A cross sectional study of p504s, CD133, and Twist expression in the esophageal metaplasia dysplasia adenocarcinoma sequence.

The incidence of esophageal adenocarcinoma has increased dramatically over recent years and Barrett's esophagus is considered the most established risk factor for its development. Endoscopic surveillance of Barrett's esophagus is therefore recommended but hinges on histological interpretation of randomly taken biopsies which is poorly reproducible. The use of biomarkers presents an opportunity to improve our ability to risk-stratify these patients.We examined three biomarkers namely p504s, CD133, and Twist in the setting of Barrett's esophagus, low-grade dysplasia, and esophageal adenocarcinoma to evaluate differential expression between benign, dysplastic, and malignant Barrett's tissue in an exploratory cross-sectional study. Twenty-five cases each of Barrett's esophagus, low-grade dysplasia, and esophageal adenocarcinoma were included along-with 25 cases of esophagectomy resections for Barrett's adenocarcinoma. The biomarkers were immunostained on automated Ventana(®) immunostainer. The biopsies were assessed for biomarker expression by two independent observers. Granular cytoplasmic staining of p504s was observed in dysplastic Barrett's biopsies and esophageal adenocarcinoma but not in Barrett's esophagus. Apical and membranous CD133 expression was also observed in dysplastic Barrett's and esophageal adenocarcinoma. Nuclear Twist expression was seen predominantly in stromal cells. There was increased p504s expression in dysplastic Barrett's esophagus and esophageal adenocarcinoma compared with controls. CD133 expression was detected for the first time in esophageal adenocarcinoma and dysplastic Barrett's esophagus. Twist expression was not convincing enough to be labeled as Barrett's biomarker. p504s and CD133 have the potential to differentiate benign from malignant Barrett's tissue in this exploratory study. Their validity should be established in prospective longitudinal studies.

Epstein-Barr virus positive inflammatory pseudo-tumour of the spleen: A case report and literature review.

INTRODUCTION: Epstein-Barr virus positive inflammatory pseudo-tumour (IPT) of the spleen is an uncommon, frequently asymptomatic entity, which is typically picked up as an incidental finding on imaging. PRESENTATION OF CASE: We present a case of EBV positive IPT of the spleen which presented as an incidental finding on CT in a patient with a history of malignancy. Splenectomy was performed. DISCUSSION: IPTs are benign spindle cell lesions of varying aetiology, which can arise in a variety of tissues, including the spleen. In situ hybridisation showed strong staining for Epstein-Barr virus RNA in our case, in common with many similar lesions described in the literature. The differential diagnosis of such spindle cell tumours is discussed. CONCLUSION: Radiologically, EBV positive spindle cell tumours are indistinguishable from malignant lesions such as lymphoma and diagnosis is made on histology, usually at splenectomy.

Integrating molecular diagnostics into histopathology training: the Belfast model.

Molecular medicine is transforming modern clinical practice, from diagnostics to therapeutics. Discoveries in research are being incorporated into the clinical setting with increasing rapidity. This transformation is also deeply changing the way we practise pathology. The great advances in cell and molecular biology which have accelerated our understanding of the pathogenesis of solid tumours have been embraced with variable degrees of enthusiasm by diverse medical professional specialties. While histopathologists have not been prompt to adopt molecular diagnostics to date, the need to incorporate molecular pathology into the training of future histopathologists is imperative. Our goal is to create, within an existing 5-year histopathology training curriculum, the structure for formal substantial teaching of molecular diagnostics. This specialist training has two main goals: (1) to equip future practising histopathologists with basic knowledge of molecular diagnostics and (2) to create the option for those interested in a subspecialty experience in tissue molecular diagnostics to pursue this training. It is our belief that this training will help to maintain in future the role of the pathologist at the centre of patient care as the integrator of clinical, morphological and molecular information.

Breast cancer in women under 40 years of age: a series of 57 cases from Northern Ireland.

BACKGROUND: There are few studies examining breast cancer in women under the age of 40 years, particularly in western European populations. Such tumours are reported to be more aggressive, possibly due to a different pathophysiology compared to older patients. METHODS: We performed a retrospective review of all women less than 40 years of age, diagnosed or treated with breast cancer, from June 2001 to June 2007 to assess pathophysiological factors that may influence clinical outcome and prognosis including patient demographics, clinical presentation, pre-operative investigations, surgical and pathological findings, treatment and outcome. RESULTS: Fifty-eight women (mean age 34.9 years, range 27-39 years) were identified. One patient was excluded due to incomplete data; 98.2% (n=56) patients presented directly to our symptomatic clinic; 89.5% (n=51) patients had a palpable lump; 71.9% (n=41) patients had no family history. Mammography was less sensitive than ultrasound (64.3% vs. 82.4%) while fine needle aspiration cytology was 92.5% sensitive for malignancy. Twenty-nine (50.9%) patients underwent breast-conserving surgery (BCS) of which 7 proceeded subsequently to completion mastectomy due to involved margins. Twenty-six (45.6%) patients required total mastectomy primarily while 2 (3.5%) patients were treated palliatively due to metastatic disease. The mean tumour size (nearest resection margin) was 2.13cm (2.58mm) for BCS and 3.95cm (6.38mm) for mastectomy. From a total of 55 primary resections, 85.5% (n=47) of tumours were invasive ductal carcinoma; 57.4% (n=31) and 40.7% (n=22) were grade II and III tumours respectively. Lymphovascular invasion was identified in 50.9% (n=28) while 40.0% (n=22) were lymph node positive for metastatic disease. 76.8% (n=43), 39.3% (n=22) and 30.2% (n=16) were oestrogen, progesterone and human epidermal growth factor receptor-2 positive respectively. The mean Nottingham prognostic index was 4.37 (range 2.2-8.4). Neo-adjuvant and adjuvant chemotherapy was administered to 9.3% (n=5) and 80.0% (n=44) of surgically treated patients respectively while 76.4% (n=42) patients received adjuvant radiotherapy. 76.4% (n=42) of patients were treated with tamoxifen. Four patients received Herceptin therapy. Statistically significant univariate factors adversely associated with overall survival were time from referral to out-patient department attendance (p=0.038), administration of neo-adjuvant treatment (p=0.019), surgical intervention (p<0.001), progesterone receptor positivity (p=0.018) and tumour recurrence (p<0.001). 86.0% (n=49) patients were alive at mean follow-up of 52 months; 82.5% (n=47) remain disease free. CONCLUSION: Our study reports a low familial trait rate combined with a high proportion of hormonally active tumours less than grade III which suggests that breast cancer in this series of young women from Northern Ireland may be less aggressive and more hormonally responsive than anticipated.

Myeloid sarcoma of the small bowel associated with a CBFbeta/MYH11 fusion and inv(16)(p13q22): a case report.

This report describes a case of aleukaemic myeloid sarcoma of the small intestine in a 50-year-old woman presenting with small bowel obstruction. Fluorescence in situ hybridisation analysis of interphase nuclei revealed a split CBFbeta signal, consistent with an underlying inversion of chromosome 16, inv(16)(p13q22). The resultant type A CBFbeta/MYH11 transcript was detected by reverse transcriptase PCR. Immunohistochemistry with the AH107 antibody to the CBFbeta-SMMHC chimeric protein showed strong nuclear staining of the tumour cell nuclei. This represents the first use of this antibody in the diagnosis of this subtype of myeloid sarcoma in the small intestine.

ALK-positive diffuse large B-cell lymphoma of the stomach associated with a clathrin-ALK rearrangement.

ALK-positive diffuse large B-cell lymphoma is a rare, recently characterized lymphoma subtype that shows granular cytoplasmic ALK expression. This report describes a primary gastric ALK-positive B-lineage lymphoma in which a clathrin (CLTC)-ALK fusion was identified by RT-PCR and direct sequencing of the breakpoint. This confirmed the presence of t(2;17)(p23;q23) involving the CLTC gene and is only the 4th report of such a translocation in this lymphoma subtype and the first to describe this tumor within the stomach. As in previous reports, immunophenotyping showed the malignant cell to be a terminally differentiated B-lineage cell characterized by the absence of B-cell antigens and expression of antigens associated with plasma cell differentiation. This case confirms the existence of such a lymphoma subtype arising in extranodal locations and underscores the importance of detailed immunophenotyping and specialized molecular genetic investigations in confirming the diagnosis.

WHO reclassification of breast lymphomas.

Fourteen cases of breast lymphoma, identified from hospital records between 1990 and 2004, were reclassified according to the World Health Organisation criteria. Primary cases occurred more frequently and all cases were of B cell origin, predominantly involving the right breast. Most primary cases were diffuse large B cell lymphomas, whereas secondary cases were heterogeneous in type and most had a poor prognosis.

Uterine serous carcinoma and endometrial intraepithelial carcinoma arising in endometrial polyps: report of 5 cases, including 2 associated with tamoxifen therapy.

Uterine serous carcinoma (USC) is the prototype of type II endometrial cancer. Endometrial intraepithelial carcinoma (EIC) is the precursor lesion of USC, Rarely, USC and EIC may arise within and be largely confined to otherwise benign endometrial polyps. This report describes 5 such cases. The patients ranged in age from 67 to 89 years, with a mean age of 75 years. In 2 of the cases there was a history of tamoxifen therapy. In 2 cases USC or EIC was confined to the endometrial polyp, and in 3 cases there was focal involvement of nonpolypoid endometrium. In 1 case there was a single small focus of extrauterine tumor within an ovarian vascular channel. In 2 cases the invasive tumor within the polyp also contained areas of endometrioid adenocarcinoma, and in 2 cases there was a component of clear cell carcinoma. In all cases USC and EIC were strongly reactive for p53 and showed a high proliferation index with MIB1. Two cases were negative with estrogen receptor, and 3 cases exhibited positive staining. The cases reported herein show that USC and EIC may rarely arise in benign endometrial polyps and that extrauterine involvement may be present without myometrial infiltration. Because 2 of the patients had been taking tamoxifen, this raises the possibility of an association between tamoxifen and the development of USC and EIC in the endometrial polyps that are characteristic of this medication.

Immunoglobulin gene rearrangement investigations in the diagnosis of lymphoid malignancies from formaldehyde-fixed biopsies.

Determination of the biologic potential of lymphoid proliferations in biopsies can be difficult by standard histological or even immunohistochemical examination. Polymerase chain reaction (PCR) has been used with increasing frequency to detect clonal rearrangements of the immunoglobulin heavy chain (IgH) in formaldehyde fixed, paraffin wax embedded tissues. Sensitivity ranges between 50 and 80%, and therefore at least 20% of neoplasms remain undetected by these approaches. Few investigators have attempted to detect immunoglobulin light chain (IgL) gene rearrangements by PCR using paraffin wax embedded samples. We studied 29 cases of B-cell neoplasms, along with 21 cases with equivocal histology and 4 reactive biopsies, using degenerate oligoprimers to amplify Ig(kappa) and Ig(lambda) light chain genes, along with IgH (Fr 1, 2 and 3) gene rearrangement analysis. The combination of these methods detected clonality in 93% of cases (27/29) with histological diagnosis of B-NHL. Fr2 and Fr3 primers detected clonality in 79% (23/29) of cases. IgL chain rearrangements detected 4 cases (14%), negative for IgH rearrangements, improving sensitivity from 79 to 93%. Clonality was detected in 52% (11/21) of histologically equivocal lymphoid proliferations, including one case detected by IgL rearrangements which was negative for IgH rearrangements. Archival material from 4 cases with reactive histology produced polyclonal results. These results confirm that PCR based immunoglobulin gene rearrangement is a sensitive and specific method for demonstrating B-cell clonality in paraffin-wax embedded sections. The addition of IgL analysis to the IgH assay allows the detection of greater than 90% of B-cell lymphoproliferative disorders from routine histological specimens with poor preservation of genomic DNA.

Mantle cell lymphoma presenting as a breast mass.

Breast lymphoma accounts for less than 1% of all non-Hodgkin's lymphomas (NHLs) and approximately 0.1% of all breast neoplasms. Most breast lymphomas are classified as diffuse large B cell or mucosa associated lymphoid tissue (MALT) lymphomas. The case of a 53 year old woman presenting with a breast mass and found to have mantle cell lymphoma is described. Core biopsy of the breast lesion showed a B cell NHL, probably of large cell type and of high grade. Morphological and immunophenotypic analysis of peripheral blood and bone marrow samples suggested a mantle cell lymphoma (MCL). This was confirmed by the detection of a t(11;14) in the bone marrow aspirate and breast tissue by polymerase chain reaction analysis. There have been no previous reports of an MCL presenting as a breast lump. Because a diagnosis of MCL has prognostic and therapeutic implications, this case highlights the need for an awareness of MCL presenting in this way, and the requirement for specialised investigations in its detection.

Induction of apoptosis by mitomycin-C in an ex vivo model of bladder cancer.

OBJECTIVE: To examine mitomycin-C (MMC)-induced apoptosis in an ex vivo model of superficial TCC, and relate it to the in vivo response to chemotherapy. Materials and methods Dose- and time-response curves were constructed to determine the optimal conditions for the induction of apoptosis by MMC in an ex vivo model of superficial bladder cancer. Subsequently, 41 individual tumours were exposed to MMC in the model and the effects assessed by measuring of apoptosis before and after chemotherapy. The relationships between tumour grade and stage and the intrinsic and induced apoptotic counts were determined. In tandem, in a clinical study, the relationship between in vivo response of a marker tumour to MMC and the ex vivo induction of apoptosis was determined. RESULTS: In the ex vivo model, apoptosis was induced at a MMC concentration of 0.5 mg/mL after an incubation time of 8 h. In 41 tumours the intrinsic apoptotic index (AI) was higher with increased grade and stage of tumour (P = 0.048). There was no correlation between the intrinsic AI and the AI after treatment with MMC (induced AI). In 21 tumours (51%) the induced AI did not increase above a predetermined response threshold and these tumours were considered resistant to MMC. Resistance to MMC was related to tumour grade (P = 0.037) with a trend for G3 pT1 tumours to be resistant to the therapy. There was a significant association between ex vivo sensitivity and in vivo marker tumour response (P = 0.02). CONCLUSIONS: Apoptosis is differentially induced in an ex vivo incubation model of superficial TCC by MMC and evidence suggests that this response matches that seen in vivo. The measurement of apoptosis before therapy does not predict the apoptotic response of a tumour to chemotherapy. The ability to undergo apoptosis correlates with clinical outcome.

Fluorescence in situ hybridisation detection of erbB2 amplification in breast cancer fine needle aspirates.

AIM: To develop a method for the detection of amplification of the erbB2 oncogene in breast cancer fine needle aspirates using fluorescence in situ hybridisation (FISH) and to compare amplification with immunohistochemical detection of the erbB2 protein. METHODS: A digoxigenin labelled probe to the erbB2 gene was hybridised to 15 aspirates prepared from operative breast cancer specimens. A chromosome 17 centromere probe was also hybridised to the aspirates either separately or in combination with the erbB2 probe. The aspirates were scored for erbB2 amplification and chromosome 17 centromere number. Subsequently, paraffin wax embedded sections of the tumours were stained with the antibody CB11 and scored for the presence of membrane staining. RESULTS: Three of the 15 tumour aspirates showed high level amplification of erbB2 detected by FISH. These three tumours also showed chromosome 17 polysomy and diffuse membrane staining by immunohistochemistry. CONCLUSIONS: FISH can be used to detect erbB2 amplification in fine needle aspirates and results correlate with conventional immunohistochemical staining. Difficulties were encountered in the visualisation of the signals in non-amplified cases without the use of specialised digital imaging.

Interphase cytogenetics of chromosomes 11 and 17 in fine needle aspirates of breast cancer.

The aims of this investigation were to compare quantitative with qualitative analysis of fluorescent in situ hybridization (FISH) centromere signals in interphase breast cancer cell nuclei and to evaluate the possible clinical utility of detecting numerical abnormalities of chromosomes 11 and 17 by FISH in the preoperative prediction of breast cancer histological grade. Commercial digoxigenin-labeled centromere probes to chromosomes 11 and 17 were hybridized to 69 malignant aspirates with histological follow-up. Aspirates were categorized as disomic or aneusomic for chromosomes 11 and 17 qualitatively; a subset of aspirates was also analyzed quantitatively. The quantitative and qualitative approaches resulted in almost identical categorisation. There was a significant association between the qualitative categorization of aspirates as aneusomic or disomic, the histological grade of the excised tumours (P = .0695, n = 69), and the cytological grade of the clinical aspirates (P = .006, n = 35). Although histological grade III tumors were almost invariably polysomic for one or both chromosomes, polysomy was also detected in grade I and II tumors. Qualitative FISH analysis was shown to be more sensitive than cytological grading in predicting histological grade III but was of lower specificity and was therefore not clinically useful.

Detection of chromosomal numerical abnormalities in clinical breast tumour fine-needle aspirations by fluorescence in situ hybridisation (FISH): refinement of a method.

This study sought to refine a fluorescence in situ hybridisation (FISH) technique for the study of numerical chromosome aberrations in cells recovered from breast tumours by fine-needle aspiration (FNA). Such techniques have been used to study numerical aberrations of chromosomes from tumours from many sites. This technique is intended for use in a routine cytopathology laboratory, and no specialised cytogenetics knowledge is necessary. Slide preparation, slide and probe denaturation, and stringency conditions were investigated. We found that the simplest slide preparation technique (direct smears) gave the best results. No difference was seen when slide and probe were denatured together or separately. Stringency conditions must be adapted to suit particular probes. Study of numerical chromosomal changes may help determine their significance in the clinical progression of the disease. Such changes may prove to have prognostic significance and therefore influence treatment of breast cancer. FISH on interphase cells is a technically straightforward, reproducible technique, suited to the study of numerical chromosome aberrations in tumour cells. The method has the potential for use in the study of malignant cells from sites other than breast, including ovary, prostate, lung and bladder.

Comparison of p53 and DNA content abnormalities in adenocarcinoma of the oesophagus and gastric cardia.

This study examined the association between 17p allelic loss, p53 gene mutation, p53 protein expression and DNA aneuploidy in a series of adenocarcinomas arising in the oesophagus and gastric cardia. 17p allelic loss was detected in 79% (15 of 19) of oesophageal and in 83% (29 of 35) of gastric adenocarcinomas. p53 mutations were detected in 70% (14 of 20) and 63% (26 of 41) of oesophageal and of gastric adenocarcinomas respectively. Both tumour types were associated with a predominance of base transitions at CpG dinucleotides. In five cases of oesophageal adenocarcinoma, the same mutation was detected both in tumour and in adjacent dysplastic Barrett's epithelium. Diffuse p53 protein expression was detected in 65% (13 of 20) and 59% (24 of 41) of oesophageal and of gastric tumours, respectively, and was associated with the presence of p53 missense mutation (Chi-squared, P < 0.0001). DNA aneuploidy was detected in 80% (16 of 20) of oesophageal and in 70% (28 of 40) of gastric tumours. No association was found between p53 or DNA content abnormalities and tumour stage or histological subtype. In conclusion, this study detected a similar pattern of p53 alterations in adenocarcinoma of the oesophagus and gastric cardia--molecular data consistent with the observation that these tumours demonstrate similar clinical and epidemiological features.

TP53 mutation in ovarian carcinoma.

This short report describes the detection of mutations of the TP53 tumour suppressor gene in sporadic ovarian carcinomas using archival paraffin-embedded tissues and automated fluorescent DNA sequencing. TP53 mutations were detected in eight tumours. Missense mutations predominated and all were transitions. Mutations were commonest in late-stage serous tumours. In three cases, where tissue was available, the mutations were homogeneous throughout several sections of the bilateral ovarian tumours and in omental metastases. These data confirm the findings of previous investigations describing TP53 mutation in ovarian carcinoma and demonstrate that archival paraffin-embedded tissues can be used for such analyses.

Validation of a rapid method to quantify apoptosis in superficial bladder cancer.

OBJECTIVES: To derive and validate a rapid method for calculating apoptotic indices in superficial transitional cell carcinoma (TCC) as a measure of chemosensitivity to mitomycin. MATERIALS AND METHODS: Apoptotic cells, identified by light microscopy in 20 superficial TCC specimens, were expressed as an index of the total tumour cell population within defined fields. For a given field, the total cell population was estimated by: (i) an exhaustive count of the total number of cells in the field and (ii) an abbreviated method in which the number of cells in a subfield was multiplied to provide an estimate of the total field number. Field and specimen estimates were compared using agreement statistics and the intra- and inter-observer reproducibility of apoptotic indices calculated. RESULTS: Cellularity and apoptotic indices obtained using method (ii) were correlated significantly with the true cell counts (P < 0.001). Agreement statistics showed that only 9.4% of counts fell outside two standard deviations (SD) from the mean in field analysis, and only 10% of counts fell outside 2 SD from the mean in specimen analysis. There was a fivefold variation in tumour cell counts among individual fields. CONCLUSIONS: The reported variation in cellularity among fields shows that the calculation of apoptosis must use the total cell population as the reference. The limits of agreement for the estimated and true cell counts are small enough to be confident that the shorter method to estimate cellularity can be used in place of counting all cells.

Extralobar sequestration and type II congenital cystic adenomatoid malformation in an infant with congenital diaphragmatic hernia.

This case report describes an infra-diaphragmatic sequestrated type II congenital cystic adenomatoid malformation (CCAM) containing striated muscle fibers in the stroma occurring in association with an extra-lobar sequestration (ELS) and a congenital diaphragmatic hernia in a female neonate. The classification and pathogenesis of CCAM and ELS are reviewed and the relationships between these lesions discussed.