A simple antigen retrieval method for the optimal demonstration of cyclin-D1 overexpression in formalin-fixed paraffin-embedded cases of mantle cell lymphoma.

Successful diagnosis of mantle cell lymphoma (MCL) by immunohistochemistry is largely dependent on the successful demonstration of cyclin D1 overexpression in cases that fit clinical, morphologic, and immunophenotypical profiles. Accurate diagnosis in these cases is important due to the aggressive progression and poor survival times associated with this type of lymphoma. However, demonstration of cyclin D1 by immunohistochemistry on formalin-fixed, paraffin-embedded tissue is often fraught with technical difficulties, chiefly poor staining intensity largely due to insufficient antigen retrieval. The authors report a simple antigen retrieval technique that combines both heat and enzymatic treatment to demonstrate cyclin D1 overexpression. Furthermore, it appears that the antigen retrieval method is the key factor in the demonstration of cyclin D1, as detection systems with differing sensitivities (labeled streptavidin biotin [ABC] and dextran polymeric conjugate [Envision]) fail to demonstrate any significant differences in their staining intensities. This isa simple, cost-effective method that can be performed manually or can easily be adapted to suit automated staining systems.

The relationship between cyclooxygenase-2 expression and characteristics of malignant transformation in human colorectal adenomas.

BACKGROUND AND AIMS: Cyclooxygenase 2 (COX-2) is a target of aspirin and other non-steroidal anti-inflammatory drugs and is implicated in the pathogenesis of colorectal cancer. The objective of this study was to evaluate the extent of COX-2 in pre-malignant colorectal polyps and to assess the relationship between COX-2 and the level of dysplasia in these lesions. METHODS: Whole polypectomy specimens were retrieved from 123 patients by endoscopic or surgical resection. Following formalin fixation and paraffin embedding, the polyps were evaluated histologically for size, type and grade of dysplasia. The extent of COX-2 expression was measured by the avidin-biotin immunohistochemical technique using a monoclonal COX-2 antibody. The extent of COX-2 expression was graded according to percentage epithelial COX-2 expression. RESULTS: The polyps were of the following histological types: 10 hyperplastic, 35 tubular adenomas, 61 tubulovillous adenomas and 17 villous adenomas. Twenty showed mild dysplasia, 65 moderate dysplasia, and 28 focal or severe dysplasia (including eight with focal invasion). The average polyp size was 1.7 cm. Nine hyperplastic polyps were COX-2-negative and one was COX-2-positive. COX-2 expression was more extensive in larger polyps and in polyps with a higher villous component. There was a significant increase in the extent of COX-2 protein with increasing severity of dysplasia. Within a polyp, there was a focal corresponding increase in COX-2 expression within epithelium showing a higher grade of dysplasia. CONCLUSIONS: COX-2 expression is related directly to colorectal adenomatous polyp size, type and grade of dysplasia. This suggests that the role of COX-2 in colorectal cancer may be at an early stage in the adenoma-to-carcinoma sequence and supports the suggestion that inhibition of COX-2 may be useful chemoprevention for this disease.

Cyclooxygenase-2 expression in stromal tumors of the gastrointestinal tract.

Cyclooxygenase (COX) is a key enzyme in the conversion of arachidonic acid to prostaglandins, prostacyclin, and thromboxane. COX-2 is expressed in many epithelial malignancies, particularly those of the gastrointestinal (GI) tract. COX-2 has been implicated in the pathogenesis of cancers and has a significant negative effect on survival. To date, little is known about the expression of COX-2 in nonepithelial tumors. The objective of this study was to evaluate the expression of COX-2 in GI stromal tumors (GISTs). We evaluated 15 GISTs using tissue microarray. Tissue blocks were retrieved and stained with hematoxylin and eosin to evaluate the histological tumor type. In addition, immunohistochemistry was performed for COX-2, the macrophage marker, CD68 (KP-1), and KIT (CD117). Two pathologists then evaluated the tissues to determine the extent and intensity of COX-2 expression. The location of CD68-positive cells, and whether these cells were COX-2 positive, was also evaluated. The results showed that 80% (12 of 15) of the tumors expressed COX-2. Expression was noted in the cytoplasm of the tumor cells, with variable intensity of staining among the tumors. COX-2 was expressed in both epithelial cell and spindle cell tumors, but appeared stronger in epithelial lesions. In mixed lesions, COX-2 was expressed to a greater extent in epithelial areas. There was a greater extent of COX-2 expression in malignant tumors and tumors located within the stomach. Tumor-infiltrating macrophages (CD68-positive cells) were identified in all of the lesions; in 80% of cases, those macrophages also expressed COX-2. This study is the first to demonstrate COX-2 expression in stromal lesions of the GI tract. The enzyme may play a role in the proliferation of these lesions, suggesting the potential use of nonsteroidal anti-inflammatory drugs in treatment.

HER-2 analysis in tissue microarrays of archival human breast cancer: comparison of immunohistochemistry and fluorescence in situ hybridization.

HER-2 gene alterations have been shown to have prognostic and predictive value for the treatment of breast cancer with therapeutic agents. As a result, the accurate evaluation of HER-2 status is crucial. HER-2 status is assessed at the protein level by immunohistochemistry and at the DNA level by fluorescence in situ hybridization (FISH). Although the best approach is to combine immunohistochemistry and FISH assays, doing so is not practical or cost-effective for routine histopathologic laboratories. The recent development of tissue microarray technology has allowed large-scale studies using formalin-fixed, paraffin-embedded material. We used this technique to assess HER-2 status in a cohort of 54 invasive breast cancer cases by immunohistochemistry and FISH assays to determine whether the results obtained were representative of the protein and gene expression patterns of the original whole tissue section. Concordance for HER-2 immunohistochemistry between the tissue microarray and full sections was 93%. Concordance for HER-2 FISH between the tissue microarray and full sections was 91%. Concordance between HER-2 FISH and HER-2 immunohistochemistry on the tissue microarray was 98%. We conclude that tissue microarrays provide highly comparable results in the assessment of HER-2 protein levels and allow large-scale analysis of the HER-2 gene by FISH.

Idiopathic cholangiopathy in a biliary cast syndrome necessitating liver transplantation following head trauma.

The development of total biliary casts is very unusual, especially in patients who have not undergone liver transplantation. The aetiology of these casts is uncertain but several factors are believed to play a role, including periods of fasting, haemolysis, cholangitis and recent surgery. Resultant bile stasis and/or gallbladder hypocontractility promote sludge and subsequent stone formation. Here we present the case of a previously well 66-year-old woman who developed a total biliary cast several weeks after being involved in a road traffic accident during which she sustained head injuries but no obvious liver insult. This cast was removed at laparotomy but the patient had resultant diffuse biliary tree abnormalities and persistent cholestasis and subsequently required a liver transplant. The possible aetiologies of biliary cast formation and subsequently cholangiopathy necessitating transplantation in this patient are described.

Disabling osteomalacic myopathy as the only presenting feature of coeliac disease.

A 59-year-old woman presented with a 3-month history of bilateral, proximal lower-limb weakness associated with disabling pain that rendered her wheelchair-bound. There were no gastrointestinal symptoms. Clinical examination showed evidence of bilateral, proximal muscle atrophy and weakness in the lower limbs. Low serum calcium and raised serum alkaline phosphatase, coupled with radiological findings, led to the diagnosis of osteomalacia. Subsequent gastroscopy and duodenal biopsy confirmed a diagnosis of coeliac disease. With adherence to a gluten-free diet, the patient's condition remarkably improved within 3 months and she could walk pain-free using a stick. Osteomalacia and myopathy may rarely be the initial and primary presentations of coeliac disease. There are very few reports of osteomalacia as the only presentation of coeliac disease and no reports that describe such a dramatic recovery 3 months after commencing a gluten-free diet. A review of the literature regarding osteomalacia and myopathy in coeliac disease is presented.

Loss of Fhit expression as a potential marker of malignant progression in preinvasive squamous cervical cancer.

OBJECTIVE: In a previous study using the same cases of squamous cervical neoplasia and microinvasive carcinoma (MICA) we found an association between FHIT gene deletion and infection with high-risk HPV (HR HPV). The purpose of this study was to evaluate Fhit protein expression by immunohistochemistry in order to determine whether FHIT gene deletion or infection with HR HPV correlated with aberrant protein expression and grade of lesion. METHODS: A total of 74 archival LLETZ biopsy cases consisting of 23 cervical intraepithelial neoplasia grade 1 (CIN1), 28 CIN3, and 23 MICA cases were selected for Fhit immunostaining. The results of this study on Fhit immunostaining were analyzed in relation to our previous findings using Epi-Info and SPSS-PC statistical analysis software. RESULTS: Fifty percent (14/28) of CIN3 lesions and 78% (18/23) of MICA lesions had a marked reduction or absence of Fhit protein expression (P = <0.001, strength of association, Cramers' V, 0.632). CIN1 lesions were found to have moderate to strong cytoplasmic expression of Fhit. Seventy percent of cases in this study with reduced/absent Fhit protein expression were also positive for FHIT gene loss of heterozygosity (LOH) (P = 0.04, strength of association, phi, 0.254). A significant statistical relationship was found between Fhit protein expression and HPV 16 infection in combined CIN1, CIN3, and MICA cases (P = <0.001). Eighty-seven percent of cases with reduced/absent Fhit protein expression were positive for HPV 16 (strength of association, phi, 0.552). Ninety percent of HPV 16 and 31 positive cases had reduced/absent Fhit expression. CONCLUSION: Our findings suggest an association between HPV infection and FHIT gene abnormalities raising the possibility of a mechanistic role for the FHIT gene as a cofactor with HPV in triggering the development of cervical cancer.