pVHL− staining pattern, 6 (15%) with a S100P+/IMP3−/pVHL− pattern, and 2 (5%) with a S100P−/IMP3+/pVHL− pattern. All 32 benign biopsies were completely negative for IMP3 with the exception of 2 cases with focal dysplasia where focal immunoreactivity was observed. Thirty benign biopsies (93.8%) were positive for pVHL with a diffuse staining pattern observed in 28 cases (93.3%). Eight benign biopsies (25%) showed focal S100P positivity. Twenty-two benign biopsies (68.8%) displayed a S100P−/IMP3−/pVHL+ staining pattern. In conclusion, an immunohistochemical panel consisting of S100P, pVHL, and IMP3 can be helpful in distinguishing adenocarcinoma from reactive epithelial changes on challenging bile duct biopsies. The findings of focal S100P and/or IMP3 expression with reciprocal loss of pVHL immunoreactivity in a few benign biopsies suggest a use of these markers in the detection of early epithelial dysplasia that may be beyond histologic recognition.

T-cell leukemia 1 expression in nodal Epstein-Barr virus–negative diffuse large B-cell lymphoma and primary mediastinal B-cell lymphoma - Corrected Proof

Gabriela Gualco, Lawrence M. Weiss, Glen N. Barber, Carlos E. Bacchi — 2010-04-12

Summary: The physiologic expression of the product of the proto-oncogene TCL1 (T-cell leukemia 1) is primarily restricted to early embryonic cells. In nonneoplastic B cells, the expression of TCL1 is determined by the differentiation step with silencing at the germinal center stage. TCL1 protein is overexpressed in a wide variety of human diseases. It has been shown that TCL1 is a powerful B-cell oncogene, which has been implicated in the pathogenesis of various types of mature B-cell lymphomas. There is no comparative information in the literature addressing the expression of TCL1 in pediatric and adult nodal diffuse large B-cell lymphoma or primary mediastinal large B-cell lymphoma. We studied 55 cases of adult and pediatric diffuse large B-cell lymphoma and primary mediastinal large B-cell lymphoma to analyze the phenotypic profile of these lymphomas, including TCL1 expression, and its relationship with clinical outcome in different age groups. The cases were analyzed by immunohistochemistry for the expression of TCL1, CD10, BCL-2, BCL-6, and MUM1. We also evaluated c-MYC translocation by fluorescence in situ hybridization. TCL1 was observed in 11 cases, 5 pediatric and 6 adult cases, all but one diffuse large B-cell lymphoma. Pediatric cases showed a significant association between TCL1 expression, high proliferative index, and presence of c-MYC translocation. TCL1 positivity was predominantly found in germinal center phenotype diffuse large B-cell lymphoma. Overall survival was worse in adult TCL1-positive cases than pediatric ones. Primary mediastinal large B-cell lymphomas infrequently expressed TCL1 in both age groups.