Tumour hypoxia is a microenvironmental factor related to poor response to radiation, chemotherapy, genetic instability, selection for resistance to apoptosis, and increased risk of invasion and metastasis. Hypoxia-regulated carbonic anhydrase IX (CA IX) has been studied in various tumour sites and its expression has been correlated with the clinical outcome. The purpose of this study was to investigate the correlation of CA IX expression with outcome in patients with invasive breast cancer. We conducted a retrospective study examining the effects of carbonic anhydrase IX (CA IX) on survival in patients with breast cancer. To facilitate the screening of multiple tissue blocks from each patient, tissue microarrays were prepared containing between two and five representative samples of tumour per patient. Immunohistochemistry was used to examine expression of CA IX in patients with breast cancer. The study includes a cohort of 144 unselected patients with early invasive breast cancer who underwent surgery, and had CA IX expression and follow-up data available for analysis. At the time of analysis, there were 28 deaths and median follow-up of 48 months with 96% of patients having at least 2 years of follow-up. CA IX was negative for 107 patients (17 deaths) and positive for 37 patients (11 deaths). Kaplan -Meier survival curves show that survival was superior in the CA IX-negative group with a 2-year survival of 97% for negatives and 83% for positives (log-rank test P ¼ 0.01). Allowing for potential prognostic variables in a Cox regression analysis, CA IX remained a significant independent predictor of survival (P ¼ 0.035). This study showed in both univariate and multivariate analysis that survival is significantly inferior in patients with tumour expressing CA IX. Prospective studies are underway to investigate this correlation in clinical trial setting.

ObjectiveUnder normoxia, non-malignant cells rely on oxidative phosphorylation for their ATP production, whereas cancer cells rely on Glycolysis; a phenomenon known as the Warburg effect. We aimed to elucidate the mechanisms contributing to the Warburg effect in human breast cancer.Experimental designLactate Dehydrogenase (LDH) isoenzymes were profiled using zymography. LDH-B subunit expression was assessed by reverse transcription PCR in cells, and by Immunohistochemistry in breast tissues. LDH-B promoter methylation was assessed by sequencing bisulfite modified DNA.ResultsAbsent or decreased expression of LDH isoenzymes 1-4, were seen in T-47D and MCF7 cells. Absence of LDH-B mRNA was seen in T-47D cells, and its expression was restored following treatment with the demethylating agent 5'Azacytadine. LDH-B promoter methylation was identified in T-47D and MCF7 cells, and in 25/ 25 cases of breast cancer tissues, but not in 5/ 5 cases of normal breast tissues. Absent immuno-expression of LDH-B protein (<10% cells stained), was seen in 23/ 26 (88%) breast cancer cases, and in 4/8 cases of adjacent ductal carcinoma in situ lesions. Exposure of breast cancer cells to hypoxia (1% O2), for 48 hours resulted in significant increases in lactate levels in both MCF7 (14.0 fold, p = 0.002), and T-47D cells (2.9 fold, p = 0.009), but not in MDA-MB-436 (-0.9 fold, p = 0.229), or MCF10AT (1.2 fold, p = 0.09) cells.ConclusionsLoss of LDH-B expression is an early and frequent event in human breast cancer occurring due to promoter methylation, and is likely to contribute to an enhanced glycolysis of cancer cells under hypoxia.

Aim: To study simultaneously the actions of maspin and CXCR4, which share several similar pathways in cancer, including apoptosis and angiogenesis. Methods: Our material consisted of 151 invasive breast carcinomas arranged in a tissue microarray setting. Maspin and CXCR4 expression was evaluated by immunohistochemistry. Microvessel density was assessed by CD34 immunodetection and apoptosis by the Tdt-mediated dUTP nick end labelling assay. Results: Maspin expression was related to CXCR4 expression, apoptosis, patient age and the Nottingham prognostic index. The expression of both maspin and CXCR4 progressively increased in high-grade tumours. In patients with lymph node negative breast cancer, maspin overexpression was associated with increased risk of death. High CXCR4 expression was associated with prolonged survival of patients with high maspin expression. Conclusions: Our results show that maspin overexpression could prove to be a potentially useful marker, especially for the clinically important group of patients with lymph node negative breast cancer. The expression of CXCR4 is of less significance in our study, but may be informative for specific patient subsets or in a longer time frame.

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Endometrial carcinomas, particularly of endometrioid type, can invade the myometrium or cervix without eliciting a stromal desmoplastic or inflammatory response and have been referred to as diffusely infiltrating endometrial carcinomas. This study describes a series of 14 endometrial carcinomas infiltrating as single "naked" glands without a stromal response. The neoplasms consisted of 12 endometrioid carcinomas, 1 mixed endometrioid and clear cell carcinoma, and 1 serous carcinoma. In all cases, there was myometrial invasion without stromal response. Seven cases exhibited cervical stromal involvement and in 2 there was involvement of both ovaries in a similar pattern. Several of the cases were seen in consultation and the pattern of infiltration raised a number of differential diagnoses, both benign and malignant, depending on the site of tumor involvement, including adenomyosis, adenomyoma, primary endocervical glandular lesions, cervical mesonephric remnants, endometriosis or tuboendometrioid metaplasia, and ovarian cortical inclusion cysts. Although this pattern of invasion has been reported previously, it continues to present diagnostic difficulties.

Evidence is accumulating to suggest that hnRNP B1 expression may be a useful tool in the early diagnosis of lung cancer. This study examined the immunohistochemical expression of hnRNP B1 in archived sections of resected lung cancers and compared the patterns of expression with those seen in similar archived sections of non-neoplastic lung. Particular attention was paid to the expression of hnRNP B1 in the benign bronchial cells in both cases, to establish if overexpression of this protein in respiratory epithelial cells is specific for malignancy. Nineteen cases of different types of non-small cell carcinoma were examined (eight squamous cell, six adenocarcinomas, two carcinosarcomas, two undifferentiated large cell carcinomas, and one mucoepidermoid carcinoma) and compared with sections from 16 open lung biopsies (three cases of cryptogenic fibrosing alveolitis, two cases of sarcoidosis, two cases of organizing pneumonia, and one case each of tuberculosis, extrinsic allergic alveolitis, non-specific interstitial pneumonitis, pneumocystis pneumonia, aspergilloma, respiratory bronchiolitis-interstitial lung disease, mineral dust disease, Sjögren's syndrome and systemic sclerosis vascular variant). All the tumours showed positive staining, with the vast majority, 16/19 (84%), showing strong diffuse nuclear staining. The background cells of these cases showed positive staining in alveolar macrophages, lymph node germinal centres, bronchial mucous glands, and bronchial epithelial cells. No significant difference was seen in the percentage of positive bronchial epithelial cells in bronchi adjacent to the tumour compared with the resection margins. In the benign lung cases, positive bronchial epithelial cells were seen in a small percentage, 3/16 (18%), of cases, but the majority of cases showed no or very focal staining. The levels of expression between benign epithelial cells of malignant cases, compared with benign, showed a significant difference when the staining was assessed in percentage of positive nuclei (p = 0.001, Fisher's exact test). The results confirm that hnRNP B1 is widely expressed in a range of lung carcinomas; that expression is seen in benign bronchial epithelial cells and inflammatory cells; and that expression in background bronchial epithelial cells appears to be higher in malignant than in benign lung disease. It is feasible that this biomarker may be of use in the detection of early lung cancer, provided that levels of expression can be accurately quantified.

BackgroundDigital pathology is now used for primary diagnostic work as well as teaching, research and consultation. In our multisite institution service reorganisation led to histopathology being located in a separate hospital from some surgical specialities. We implemented remotely supervised specimen sampling and frozen section diagnosis using digital pathology. In this study we assessed the concordance of glass and digital slide diagnosis using this system.MethodsWe reviewed cases from the first 2 years of digital frozen section reporting at our institution. Cases with potential digital to glass slide discordance were reviewed by three experienced thoracic histopathologists. The reasons for discordance were determined and common themes identified. We also reviewed critical incidents relating to digital pathology during the study period.ResultsThe study population comprised 211 cases. Frozen section to final diagnosis concordance between digital and glass slide diagnosis was found in 196 (92.6%) cases. The 15 potentially discordant cases were reviewed. Intraobserver concordance between glass and digital slide review ranged from 9/15 to 12/15 cases across the three pathologists. Glass slide review diagnosis showed better concordance with ground truth in two cases; digital slide review was more accurate in two cases. One relevant critical incident was identified during the study period.DiscussionThis is the largest study to examine digital pathology for thoracic frozen section diagnosis and shows that this is a safe and feasible alternative to glass slide diagnosis. Discordance between digital and glass slide diagnoses were unrelated to the processes of whole slide imaging and digital microscopy.

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