Virtual electron microscopy: a simple implementation creating a new paradigm in ultrastructural examination.

Ultrastructural examination is a time-consuming and tiring process, requiring search for diagnostic features on a low-contrast screen in a dim environment. This article describes a method to circumvent these problems through the creation of a virtual ultrathin slide. This can be achieved by automated capturing of hundreds of images at high magnification and stitching them together into a digital image with a resolution of 4 nm/pixel. The pathologist can then navigate the virtual slide at his/her workstation computer. The image shows good contrast and resolution for diagnostic purposes, and most important, the pathologist can precisely note where the specific ultrastructural features are located. The setup required to implement virtual electron microscopy includes a transmission electron microscope equipped with motorized stage and automated digital image capture function, 2 free software components, self-developed software, and a desktop-grade computer. Besides use in daily diagnosis, virtual electron microscopy can open up many new applications such as undergraduate teaching, pathology resident training, external quality assurance program, and expert consultation.

Renal cell carcinoma of native kidney in Chinese renal transplant recipients: a report of 12 cases and a review of the literature.

OBJECTIVES: To present and discuss the epidemiological and clinical aspects, as well as therapeutic options and outcome of de novo renal cell carcinoma (RCC) of the native kidneys in a series of Chinese renal transplant recipients. PATIENTS AND METHODS: A retrospective, cohort study examining all renal transplant recipients with the diagnosis of RCC of native kidney followed up in two major regional hospitals in Hong Kong between January 2000 and December 2009. Clinical data included age, gender, cause of renal failure, symptoms at presentation, duration of transplantation, immunosuppressive therapy, and history of acquired cystic kidney disease (ACKD). Laboratory, radiographic, operative, and pathology reports were used to assess the tumor extent. RESULTS: Among the 1,003 renal transplant recipients recruited, 12 transplant recipients had a nephrectomy for a total of 13 RCC. The prevalence of de novo RCC was 1.3%. The mean age at diagnosis of RCC was 48.4 years, and the median time from transplantation to diagnosis was 6.1 years. ACKD was found in 6 (50%) of the patients. All patients except one were asymptomatic. pT1 disease was found in ten patients with a mean tumor size of 3.2 cm. All patients were treated successfully with radical nephrectomy. After a median follow-up of 38 months, two patients (16.7%) died. One died of sepsis, and the other died of metastatic carcinoma. CONCLUSIONS: With increasing data showing a better prognosis if RCC is detected early by screening, it is time to consider screening all kidney transplant recipients for ACKD and RCC.

IgG4-related Sclerosing disease: a potential new etiology of cutaneous pseudolymphoma.

IgG4-related sclerosing disease is a syndrome characterized by the involvement of a wide variety of tissues by lymphoplasmacytic infiltrates and sclerosis, elevated serum IgG4 titer, and increased IgG4+ plasma cells in tissues. This report describes 2 cases with skin involvement, a feature not well documented in the literature. The patients had plaques or nodules in the skin of the cheek, temporal or periauricular region. Histologically, there was dermal and subcutaneous involvement by a nodular lymphoid infiltration often interspersed with lymphoid follicles and sclerotic stroma. The infiltrate was rich in plasma cells, small lymphocytes, and sometimes plasmablasts. The IgG4+ cell count was high (342 to 425 per high power field), with an IgG4/IgG proportion from 68% to 100%. As the morphology was compatible with pseudolymphoma, 14 cases of cutaneous pseudolymphoma were retrieved from the archives for IgG4 and IgG immunostaining. Two cases exhibited marked increase in IgG4+ cells, and showed many similarities with cutaneous manifestation of IgG4-related sclerosing disease, but the limited available clinical information precluded a conclusion on their nosologic nature. In summary, IgG4-related sclerosing disease can manifest with skin lesions, and is also one of the potential etiologies of cutaneous pseudolymphomas.

Isolated tumor cells and circulating CK20 mRNA in pN0 colorectal cancer patients.

Micrometastases in lymph nodes and blood may provide important prognostic information. In this study, cytokeratin 20 (CK20) positive cells in lymph nodes and circulating CK20 mRNA were studied using 57 paraffin-embedded lymph node specimens and blood from 24 patients with pN0 colorectal cancer (CRC), respectively. Results showed that 29 out of 56 (52%) lymph node specimens had CK20-positive cells (range: 1-35). Follow-up of the patients for 12 months indicated that 4 patients (7%) had CRC metastases to liver, lung, and bone. In addition, 8 out of 24 (33%) samples had at least 2-fold circulating CK20 mRNA expression higher than the pooled normal sample. This study provides evidence that CK20-positive cells were found in the lymph nodes and differentially expressed circulating CK20 mRNA was also detected in the blood from patients with pN0 CRC. Long-term follow-up is necessary to study their prognostic use in patients with non-metastatic CRC.

Protein chip array profiling analysis in patients with severe acute respiratory syndrome identified serum amyloid a protein as a biomarker potentially useful in monitoring the extent of pneumonia.

BACKGROUND: A new strain of coronavirus (CoV) has caused an outbreak of severe acute respiratory syndrome (SARS), with 8098 individuals being infected and 774 deaths worldwide. We carried out protein chip array profiling analysis in an attempt to identify biomarkers that might be useful in monitoring the clinical course of SARS patients. METHODS: We performed surface-enhanced laser desorption ionization time-of-flight mass spectrometry on 89 sera collected from 28 SARS patients, 72 sera from 51 control patients with various viral or bacterial infections, and 10 sera from apparently healthy individuals. RESULTS: Nine significantly increased and three significantly decreased serum biomarkers were discovered in the SARS patients compared with the controls. Among these biomarkers, one (11,695 Da) was identified to be serum amyloid A (SAA) protein by peptide mapping and tandem mass spectrometric analysis. When we monitored the SAA concentrations longitudinally in 45 sera from four SARS patients, we found a good correlation of SAA concentration with the extent of pneumonia as assessed by a serial chest x-ray opacity score. Increased SAA occurred in three of four patients at the time of extensive pneumonia as indicated by high x-ray scores. Over the course of gradual recovery in two patients, as assessed clinically and radiologically, SAA concentrations gradually decreased. In the third patient, the concentrations were initially increased, but were further increased with superimposed multiple bacterial infections. SAA was not markedly increased in the fourth patient, who had low x-ray scores and whose clinical course was relatively mild. CONCLUSIONS: Protein chip array profiling analysis could be potentially useful in monitoring the severity of disease in SARS patients.

Quantification of plasma beta-catenin mRNA in colorectal cancer and adenoma patients.

PURPOSE: Colorectal cancer is an important cause of cancer deaths. Here, we focused our investigation on the beta-catenin gene which is implicated in colorectal carcinogenesis and tested whether beta-catenin mRNA is detectable in the plasma of colorectal carcinoma and adenoma patients using quantitative reverse transcriptase-PCR. EXPERIMENTAL DESIGN: Plasma beta-catenin mRNA was measured from 58 colorectal carcinoma patients, 49 colorectal adenoma patients, and 43 apparently normal subjects using intron-spanning primers and Taqman probes. Five clinicopathological parameters were studied and correlated with plasma beta-catenin mRNA concentration. Additionally, 19 colorectal carcinoma patients after tumor removal were also recruited for plasma beta-catenin mRNA measurement to further demonstrate the clinical usefulness of this test. RESULTS: beta-catenin mRNA was detected with median concentrations of 8737 (range: 1480-933100), 1218 (range: 541-2254) and 291 (range: 0-1366) copies/ml plasma in colorectal carcinoma, colorectal adenoma, and apparently normal subjects, respectively. Statistical analysis demonstrated that plasma beta-catenin mRNA concentration was correlated to tumor stage but not sex, age, lymph node status, and degree in differentiation. Moreover, plasma beta-catenin mRNA concentration decreased significantly after tumor removal in 16 of 19 (84%) colorectal carcinoma patients. CONCLUSIONS: We conclude that plasma beta-catenin mRNA may potentially serve as a marker for colorectal cancer.

Prognostic and diagnostic significance of beta-catenin nuclear immunostaining in colorectal cancer.

In the present study, we investigated the prognostic and diagnostic significance of beta-catenin nuclear immunostaining in 60 specimens of normal colorectal tissue; 180 specimens of colorectal polyps, adenomas, and carcinomas; and 40 specimens from patients with the simultaneous occurrence of polyps, adenomas, and carcinomas. Additional specimens from 59 patients with colorectal carcinoma and 14 patients with adenoma who subsequently developed carcinoma were examined for possible survival study. Immunohistochemical staining showed that the occurrence of nuclear beta-catenin correlated with the sequential stages in colorectal carcinogenesis, in which positive staining was observed in 0% of normal tissues, 8% of polyps, 92% of adenomas, and 100% of carcinomas. High immunohistochemical scores in colorectal carcinoma were significantly associated with lymph node metastasis and poor survival. Adenomas associated with synchronous or metachronous carcinomas showed significantly higher levels of nuclear beta-catenin compared with adenomas without associated carcinomas. Nuclear translocation of beta-catenin was rare or absent in other types of cytokeratin 20 positive adenocarcinomas examined (99 cases). Thus, it was positive in only 7% of colonic mucinous adenocarcinomas, 3% of pancreatic adenocarcinomas, 8% of ovarian mucinous cystadenocarcinomas, and 0% of gastric adenocarcinomas. However, 100% of primary and metastatic colorectal adenocarcinomas were positive for nuclear staining for beta-catenin. Thus, nuclear staining for beta-catenin may serve as an additional parameter to help distinguish colorectal adenocarcinomas from adenocarcinomas of other tissue sites. Collectively, the present large-scale study has clearly addressed the clinical significance of beta-catenin nuclear translocation with respect to tumor progression, survival, and differential diagnosis.

Perianal ulceration in a "healthy" Chinese man with disseminated tuberculosis.

Orificial tuberculosis (OTB) is a rare form of cutaneous Mycobacterium tuberculosis infection affecting the mucosa and skin around orifices in patients with advanced internal tuberculosis and poor general health. We report a 72-year-old Chinese man who had a 10-year history of OTB with disseminated tuberculosis infection of the lungs and urinary tract. He appeared surprisingly healthy and had been free from systemic symptoms all along despite widespread tuberculosis. The diagnosis of OTB was established by the microscopic presence of acid-fast bacilli (AFB) in the tissue section and was rapidly confirmed by polymerase chain reaction (PCR) to be Mycobacterium tuberculosis. PCR shortens the time of diagnosing rare presentations of cutaneous tuberculosis and prevents delays in treatment. Conventional culture is still important in confirming the diagnosis and screening for drug resistance.

Expression of frizzled-related protein and Wnt-signalling molecules in invasive human breast tumours.

Frizzled-related protein (Frp) is a new family of secreted proteins that contain a region homologous to the extracellular cysteine-rich domain (CRD) of the frizzled family proteins. The role of Frp protein is far from clear. To explore the role of Frp and its relationship to the Wnt-signalling pathway in breast cancer, in situ hybridization and immunohistochemical analyses of Frp, Wnt-1, APC, beta-catenin, and its target genes c-myc and cyclin D1 were conducted in 70 specimens of invasive ductal carcinomas of the human breast. Frp mRNA was down-regulated in 62 and elevated in eight tumour specimens, compared with adjacent normal tissues. In the course of tumour progression, however, Frp mRNA steadily increased in both tumour and the adjacent tissues. Interestingly, the number of cases with axillary lymph node metastasis was significantly lower in the group with elevated Frp than in the group with decreased Frp, suggesting that Frp may contribute as a prognostic factor in invasive breast cancer. Wnt-1, a gene implicated in human breast cancer, was markedly elevated in grade 1 tumours, but declined as tumour grade declined. The level of Wnt-1 was linearly correlated with its downstream target beta-catenin (p<0.05), but was inversely correlated with Frp (p<0.05), suggesting a possible negative regulatory role of Frp with regard to Wnt-1. APC was inversely correlated with beta-catenin (p<0.05). Beta-catenin, a key transcriptional activator responsible for the activation of both c-myc and cyclin D1 in colorectal tumours, was detected at high levels in the plasma membranes of cells in normal tissue. In tumour masses, however, beta-catenin lost its tight association with the membrane and diffused into the cytoplasm. Surprisingly, it clearly did not penetrate the nuclei, despite the fact that both c-myc and cyclin D1 were markedly elevated in all tumour tissues. As revealed in this study, Wnt-1/beta-catenin plays very different roles in the oncogenesis of breast and colon cancers. This first systemic analysis of the Frp and the Wnt-signalling pathway in human breast cancer provides a springboard for further work on the role of Frp in the development of breast cancer.