391 P53 AS A UTILITY MARKER FOR RISK ASSESSMENT IN BARRETT’S OESOPHAGUS

Staging and prognosis of a case of Barrett's oesophagus is done by identifying the presence and grade of dysplasia. However, this method is highly subjective and can yield inaccurate diagnoses and prognoses. Demonstration of accumulation of p53 protein may be the most accurate and definitive approach to prognosticating cases of Barrett's oesophagus. This study focuses on p53 protein as a biomarker by immunohistochemistry staining in slides of different progression and demonstrates its promising clinical application. Methods Of 6175 endoscopies, from 970 cases of oesophagitis, a pilot study was performed on 6 cases of pathologically proven Barrett’s oesophagus, including a case with low-grade dysplasia, a case with high grade dysplasia and one case of poorly differentiated adenocarcinoma. Formalin fixed paraffin embedded tissue sections of the above cases were stained for anti p53 antibodies. The elevated immunohistochemical expression was quantified microscopically and a comparative analysis was done. Results p53 staining analysis showed significant immunoreactivity in the case of adenocarcinoma of oesophagus with diffuse strong nuclear staining. Low grade dysplasia and high grade dysplasia showed strong nuclear staining in the involved epithelium with focal and variable intensity positivity in the metaplastic epithelium. Those of reflux oesophagitis showed no nuclear staining in the epithelium. This pattern of staining with histological correlation is in concordance with other similar studies done supporting p53 as one of the crucial biomarkers for increased risk for carcinoma. Conclusion Increased p53 expression shows association with the higher risk of histological progression in the Barrett’s- carcinoma sequence. With the available literature p53 immunohistochemical staining along with other biomarkers will help us to predict the high risk patients and help treating clinician follow up the high risk patients. p53 shows promising utility as a part of panel for Barrett’s oesophagus and the spectrum.

Diagnostic Utility of BCOR Antibody in Clear Cell Sarcomas of Kidney

Purpose. Clear cell sarcoma of the kidney (CCSK) is an uncommon malignant renal tumor. It is the second most common renal pediatric renal malignancy after Wilms tumor. It exhibits a heterogeneous morphology, with overlapping features with its close differentials, which results in diagnostic challenges. There was no specific immunohistochemical marker in the past, to help in this regard. BCOR antibody has recently been suggested to be helpful in the differential diagnosis. We aim to study the utility of the BCOR antibody in the diagnosis of CCSK. Methods. We selected a total of 27 cases of CCSK (n = 12), Wilms tumor (n = 12), and congenital mesoblastic nephroma (n = 3). All cases were evaluated for the extent and intensity of nuclear labeling for BCOR antibody by immunohistochemistry (IHC). Results. We found that BCOR IHC was positive in 11 out of 12 cases with diffuse and strong staining in 8 of the cases. None of the cases of Wilms tumor and congenital mesoblastic nephroma were positive. Only 2 cases of Wilms tumor showed minimal and weak staining in <5% of cells. Conclusion. Diffuse and strong nuclear staining for the BCOR antibody is highly specific for CCSK among common pediatric renal malignancies. Our study confirms that BCOR IHC is a good IHC marker for the diagnosis of CCSK.

Elevated Foxp3/CD8 Ratio in Lung Adenocarcinoma Metastatic Lymph Nodes Resected by Transcervical Extended Mediastinal Lymphadenectomy

A balance between tumor invasion and immune defence system is widely investigated. Objective. The aim of this study was to evaluate lymphocyte phenotype in lymph nodes (LNs) of patients with lung cancer in relation to the presence of metastases. Methods. We investigated 364 LNs resected by transcervical extended mediastinal lymphadenectomy (TEMLA) of 49 patients with squamous cell carcinoma (SCC) or adenocarcinoma (AD) with (A) and without metastases (B). Expression of CD4, CD8, CD25, CTLA-4, and Foxp3 was assessed by immunohistochemical staining. Results. We observed a strong nuclear staining for Foxp3 in lymphocytes and cancer cells and strong membranous/cytoplasmatic reaction for CD4 and CD8, but low for CD25 and CTLA-4. There were significantly higher proportions of CD8+ cells in AD (B) versus AD (A) LNs (80% versus 52.5%, p<0.05). The Foxp3/CD8 ratio was higher in AD (A) versus AD (B) LNs (0.4 versus 0.25, p<0.05). No significant differences in the cell markers expression in SCC LNs were found. Conclusion. Significant differences in lymphocyte phenotype in AD may indicate an exceptional biology of this type of lung cancer. TEMLA resected LNs may serve as valuable samples for evaluation of immune status in lung cancer patients.

Clinical outcome in patients with endometrial papillary serous and endometrioid carcinomas as related to WT-1, Pax-2, and p16 immunohistochemical expression profiles

e16547 Background: Endometrial papillary serous carcinoma (EPS) can be difficult to differentiate histologically from high grade endometrial endometrioid carcinoma (EE). Both of these tumors often present with solid growth pattern with few papillary features. Recurrences have been found to be more frequent in EPS than high grade EE. These differences were found despite the same preoperative and postoperative radiotherapy and chemotherapy, indicating more aggressive tumor biology. To date, there are no well defined immunophenotypic or molecular methods to differentiate these two tumors or their clinical behavior. Methods: Eleven cases of EE and twenty-three cases of EPS were retrieved from our departmental archives and stained using Pax-2, WT-1 and p16 antibodies. For all three antibodies, a strong nuclear staining is considered positive. The intensity and proportion of cells positive are noted. Seven cases from each group were further examined for their staging and clinical outcome in terms of recurrence, metastasis and treatment. Results: WT-1 is negative in 100% of EE cases. p16 is positive in 95% of EPS cases, and only 55% of EE cases. In addition, out of the seven EPS patients whose clinical information was obtained, the three EPS patients who had strong Pax-2 staining are still alive more than two years status post surgical intervention, while four other EPS patients are deceased. The three patients with the strongly staining PAX-2 EPS tumors all had varying tumor characteristics, recurrence and adjuvant therapies. This finding is now being evaluated in the additional cases. Conclusions: WT-1, Pax-2 and p16 are useful to differentiate high grade EE and EPS cases. Strong Pax-2 staining in three cases of EPS with better outcome is an interesting finding that deserves further investigation. No significant financial relationships to disclose.

Histone H4 Acetylation Is Associated with Improved Relapse-Free Survival in Newly Diagnosed Patients with Acute Lymphoblastic Leukemia (ALL) Less Than 60 Years of Age without Poor Risk Cytogenetics.

Imbalances in histone acetylation can lead to changes in transcriptional dysregulation of genes involved in cell cycle progression and/ or apoptosis. Histone deacetylase inhibitors represent a novel antitumor therapy. We evaluated the acetylation of histone H4 in patients with newly diagnosed ALL, and evaluated the prognostic impact of acetylation on time to relapse and overall survival (OS). Methods: Patients with newly diagnosed ALL and an available diagnostic bone marrow biopsy performed at the Cleveland Clinic were evaluated between 1998 and 2005. B5-fixed bone marrow core biopsies were reviewed for areas with the highest concentration of blasts. A tissue microarray was constructed using 1 mm tissue cores. The cores were arrayed in duplicate in the majority of samples. Immunohistochemistry was performed for acetyl-H4 (1:200 dilution; polyclonal; Upstate Biotech, Lake Placid, NY) using automated stainers and heat induced epitope retrieval. Five hundred blasts were counted in each case and only strong nuclear staining was classified as positive. Based on the distribution of cell counts, there was a clear separation of cases at 40% blasts with strong nuclear staining. Therefore, cases were classified as positive (“acetylated”) if strong nuclear staining occurred in ≥ 40% of the blasts. Cox proportional hazards analysis was used to identify univariate and multivariate risk factors for relapse-free survival (RFS), OS, and CR rate. Results: Thirty-four patients had adequate tissue and clinical data for analysis. The median age was 40 yrs (range 18–74). Twelve patients (35%) had poor risk cytogenetics (CG), 6 (18%) normal karyotype, 9 (26%) miscellaneous abnormalities, and 7 (21%) unknown CG as defined by CALGB criteria. Thirty-one patients (91%) had precursor B-cell ALL, 1 (3%) mixed lineage, and 2 (6%) had precursor T cell ALL. The median white count was 41.7 × 109/ L (range 0.93–278) and LDH 1033 U/L (range 155–4608). Twenty-three (68%) patients had high levels of acetylated histone H4. Patients received a variety of induction and post-remission therapies. On univariate analysis, acetylation of H4 was associated with an improved RFS [HR 0.19, 95% CI (0.05–0.69), p=0.012] and a trend towards improved OS [HR 0.34, 95% CI (0.11–1.05), p=0.06] and increased CR rate [benefit ratio (BR) 4.12, 95% CI (0.88–19.3), p=0.07] in patients less than 60 years of age without poor risk CG. Using WBC and age at diagnosis in multivariate analysis, histone acetylation was associated with a better RFS [HR 0.20, 95% CI (0.05–0.77), p=0.019] and a trend towards an improved CR rate [BR 3.81, 95% CI (0.80–18.1), p=0.09] and OS [HR 0.33, 95% CI (0.10–1.11), p=0.07] in this same group of patients. Conclusions: Acetylation of histone H4 is associated with a better RFS and a trend towards an improved OS and CR rate in newly diagnosed patients with ALL less than 60 years of age without poor risk CG. These results should be confirmed in an independent data set and may provide rationale for the design of novel regimens incorporating histone deacetylase inhibitors, especially in patients less than 60 years of age.

Testicular Interstitial Cell Tumor and Gynecomastia in a Rabbit

Unilateral testicular interstitial (Leydig) cell tumor and gynecomastia were diagnosed in an adult male rabbit. The interstitial cell tumor was a well-circumscribed, 2-mm diameter, pale tan nodule composed of a uniform population of polygonal cells. Neoplastic interstitial cells exhibited diffuse, granular cytoplasmic staining with Melan A, a marker of steroid-producing cells in humans and dogs. Multiple subcutaneous masses in the caudal abdomen were associated with enlarged nipples and consisted of hyperplastic mammary gland tissue with proliferation of ducts and alveoli, marked lobule formation, and pseudolactational hyperplasia. Many epithelial cells lining the hyperplastic ducts and alveoli exhibited intense nuclear expression of progesterone receptor antigen, whereas myoepithelial cells showed strong nuclear staining for p63 antigen. This is the first report of concurrent interstitial cell tumor and gynecomastia in a rabbit and also the first description of gynecomastia in this species.

Differential expression of menin in sporadic pituitary adenomas.

Pituitary adenomas represent one of the key features of multiple endocrine neoplasia type 1. The gene involved in this syndrome (MEN1) is a putative tumor suppressor, that codes for a 610-amino acid nuclear protein termed 'menin'. Analyses of sporadic pituitary adenomas have so far failed to reveal MEN1 mutations or defects in MEN1 transcription in these tumors. In the present study we detected menin protein expression in a panel of normal and tumoral pituitary tissues, using a monoclonal antibody against the carboxy-terminus of menin. In the normal human pituitary gland, strong nuclear staining for menin was detectable in the majority of the endocrine cells of the anterior lobe, without a clear association with a particular hormone-producing type. In sporadic pituitary adenomas, menin expression was variable, with a high percentage of cases demonstrating a significant decrease in menin immunoreactivity when compared with the normal pituitary. Interestingly, metastatic tissues derived from one pituitary carcinoma had no detectable menin levels. Altogether, our data provide the first information regarding the status of menin expression in human normal and neoplastic pituitary as determined by immunohistochemistry (IHC).

Immunohistochemical Analysis of Nuclear Versus Cytoplasmic Staining of WT1 in Malignant Mesotheliomas and Primary Pulmonary Adenocarcinomas

Context.—Previous studies have indicated certain immunohistochemical markers, including WT1, may be helpful in distinguishing adenocarcinomas from mesotheliomas, but to date there are no reliable, widely accepted, commercially available antibodies positive in mesotheliomas and negative in adenocarcinomas. We compared the nuclear and cytoplasmic staining patterns of WT1 in these 2 malignancies using a commercially available antibody and examined the expression of 2 other previously reported positive markers, calretinin and thrombomodulin. Methods.—Sixty-seven mesotheliomas and 51 adenocarcinomas, all paraffin embedded, were retrieved from recent case files. The diagnosis of mesothelioma was based on typical clinical and morphologic features, as well as immunohistochemistry; electron microscopy had been performed on 16 cases. The diagnosis of adenocarcinoma was based on typical light microscopic findings and a positive stain for mucin. Commercially available antibodies to WT1, thrombomodulin, and calretinin were applied. Because of the conflict surrounding calretinin, 2 anticalretinin antibodies (from Chemicon Inc and Zymed Laboratories) were utilized. Results.—Fifty of 67 mesotheliomas showed strong nuclear staining with WT1. No adenocarcinomas (0/51) showed nuclear staining. Twenty-three of 67 mesotheliomas were positive for thrombomodulin, and 35 of 67 mesotheliomas were positive for calretinin with the Chemicon antibody. Nine of 15 mesotheliomas were positive for calretinin with the Zymed antibody. Conclusions.—Thrombomodulin and calretinin did not prove useful in discriminating between mesotheliomas and adenocarcinomas. The degree of positivity with calretinin may be dependent on the specific antibody utilized. Nuclear staining for WT1 is highly specific for mesothelioma and, in the appropriate clinical setting, can be a helpful adjunct in the distinction between adenocarcinomas and mesotheliomas.