Epstein-Barr virus-positive mucocutaneous ulcer resulting in severe methotrexate intoxication: a case report.

BACKGROUND: Epstein-Barr virus-positive mucocutaneous ulcer is one of the mature B-cell lymphoproliferative diseases occurring in patients with immune dysfunction including those with immunosuppressive treatment such as methotrexate. CASE PRESENTATION: A Japanese elderly man in his 80s with rheumatoid arthritis on methotrexate was admitted to our hospital complaining persistent pharyngeal pain. Laboratory tests revealed severe pancytopenia, elevated C-reactive protein, and increased creatinine levels. An otolaryngological examination showed ulceration of the right tonsil, from which diagnostic biopsy was performed. The diagnosis of Epstein-Barr virus-positive mucocutaneous ulcer was made and bone marrow aspiration revealed hypocellularity and megaloblastic changes. Pancytopenia was improved after discontinuing methotrexate, and repeated bone marrow aspiration test revealed recovery of normal cellularity and disappearance of dysplasia, confirming the diagnosis of methotrexate intoxication. Tonsil ulcer was improved only with discontinuation of methotrexate, which strongly supported the diagnosis of EBV-MCU. CONCLUSION: Our case suggested that even this best prognosis form of lymphoproliferative disease could lead to fatal complications if not appropriately managed.

Complete remission of primary membranous nephropathy following hepatitis E infection.

Primary membranous nephropathy (PMN) is a major cause of nephrotic syndrome in adults. Studies have shown that one-third of PMN cases undergo spontaneous remission, among which are some cases of infection-related complete remission. Herein, we report the case of a 57-year-old man who achieved complete remission of PMN shortly after the onset of acute hepatitis E infection. At the age of 55 years, the patient developed a nephrotic syndrome, and renal biopsy revealed membranous nephropathy, Ehrenreich-Churg stage 1. Treatment with prednisolone (PSL) reduced urinary protein from 7.8 g/gCre to approximately 1 g/gCre but did not lead to complete remission. However, 7 months after starting treatment, he developed an acute hepatitis E infection after consuming wild boar meat. Immediately after the onset of acute hepatitis E, the patient's urinary protein levels decreased to < 0.3 g/gCre. The PSL dose was subsequently reduced and discontinued after 2 years and 8 months, and complete remission was maintained thereafter. We considered that an increase in the number of regulatory T cells (Tregs) caused by acute hepatitis E infection was associated with PMN remission in this patient.

Surgical resection of late solitary locoregional gastric cancer recurrence in stomach bed.

BACKGROUND: Late-onset and solitary recurrence of gastric signet ring cell (SRC) carcinoma is rare. We report a successful surgical resection of late solitary locoregional recurrence after curative gastrectomy for gastric SRC carcinoma. CASE REPORT: The patient underwent total gastrectomy for advanced gastric carcinoma at age 52. Seven years after the primary operation, he visited us again with sudden onset of abdominal pain and vomiting. We finally decided to perform an operation, based on a diagnosis of colon obstruction due to the recurrence of gastric cancer by clinical findings and instrumental examinations. The laparotomic intra-abdominal findings showed that the recurrent tumor existed in the region surrounded by the left diaphragm, colon of splenic flexure, and pancreas tail. There was no evidence of peritoneal dissemination, and peritoneal lavage fluid cytology was negative. We performed complete resection of the recurrent tumor with partial colectomy, distal pancreatectomy, and partial diaphragmectomy. Histological examination of the resected specimen revealed SRC carcinoma, identical in appearance to the previously resected gastric cancer. We confirmed that the intra-abdominal tumor was a locoregional gastric cancer recurrence in the stomach bed. The patient showed a long-term survival of 27 months after the second operation. CONCLUSIONS: In the absence of effective alternative treatment for recurrent gastric carcinoma, surgical options should be pursued, especially for late and solitary recurrence.

Fluorescence in situ hybridization analysis with a tissue microarray: 'FISH and chips' analysis of pathology archives.

Practicing pathologists expect major somatic genetic changes in cancers, because the morphological deviations in the cancers they diagnose are so great that the somatic genetic changes to direct these phenotypes of tumors are supposed to be correspondingly tremendous. Several lines of evidence, especially lines generated by high-throughput genomic sequencing and genome-wide analyses of cancer DNAs are verifying their preoccupations. This article reviews a comprehensive morphological approach to pathology archives that consists of fluorescence in situ hybridization with bacterial artificial chromosome (BAC) probes and screening with tissue microarrays to detect structural changes in chromosomes (copy number alterations and rearrangements) in specimens of human solid tumors. The potential of this approach in the attempt to provide individually tailored medical practice, especially in terms of cancer therapy, is discussed.

Copy number estimation algorithms and fluorescence in situ hybridization to describe copy number alterations in human tumors.

The platforms of high-resolution genetic analysis of human tumors have become popular, and several copy number estimation algorithms have been applied to the data generated by single-nucleotide polymorphism microarrays. Although comparisons have been made between several different platforms or methodologies, there has never been a robust comparison of different copy number estimation algorithms, and the validity of the estimations in comparison with multiple fluorescence in situ hybridization (FISH) data in tumors has rarely been addressed. In the present study the dataset that the Affymetrix 250K Nsp array generated in two cancer cases was used to compare the two widely used algorithms for estimating copy number alterations (CNA): the genotyping microarray-based copy number variation (CNV) analysis (GEMCA) algorithm and the copy number analyzer for Affymetrix Genechip mapping (CNAG) algorithm. Considerable differences were noticed between the estimations by these two algorithms, because of the difference in the formula used to calculate the threshold values. Both algorithms yielded highly consistent data with the FISH results, but CNAG was more stringent for detecting loss. There were areas in which both algorithms provided gains, but FISH showed no change. It will be interesting to pursue the reasons for these remaining discrepancies.

Inhibitory Effects of S-carboxymethylcystein on Goblet Cell Proliferation in Cultured Epithelium.

BACKGROUND: The influence of S-carboxymethylcystein (S-CMC) on the proliferation ability of goblet cells in nasal polyp epithelium in response to inflammatory stimulation was examined. METHODS: The subjects were patients with chronic paranasal sinusitis. An epithelial cell culture system was established using nasal polyp mucosa excised during endoscopic paranasal sinus surgery. The samples were divided into 4 groups (group a: control group, group b: 10 ng/mL tumor necrosis factor-α (TNF-α) treatment group, group c: 10-7 M S-CMC and 10 ng/mL TNF-α treatment group, group d: 10-5 M S-CMC and 10 ng/mL TNF-α treatment group). The total number of epithelial cells and number of goblet cells were measured under a microscope, and the ratio of goblet cells to the total number of epithelial cells was calculated. RESULTS: In group b, 10 ng/mL of TNF-α significantly increased the number of goblet cells compared with group a, suggesting involvement of TNF-α in goblet cell proliferation. In addition, the number of goblet cells significantly decreased in group d compared with that in group b, and it also decreased in group c compared with that in group b, although the difference was not significant, and the decrease was smaller than that in group d, suggesting that S-CMC inhibited goblet cell proliferation in a concentration-dependent manner. CONCLUSION: TNF-α promoted goblet cell proliferation in nasal polyps, suggesting its influence on nasal polyp formation. As S-CMC inhibited inflammatory stimulation-induced goblet cell proliferation in nasal polyp epithelium, it may be useful for the treatment of sinusitis.

Effect of loss of heterozygosity of the c-kit gene on prognosis after hepatectomy for metastatic liver gastrointestinal stromal tumors.

The authors have previously reported that loss of heterozygosity (LOH) of the c-kit gene could be responsible for the gain in high proliferative activity in some gastrointestinal stromal tumors (GIST), resulting in enhanced metastatic potential. In the present study, an attempt was made to identify the factors that might predict the postoperative prognosis of patients with metastatic liver GIST. The clinicopathologic or genetic features of resected liver GIST in 14 patients who had undergone a hepatectomy for metachronous liver metastases and who had not received adjuvant imatinib treatment were examined. LOH of the c-kit gene was observed in seven of 12 metastatic liver GIST (58.3%), of which DNA suitable for testing could be extracted. Ten patients had recurrence after hepatectomy and four had none. The median post-recurrent disease-free survival (PRDFS) after hepatectomy was 27.5 months (range 8-104). The tumor-specific PRDFS was examined using clinicopathologic features, c-kit mutation and LOH of the c-kit gene. No single clinicopathologic or genetic finding was significantly associated with PRDFS. However, patients with 'Ki67 labeling index <5% and LOH(-)' had a significantly longer PRDFS than those with 'Ki67 >/=5% or LOH(+)' (P = 0.032), and there was no correlation between the presence of LOH of the c-kit gene and the Ki67 labeling index. LOH of the c-kit gene in metastatic liver seems to be a common event, and LOH of the c-kit gene in resected liver GIST may be a helpful factor in the prediction of the post-recurrent prognosis of patients with liver metastasis.

Usefulness of Color Coding Resected Samples from a Pancreaticoduodenectomy with Tissue Marking Dyes for a Detailed Examination of Surgical Margin Surrounding the Uncinate Process of the Pancreas.

BACKGROUND: Characteristics of a cancer-positive margin around a resected uncinate process of the pancreas (MUP) due to a pancreticoduodenectomy are difficult to understand by standardized evaluation because of its complex anatomy. The purposes of this study were to subclassify the MUP with tissue marking dyes of different colors and to identify the characteristics of sites that showed positivity for cancer cells in patients with pancreatic head carcinoma who underwent circumferential superior mesenteric arterial nerve plexus-preserving pancreaticoduodenectomy. Results of this evaluation were used to review operation procedures and perioperative methods. METHOD: We divided the MUP into 4 sections and stained each section with a different color. These sections were the pancreatic head nerve plexus margin (Area A), portal vein groove margin (Area B), superior mesenteric artery margin (Area C), and left of the superior mesenteric artery margin (Area D). The subjects evaluated were 45 patients who had carcinoma of the pancreatic head and were treated with circumferential superior mesenteric arterial nerve plexus-preserving pancreaticoduodenectomy. RESULTS: Of the 45 patients, nine cases (90%) of incomplete resection showed cancer-positivity in the MUP. Among the 4 sections of the MUP, the most cases of positive results [MUP (+) ] were found in Area B, with Area A (+), 0 case; Area B (+), 6 cases; Area C (+), 2 cases; and Area D (+), 3 cases (total, 11 sites in 9 patients). Relapse occurred in 7 of the 9 patients with MUP (+). Local recurrence was observed as initial relapse in all 3 patients with Area D (+). In contrast, the most common site of recurrence other than that in patients with Area D (+) was the liver. CONCLUSION: By subclassifying the MUP with tissue marking dyes of different colors, we could confirm regional characteristics of MUP (+). As a result, circumferential superior mesenteric arterial nerve plexus-preserving pancreticoduodenectomy was able to be performed in R0 operations in selected patients while a better postoperative quality of life was maintained. Furthermore, Area D (+) represents an extension beyond the limit of the local disease and may indicate the need for early aggressive adjuvant chemotherapy.

[Ultrasound-mediated treatment of pathology archival samples].

Over the past 30 years, drastic changes in molecular biology have accelerated technical advancements in pathological practice. Microwave irradiation was first applied to pathology archival samples for improved of tissue fixation, immunoreactions, in situ hybridizations, PCR reactions and other molecular assays. Compared to microwave irradiation, ultrasound processing allows rapid cross-linking of formalin-fixed tissue and uniformity of chemical reactions in tissues. In addition, temperature control is easier and tissue damage is minimized. Ultrasound was first applied to fixation solutions to improve the efficiency of tissue fixation. Many phenomena, including cavitation, and thermal and mechanical effects, are believed to play an important role in the ultrasound-mediated enhancement of fixation, dehydration, paraffin penetration, immunological reactions, hybridization, and clearing and impregnating tissue in an extremely short time. We found that to use ultrasound successfully for rapid tissue fixation and processing without tissue damage, it was critical to maintain ultrasound at low frequency and high intensity (40 KHz, 200 W/cm2). This ultrasound-mediated high-speed biological reaction and tissue processing also allows antigen-antibody reactions or nucleic acid hybridizations to occur rapidly with high specificity and with a very low or no background noise. Furthermore, we applied this ultrasound-assisted technique to decalcify and remove fat from tissue for rapid diagnosis, and compared the results to those obtained using conventional methods. In this study, we describe and highlight the advantages of ultrasound-mediated rapid tissue processing for routine pathological work and current molecular pathological applications.

Nonrandom chromosomal numerical abnormality as a new molecular cytogenetic tumor marker--a retrospective study of 60 gastric cancer cases.

Chromosomal numerical abnormality (CNA) is one of the distinct characteristics of human cancers, though the mechanisms and tumor specificity of this phenomenon have not been adequately analyzed. Recently, we developed a new sensitive fluorescence in situ hybridization (FISH) method that involves short-term microwave (MW) treatment for hybridization. In this study, we applied this modified FISH technique to investigate the CNA of 60 gastric cancer cases with a panel of 18 chromosome-specific alpha-satellite probes (for chromosome 1-4, 6-12, 15-18, 20, X, and Y) and region-specific probes (c-myc, p53, and Her-2/neu) to enumerate the respective chromosome numbers in interphase nuclei of formalin-fixed paraffin-embedded sections. The numerical aberrations of chromosome 1, 3, 8, 17, 20, and X were frequent regardless of histologic types, whereas aberrations of chromosomes 10, 15, and 18 occurred less frequently (p<0.001). From a histopathological standpoint, the mucocellular type of carcinoma had stable CNA in comparison with the tubular type of carcinoma (21.7+/-9.63% vs. 58.3+/-12.32%, p<0.001) and, of note, there was less extensive CNA in female cases. A dramatic difference in patient outcome was detected according to the involvement of chromosomes 3, 10, 11, 12, 17, and Y; cases with CNA of these chromosomes had a worse prognosis (p<0.001). A two-step analysis of the CNA of 6 chromosomes and locus specific gene abnormalities successfully divided gastric cancer cases into those with a good outcome and those with a poor outcome. This analysis allows one to more accurately predict prognosis than by using a simple classification based on conventional clinicopathological diagnosis.

Centromere numerical abnormality in the papillary, papillotubular type of early gastric cancer, a further characterization of a subset of gastric cancer.

Papillary adenocarcinoma of the stomach is a relatively uncommon histological type, and it is often detected in the early stage. We recently characterized the papillary type of gastric cancer and found frequent microsatellite instability and associated mutations. In this study we analyzed the centromere numerical abnormality (CNA) of 18 chromosomes (chromosomes 1-4, 6-12, 15-18, 20, X, and Y) in the papillary and papillotubular types of gastric cancer by a modified fluorescence in situ hybridization technique with microwave treatment. All 3 cases (100%) of papillary adenocarcinoma had high microsatellite instability (MSI-H), and low CNA, and 41% (7 cases) of the 17 cases of papillotubular adenocarcinoma exhibited MSI-H and all 7 cases had low CNA. Further 8 cases (47%) had extensive CNA. In these 15 cases, all the MSI-H cases had lower CNA, and low microsatellite instability (MSI-L) and MSS cases had higher CNA. The remaining two cases showed low CNA and MSI-L and MSS. These profiles were different from those of tubular type gastric cancer, which always had extensive CNA and no MSI. Although the numbers of the cases in this series are limited, our data may suggest that a modest CNA may be another characteristic of gastric cancer with papillary structure.

Peripheral lung adenocarcinomas: 10 mm or less in diameter.

BACKGROUND: Few reports have been published regarding peripheral lung adenocarcinomas that are 10 mm or less in diameter. This is considered to be the smallest tumor size detectable by present diagnostic modalities. METHODS: Clinicopathologic studies were performed in 57 patients with peripheral lung adenocarcinomas of 10 mm or less in diameter. Outcomes were compared with two other groups that consisted of 32 patients with adenocarcinomas between 11 and 15 mm in diameter and 35 patients with adenocarcinomas between 16 and 20 mm in diameter. Tumors were curatively resected between 1992 and 2002. RESULTS: The mean age was 61.7 years. The following three features were more frequent: female sex (78.9%), nonsmokers (77.2%), and cases with carcinoma detected by computed tomography despite negative chest radiography (96.5%). Negative lymphatic invasion (94.7%) was significantly higher. Three cases showed lymphatic invasion that was classified as types E or F, according to Noguchi's classification. There were no cases of lymph node metastasis, pleural involvement, or intrapulmonary metastasis. Well-differentiated type was in 93.0%. Types A and B, which are noninvasive alveolar replacement-type adenocarcinomas, were significantly dominant (86.0%). The 5-year postoperative survival rate was 97.3%, which was significantly better than in the other two groups (75.5%, 78.1%). CONCLUSIONS: Histopathologic features of most peripheral lung adenocarcinomas of 10 mm or less in diameter were types A and B. Types A and B were considered fundamentally indicated for thoracoscopic wedge resections. However, the other types required the standard operation.

Local recurrence of lung adenocarcinoma 10 years after left upper lobectomy resembling pseudomesotheliomatous adenocarcinoma: a case report.

The form and timing of the local recurrence of lung cancer can be unpredictable and unexpected. Pseudomesotheliomatous adenocarcinoma is a rare tumor that mimics malignant pleural mesothelioma both clinically and pathologically. Distinguishing pseudomesotheliomatous adenocarcinoma from malignant pleural mesothelioma on the basis of clinical findings can be difficult; therefore, a biopsy is usually required for diagnosis. Here we report on a 73-year-old Japanese man who presented with extensive dissemination along the pleural surfaces and clinical findings similar to those of pseudomesotheliomatous lung cancer 10 years after undergoing left upper lobectomy for lung adenocarcinoma. This report provides information that will help physicians establish an accurate diagnosis in similar cases.

Case of IgG4-related sclerosing cholangitis with a normal serum IgG4 level: report of a case.

Although hilar cholangioma is the most common cause of stricture of the hilar bile duct, several diseases can contribute to stenosis. Here, we report on a patient with immunoglobulin (Ig) G4-related sclerosing cholangitis (IgG4-SC) of the hilar bile duct arising from obstructive jaundice. The patient had undergone laparoscopic cholecystectomy for the removal of gallstones. The differential diagnosis for icterus included hilar cholangiocarcinoma, primary sclerosing cholangitis, IgG4 sclerosing cholangitis, ischemic bile duct stenosis, a complication of cholecystitis, amputation neuroma, and iatrogenic stenosis. Numerous examinations were performed, but a definite diagnosis remained elusive. Because cholangiocarcinoma could not be ruled out, we proposed surgical resection. The patient subsequently underwent extended right liver lobectomy and intrahepatic cholangiojejunostomy. Pathological examination revealed numerous inflammatory cell infiltrates resembling IgG4-positive antibody plasma cells in the stromal layer of the stenotic bile duct walls. Hypertrophy of the nerve fiber fascicles was not observed. The serum IgG4 level of the patient was within the normal range. Few reports of IgG4-SC with a normal serum IgG4 level have been published. When this condition presents as it did in the present case, establishing a definite diagnosis can be difficult.

A case of anaplastic carcinoma of the pancreas producing granulocyte-colony stimulating factor.

We report a case of anaplastic carcinoma of the pancreas with production of granulocyte-colony stimulating factor (G-CSF) in a 59-year-old male. He was referred to our hospital with a chief complaint of epigastralgia and suffered from leukocytosis. Differential diagnosis included pancreatic tumors and submucosal tumor of the stomach, but definite preoperative diagnosis could not be made. He underwent distal pancreactomy, total gastrectomy with Roux-en-Y reconstruction and splenectomy. He recovered uneventfully postoperatively and was discharged from hospital on the 14th postoperative day. Histological examination showed anaplastic carcinoma of the pancreas. Since the peripheral leukocyte count was sharply decreased after the operation, we suspected the tumor would be producing G-CSF. Then immunohistochemistry showed a positive stain in the tumor. Therefore, we diagnosed the tumor as anaplastic carcinoma of the pancreas producing G-CSF. Three months after the resection, local recurrence was detected by abdominal computed tomography. The patient died of hemorrhagic shock due to tumor invasion of the intestine 8 months after the operation.

Chromosomal numerical abnormalities in early stage lung adenocarcinoma.

Chromosomal numerical abnormalities (CNA) are ubiquitous in human cancers. However, the question of when a CNA occurs in the course of tumor generation and progression, is controversial. Recent radiological scrutiny has enabled the identification of small peripheral lesions in the lung. A chromosome-wide investigation encompassing almost all the chromosomal centromeres was performed using modified fluorescence in situ hybridization on the archived pathological samples of 16 atypical adenomatous hyperplasia (AAH) and 30 lung adenocarcioma (AdCa) specimens including those smaller than 1 cm in size. The prevalence of the gain was more extensive in male than in female patients, and in non-smokers than in smokers. It tended to be greater in poorly differentiated AdCa, in moderately differentiated AdCa, and in well-differentiated AdCa cases, in that order. Most AAH had non-specific gains affecting all the examined chromosomes. The prevalence of the gain differed significantly between AAH and bronchioloalveolar carcinoma (BAC) 1 cm. It is proposed that the CNA is a distinct phenomenon occurring in the early or premalignant stage of lung AdCa, and that the CNA itself may not be a sequel in the carcinogenetic process, but a driving factor in carcinogenesis.

Repeated fluorescence in situ hybridization by a microwave-enhanced protocol.

A novel re-hybridization protocol for pathology archive sections that uses microwave-assisted fluorescence in situ hybridization (FISH) is described. Stripping the probe from the pathology archive sections with HCl and re-hybridizing with the next probe by intermittent microwave irradiation generated clear signals without background noise. Repeated stripping and hybridization with numerous bacterial artificial chromosome (BAC)-derived probes would identify the profile of genome-wide changes in small lesions on sections.

Chromosomal numerical abnormality profiles of gastrointestinal stromal tumors.

BACKGROUND: Biological variations in and the heterogeneity of gastrointestinal stromal tumors (GISTs) are well known, but chromosomal numerical abnormality (CNA) has not been fully examined especially in this context. The aim of this study is to test CNA as a possible biological predictor of biological behavior of GISTs. METHOD: We applied microwave-assisted FISH protocol to pathological archives of GIST tumors displaying different clinical features to characterize the CNA profile of these tumors. A panel of 18 centromere enumeration probes (CEP) and 24 bacterial artificial chromosome (BAC) or P1-derived artificial chromosome (PAC) probes containing genes like Aurora kinases (AURKs) and other candidate genes involved in human carcinogenesis were used. CNA profiles, histopathological risk categorization and Ki-67 labeling indexes of 23 primary and/or metastatic GIST tumors of 12 subjects (both primary and metastatic in 7 subjects) were compared between primary GIST with and without metastases, and between metastatic and primary portions in 7 individuals. RESULTS: CNA in the primary sites was more extensive in the GISTs with recurrence and metastasis than in those without, especially as to the loss of chromosome 20 and genomic imbalance of AURKA-containing BAC probe on 20q in the cases with metastasis. The consistent loss of one allele of chromosome 14q was also noted. Interestingly, both primary and metastatic tumors in identical individuals had similar CNA profiles. CONCLUSION: The extent of CNA differed between GISTS with and without recurrence or metastasis; thus, FISH analysis of specimens from the primary sites may predict the biological behavior of this tumor.

Modified fluorescence in situ hybridization methods using pathology archives.

Brain tumors, like many other common tumors, are frequently associated with chromosomal numerical abnormalities. However, as the identification of abnormal characteristics by conventional cytogenetic or molecular methods has been hampered by technical difficulties, minimal information has been available about specific chromosomal or locus-specific gene alterations. Recently, fluorescence in situ hybridization (FISH) has emerged as a powerful clinical and research tool for the assessment of genomic instability within interphase nuclei. Here, we developed a modified FISH protocol including short-term microwave treatment to analyze specimens from the pathology archives that had been routinely processed and stored. The FISH signals obtained using this modified method showed a significant improvement compared with those obtained using the standard FISH method. This new technique thus enables the analysis of various paraffin-embedded tissue sections of intracranial tumors obtained under inappropriate fixation conditions. We highlight the advantages of this modified FISH procedure on a tissue microarray of archival materials for current diagnostic and investigative neuropathology applications.