The relationship between tumour budding, the tumour microenvironment and survival in patients with invasive ductal breast cancer.

BACKGROUND: Tumour budding has previously been reported to predict survival in several solid organ tumours, including breast; however, whether this is independent of other aspects of the tumour microenvironment is unknown. In the present study, the relationship between tumour budding, the tumour microenvironment and survival was examined in patients with invasive ductal breast cancer. METHODS: Patients presenting between 1995 and 1998 were studied (n=474). Using routine pathological sections, tumour budding was measured at the invasive margin and its association with clinicopathological characteristics and cancer-specific survival (CSS) was examined. RESULTS: Tumour budding was associated with several adverse pathological characteristics, including lymph node involvement, lymph vessel invasion (LVI), increased tumour stroma percentage (TSP) and weaker local inflammatory infiltrative. Tumour budding was associated with reduced CSS (hazard ratio (HR) 2.08, 95% confidence interval (CI) 1.14-3.09, P=0.004), independent of nodal status, molecular subtypes, tumour necrosis, CD8+, CD138+, LVI, blood vessel invasion and TSP. Further, tumour budding was independently associated with reduced CSS in node-negative patients (HR 2.63, 95% CI 1.16-5.92, P=0.020) and those who have low TSP (HR 1.98, 95% CI 1.09-3.57, P=0.024) and high-grade local inflammatory infiltrative (HR 2.27, 95% CI 1.35-5.36, P=0.014). CONCLUSIONS: Tumour budding was a significant predictor of survival in patients with invasive ductal breast cancer, independent of adverse pathological characteristics and components of tumour microenvironment. The present study further confirms the clinical utility of both tumour and host-based factors of tumour microenvironment.

The relationship between the tumour stroma percentage, clinicopathological characteristics and outcome in patients with operable ductal breast cancer.

BACKGROUND: The percentage of tumour stroma (TSP) has recently been reported to be a novel independent predictor of outcome in patients with a variety of common solid organ tumours. The aim of this study was to examine the relationship between TSP, clinicopathological characteristics and outcome in patients with invasive ductal breast cancer, in particular node negative and triple negative disease. METHODS: A total of 361 patients with primary operable invasive ductal breast cancer were included in this study. The TSP was assessed visually on the haematoxylin and eosin-stained tissue sections. With a cutoff value of 50% TSP, patients with ≤ 50% stroma were classified as the low-TSP group and those with >50% stroma were classified as the high-TSP group. RESULTS: A total of 109 (30%) patients had high TSP. Patients with high TSP were old age (P=0.035), had more Her-2-positive tumours (P=0.029), low-grade tumour inflammatory infiltrate (P=0.034), low CD68+macrophage infiltrate (P<0.001), low CD4+ (P=0.023) and low CD8+ T-lymphocytes infiltrate (P=0.017), tumour recurrence (P=0.015) and shorter cancer-specific survival (P<0.001). In node-negative patients (n=207), high TSP was associated with low CD68+macrophage infiltrate (P=0.001), low CD4+ (P=0.040) and low CD8+ T-lymphocytes infiltrate (P=0.016) and shorter cancer-specific survival (P=0.005). In triple negative patients (n=151), high TSP was associated with high tumour grade (P=<0.001), lymph node positivity (P=0.027), low CD68+macrophage infiltrate (P=0.011) and shorter cancer-specific survival (P=0.035). The 15-year cancer-specific survival rate was 79% vs 21% in the low-TSP group vs high-TSP group. In multivariate survival analysis, a high TSP was associated with reduced cancer-specific survival in the whole cohort (P=0.001), node-negative patients (P=0.007) and those who received systemic adjuvant therapy (P=0.021), independent of other pathological characteristics including host inflammatory response. However, TSP was not an independent prognostic factor for triple negative patients (P=0.151). CONCLUSIONS: A high TSP in primary operable invasive ductal breast cancer was associated with recurrence and poorer long-term survival. The inverse relation with the tumour inflammatory infiltrate highlights the importance of the amount of tumour stroma on immunological response in patients with primary operable ductal breast cancer. Implementing this simple and reproducible parameter in routine pathological examination may help optimise risk stratification in patients with invasive ductal breast cancer.

The relationship between lymphocyte subsets and clinico-pathological determinants of survival in patients with primary operable invasive ductal breast cancer.

BACKGROUND: The importance of lymphocyte subtypes in determining outcome in primary operable ductal invasive breast cancer remains unclear. The aim of present study was to examine the relationship between tumour lymphocyte subsets infiltrate and standard clinico-pathological factors and survival in patients with primary operable invasive ductal breast cancer. METHODS: The analysis of the inflammatory cell infiltrate, including lymphocyte subtypes, was undertaken using immunohistochemical techniques and visual quantitative and semi-quantitative techniques in 338 patients with ductal breast cancer. RESULTS: The majority (91%) of patients had high grade inflammatory cell infiltrate. The median follow-up of the survivors was 164 months. During this period, 65 died of their cancer. On univariate analysis, tumour inflammatory cell infiltrate, macrophages infiltrate (P<0.05), lymphocytic infiltrate (P<0.001) and CD8+ T-lymphocytic infiltrate (P<0.01) were associated with improved cancer-specific survival, whereas neutrophil (P<0.05) and CD138+ B-lymphocytic infiltrate (P<0.001) were associated with poorer cancer-specific survival. On multivariate analysis, tumour lymphocytic infiltrate (P<0.001), macrophage infiltrate (P<0.05), CD8+ T-lymphocytic infiltrate (P<0.01) and CD138+ B-lymphocytic infiltrate (P<0.001) were independently associated with cancer survival. When the significant inflammatory cell types were included with tumour-based factors in multivariate analysis only tumour size (Hazard ratios (HR): 2.55, 95% confidence interval (CI): 1.53-4.27, P<0.001), Ki-67 index (HR: 2.08, 95% CI: 1.08-4.00, P<0.05), lymphovascular invasion (HR: 4.40, 95% CI: 2.07-9.35, P<0.001), macrophage infiltrate (HR: 0.49, 95% CI: 0.33-0.73, P<0.001), lymphocytic infiltrate (HR: 0.11, 95% CI: 0.05-0.23, P<0.001), CD8+ T-lymphocytic infiltrate (HR: 0.57, 95% CI: 0.38-0.87, P<0.001) and CD138+ B-lymphocytic infiltrate (HR: 2.86, 95% CI: 1.79-4.56, P<0.001) were independently associated with cancer survival. CONCLUSION: The majority of patients with invasive ductal breast cancer had high-grade inflammatory cell infiltrate. In these patients, inflammatory cells including macrophage and lymphocytic infiltrate, and subsets CD8+ T-lymphocytic infiltrate and CD138+ B-lymphocytic infiltrate had superior prognostic value, compared with hormone status and lymph node involvement in patients with primary operable invasive ductal breast cancer.

The relationship between components of tumour inflammatory cell infiltrate and clinicopathological factors and survival in patients with primary operable invasive ductal breast cancer.

BACKGROUND: The importance of the components of host local inflammatory response in determining outcome in primary operable ductal invasive breast cancer is not clear. The aim of this study was to examine the relationship between components of the tumour inflammatory cell infiltrate and standard clinicopathological factors including hormone status (oestrogen receptor (ER), progesterone receptor (PR) and human epidermal growth factor receptor (HER)-2), Ki-67 and survival in patients with primary operable invasive ductal breast cancer. METHODS: Tumour inflammatory cell infiltrate, hormone status (ER, PR and HER-2), Ki-67 and standard clinicopathological factors were determined using routine pathological and immuno-histochemical techniques in 468 patients. RESULTS: The large majority (94%) of ductal tumours had evidence of inflammatory cell infiltrate. The general inflammatory cell infiltrate was positively associated with high grade (P<0.001), the absence of ER (P<0.001), the absence of PR (P<0.01), the presence of vascular invasion (P<0.05) and high lymphocytic infiltrate, plasma cell infiltrate, other inflammatory cell infiltrate and macrophage infiltrate (all P<0.001). The median follow-up of the survivors was 165 months. During this period, 93 patients died of their cancer. On univariate analysis, stratified for ER status, tumour size (P<0.01), lymph node involvement (P<0.001), tumour plasma cell infiltrate (P<0.001), other inflammatory cell infiltrate (P<0.05) and treatment (P<0.05) were associated with poorer cancer-specific survival whereas lymphocyte infiltrate (P<0.001) was associated with improved cancer-specific survival. On multivariate analysis, stratified for ER status, lymph node involvement (P<0.05) was independently associated with poorer cancer-specific survival whereas increased tumour lymphocyte infiltrate (P<0.001) was independently associated with improved cancer-specific survival. CONCLUSION: The results of this study show that, using routine histology, the general inflammatory cell infiltrate was a common feature and was positively associated with high grade, the absence of ER, the absence of PR, the presence of vascular invasion and high-grade infiltration of lymphocytes, plasma cells, other inflammatory cells and macrophages. Also, that within a mature cohort of patients, a high lymphocytic infiltrate was associated with improved survival, independent of clinicopathological characteristics including ER status, in primary operable ductal invasive breast cancer. These results rationalise previous work and provide a sound basis for future studies in this important area of breast cancer research.

The relationships between cellular components of the peritumoural inflammatory response, clinicopathological characteristics and survival in patients with primary operable colorectal cancer.

BACKGROUND: The host inflammatory response is an important determinant of cancer outcome. We examined different methods of assessing the local inflammatory response in colorectal tumours and explored relationships with both clinicopathological characteristics and survival. METHODS: Cohort study of patients (n=130) with primary operable colorectal cancer and mature follow-up. Local inflammatory response at the invasive margin was assessed with: (1) a semi-quantitative assessment of peritumoural inflammation using Klintrup-Makinen (K-M) grading and (2) an assessment of individual immune cell infiltration (lymphocytes, plasma cells, neutrophils, macrophages and eosinophils). RESULTS: The peritumoural inflammatory response was K-M low grade in 48% and high grade in 52%. Inflammatory cells were primarily macrophages, lymphocytes and neutrophils with relatively few plasma cells or eosinophils. On univariate analysis, K-M grade, lymphocyte infiltration and plasma cell infiltration were associated with cancer-specific survival. On multivariate analysis, only systemic inflammatory response, TNM (tumour, node and metastases) stage, venous invasion, tumour necrosis and K-M grade were independently associated with cancer-specific survival. There was no relationship between local infiltration of inflammatory cells and a systemic inflammatory response. However, high K-M grade, lymphocyte infiltration and plasma cell infiltration were associated with a number of favourable pathological characteristics, including an absence of venous invasion. CONCLUSION: Infiltration of inflammatory cells in the invasive margin of colorectal tumours is beneficial to survival. The adaptive immune response appears to have a prominent role in the prevention of tumour progression in patients with colorectal cancer.

The relationship between tumour site, clinicopathological characteristics and cancer-specific survival in patients undergoing surgery for colorectal cancer.

AIM: It is recognised that colorectal cancer may arise from different genomic instability pathways. There is evidence to suggest that colon and rectal cancers exhibit different clinicopathological features. We examined the relationship between tumour site, clinicopathological characteristics and cancer-specific survival in patients undergoing potentially curative resection for colorectal cancer. METHOD: Four hundred and eleven patients who underwent surgery. Clinicopathological data including components of the Peterson index, Klintrup scores, haemoglobin and the modified Glasgow Prognostic Score (mGPS) were studied. RESULTS: There were 134 (33%) right sided, 125 (30%) left sided and 152 (37%) rectal tumours. Emergency presentation (P < 0.001), anaemia (P < 0.001), higher mGPS (P < 0.001), advanced T stage (P < 0.001), poor differentiation (P < 0.001) and older age (P < 0.05) were more commonly observed in right sided cancer. The mean follow-up was 94 months (minimum 36 months) and 114 patients died of cancer. There was no difference between tumour site and survival (P = 0.427). On multivariate analysis older age (P = 0.015), lymph node ratio (P < 0.001), mGPS (P = 0.028), Peterson Index (P < 0.001) and Klintrup score (P = 0.008) were independently related to cancer-specific survival. Klintrup score was only associated with poor cancer-specific survival in rectal cancer (P = 0.009). CONCLUSION: The study suggests that colorectal cancer is a group of heterogeneous tumours with different clinicopathological features. Despite this, there was no difference between tumour site and survival. The prognostic role of clinicopathological factors in tumours arising from different genomic instability pathways requires further study.

Prospective study of the role of inflammation in renal cancer.

BACKGROUND: The local and systemic inflammatory responses provide prognostic information in cancer. The modified Glasgow Prognostic Score (mGPS) provides additional prognostic information than C-reactive protein (CRP) alone when assessing the systemic inflammation in cancer. The aim of this study was to determine the role of local and systemic inflammation in renal cancer. METHODS: The cohort consisted of 79 patients who had undergone potential curative resection. Systemic inflammation, mGPS, was constructed by measuring preoperative CRP and albumin concentrations and the Klintrup-Makinen score was evaluated histologically for the local inflammatory response. Pathological parameters such as T stage, grade and tumour necrosis were also assessed. The local inflammatory response was assessed by examining all inflammatory cells at the tumour edge on diagnostic haematoxylin and eosin slides. RESULTS: On univariate analysis, T stage (p < 0.001), grade (p = 0.044) and mGPS (p < 0.001) were significant predictors of cancer-specific survival. On multivariate analysis, mGPS (hazard ratio 8.64, 95% confidence interval 3.5-21.29, p < 0.001) was the only significant independent predictor of cancer-specific survival. CONCLUSION: A preoperative systemic inflammatory response as measured by the mGPS is an independent predictor of poor cancer-specific survival in renal cancer in patients undergoing potential curative resection.

The relationship between tumour necrosis, tumour proliferation, local and systemic inflammation, microvessel density and survival in patients undergoing potentially curative resection of oesophageal adenocarcinoma.

BACKGROUND: There is increasing evidence that the local and systemic inflammatory responses are associated with survival in oesophageal cancer. The aim of this study was to examine the relationship between tumour necrosis, tumour proliferation, local and systemic inflammation and microvessel density and survival in patients undergoing potentially curative resection of oesophageal adenocarcinoma. METHODS: The interrelationship between tumour necrosis, tumour proliferation, local inflammatory response (Klintrup-Makinen criteria, intra-tumoural CD8+ lymphocyte and macrophage infiltration), systemic inflammatory response (modified Glasgow Prognostic score (mGPS)), and microvessel density was examined in 121 patients undergoing potentially curative resection for oesophageal adenocarcinoma (including type I and II tumours of the gastro-oesophageal junction). RESULTS: Tumour necrosis was not significantly associated with any tumour measure other than the degree of differentiation. On multivariate analysis, only age (HR 1.93, 95% CI 1.23-3.04, P=0.004), mGPS (HR 2.91, 95% CI 1.51-5.62, P=0.001), positive to total lymph node ratio (HR 2.38, 95% CI 1.60-3.52, P<0.001) and macrophage infiltration (HR 1.49, 95% CI 1.02-2.18, P=0.041) were independently associated with cancer-specific survival in oesophageal adenocarcinoma. Intra-tumoural macrophages were associated with tumour proliferation (P<0.001) and CD8+ lymphocytes infiltration (P<0.01). CONCLUSION: The results of this study suggest that tumour necrosis does not link local and systemic inflammatory responses and is not significantly associated with survival. In contrast, tumour macrophage infiltration appears to have a central role in the proliferative activity and the coordination of the inflammatory cell infiltrate and is independently associated with poorer survival in patients with oesophageal adenocarcinoma.

Comparison of visual and automated assessment of Ki-67 proliferative activity and their impact on outcome in primary operable invasive ductal breast cancer.

BACKGROUND: Immunohistochemistry of Ki-67 protein is widely used to assess tumour proliferation, and is an established prognostic factor in breast cancer. There is interest in automating the assessment of Ki-67 labelling index (LI) with possible benefits in handling increased workload, with improved accuracy and precision. PATIENTS AND METHODS: Visual and automated assessment of Ki-67 LI and survival were examined in patients with primary operable invasive ductal breast cancer. Tissue microarrays (n=379 patients) immunostained for Ki-67 were scored visually and automatically with the Slidepath Tissue IA system. RESULTS: Visual and automated Ki-67 LI were in excellent agreement (ICCC=0.96, P<0.001). On univariate analysis, visual (P<0.001) and automated Ki67 LI (P<0.05) were associated with cancer-specific survival in patients with invasive ductal breast cancer overall and in patients who received endocrine therapy (Tamoxifen) (P<0.01 for visual and P<0.05 for automated scoring). CONCLUSION: Automated assessment of Ki-67 LI would appear to be comparable to visual Ki-67 LI. However, automated Ki-67 LI assessment was inferior in predicting cancer survival in patients with breast cancer, including patients who received Tamoxifen.

Is the presence or absence of tumour necrosis a significant predictor of survival in renal cell cancer?

OBJECTIVE: Currently when renal cancer pathology is assessed the presence or absence of necrosis is simply reported. It has been suggested that a presence or absence response ignores heterogeneity and a classification based on the extent of necrosis involvement would aid prognostic value in cancer-specific survival. The aim of this study was to determine whether a quantitative assessment of tumour necrosis would provide additional prognostic information. METHODS: We studied the pathological features and cancer-specific survival of 47 patients with renal cancer undergoing surgery with curative intent. A quantitative assessment of tumour necrosis was compared to the presence or absence of necrosis. RESULTS: Tumour necrosis was present in 27 of 47 cases. A simple assessment of the presence or absence was not associated with cancer-specific survival (p = 0.052). When assessed quantitatively, tumour necrosis was associated with decreased cancer-specific survival (p < 0.001). A 2-tiered assessment, <25% and >25% involvement of necrosis, was further utilised and shown to predict cancer-specific survival (p < 0.001). On multivariate analysis, using this 2-tiered assessment of <25% and >25% involvement of necrosis was retained as a significant independent factor for cancer-specific survival (HR 11.84, 95% CI 3.81-36.75, p < 0.001). CONCLUSION: A simple assessment of the presence/absence of tumour necrosis is reported to be a prognostic factor in renal cell cancer. In this study, the presence/absence was not shown to be a significant prognostic marker of cancer-specific survival. However, a more accurate quantitative assessment of tumour necrosis, whereby a 2-tiered response is still utilised, but basing this on <25% and >25% involvement of necrosis was statistically significant and independent in predicting cancer-specific survival.

Still waiting for predictive biomarkers in Barrett's oesophagus.

Barrett's oesophagus is important as a precursor of oesophageal adenocarcinoma via a metaplasia-dysplasia-carcinoma sequence. It is often detected on upper gastrointestinal endoscopy. In the absence of glandular dysplasia the risk of progression to cancer is low but ascertainment of dysplasia is not always straightforward. Sparse mucosal sampling may miss dysplasia, or reactive changes may be overinterpreted due to inter and intraobserver variation. Low-grade and even high-grade dysplasia do not necessarily progress, provided prevalent cancer has been rigorously excluded. This indeterminacy motivates an ongoing search for clinically useful predictive biomarkers. Although many genetic and epigenetic abnormalities have been associated with neoplastic progression in Barrett's mucosa no molecular tests have as yet been accepted into routine pathology practice. Challenges of assay definition remain and many marker studies lack statistical power or have other methodological flaws. Even where strong evidence of clinically relevant predictive value does exist (in the case of ploidy analysis by flow or image cytometry) adoption has been minimal, likely reflecting technological and possible reimbursement obstacles. Well designed multicentre studies are likely to be required to translate improved knowledge of Barrett's carcinogenesis into clinically significant progress on predictive testing, and will require a degree of cooperation not so far widely seen in the field.

Aberrant expression of TopBP1 in breast cancer.

AIMS: The TopBP1 protein includes eight BRCT domains (originally identified in BRCA1) and has homology with BRCA1 over the carboxyl terminal half of the protein. The aim of this study was to determine whether TopBP1 is aberrantly expressed in breast cancer. METHODS AND RESULTS: Sixty-one breast carcinomas from an unselected consecutive patient cohort were studied along with 12 samples of breast tissue from cosmetic breast reduction surgery; these were analysed immunohistochemically for TopBP1 expression using a rabbit polyclonal antibody. This antibody was validated in immunoprecipitation and immunofluorescence experiments. Immunohistochemical analysis demonstrated that TopBP1 was expressed almost exclusively in the nuclei of the normal breast epithelium. However, in a significant number of breast carcinomas TopBP1 was aberrantly expressed, as it was detected in the cytoplasm and nucleus of some tumours and exclusively in the cytoplasm of others. In two out of 61 carcinomas investigated, no TopBP1 expression was detected. CONCLUSIONS: For the first time this report demonstrates aberrant expression of the TopBP1 protein in breast carcinoma. We propose TOPBP1 as a breast cancer susceptibility gene.

Metastatic recurrence of early-stage colorectal cancer is linked to loss of heterozygosity on chromosomes 4 and 14q.

OBJECTIVE: To investigate the prognostic value for loss of heterozygosity (LOH) of chromosomes 4 and 14q in early-stage colorectal cancer (CRC). METHODS: A total of 70, largely microsatellite stable, tumours and their corresponding normal mucosa were subjected to microdissection and analysed for LOH at chromosomes 4 and 14q by using 13 highly polymorphic microsatellite markers. LOH was correlated with the survival of the patients, using univariate, multivariate and Kaplan-Meier's survival curves. RESULT: LOH at D4S2935, D4S1579 and D4S1595 on chromosome 4 was significantly associated with metastatic recurrence of early-stage CRC. For chromosome arm 14q, two minimal regions of deletion were associated with metastatic recurrence and mapped to neighbouring markers D14S275/D14S49 at 14q12-13 and D14S65/D14S250 at 14q32. High-level loss (loss of five to eight of the informative microsatellite markers) on both chromosomes 4 and 14q, to be an independent prognostic indicator in early-stage CRC was shown by multivariate analysis. CONCLUSION: Determining the LOH of chromosomes 4 and 14q and their extent in primary tumours of patients with early-stage CRC may constitute a molecular signature of metastatic recurrence. This may be achieved if new finding sheds light on the treatment of this subgroup of patients that have been largely ignored.

An elevated C-reactive protein concentration, prior to surgery, predicts poor cancer-specific survival in patients undergoing resection for gastro-oesophageal cancer.

There is increasing evidence that the presence of an ongoing systemic inflammatory response is associated with poor outcome in patients undergoing resection for a variety of tumours. The aim of the present study was to examine the relationship between clinico-pathological status, preoperative C-reactive protein concentration and cancer-specific survival in patients undergoing resection for gastro-oesophageal cancer. One hundred and twenty patients attending the upper gastrointestinal surgical unit in the Royal Infirmary, Glasgow, who were selected for potentially curative surgery, were included in the study. Laboratory measurements of haemoglobin, white cell, lymphocyte and platelet counts, albumin and C-reactive protein were carried out at the time of diagnosis. All patients underwent en-bloc resection with lymphadenectomy and survived at least 30 days following surgery. On multivariate analysis, only the positive to total lymph node ratio (hazard ratio (HR) 2.02, 95% confidence interval (CI) 1.44-2.84, P<0.001) and preoperative C-reactive protein concentration (HR 3.53, 95% CI 1.88-6.64, P<0.001) were independent predictors of cancer-specific survival. The patient group with no evidence of a preoperative systemic inflammatory response (C-reactive protein < or =10 mg l(-1)) had a median survival of 79 months compared with 19 months in the elevated systemic inflammatory response group (P<0.001). The results of the present study indicate that in patients selected to undergo potentially curative resection for gastro-oesophageal cancer, the presence of an elevated preoperative C-reactive protein concentration is an independent predictor of poor cancer-specific survival.

IHC for Her2 with CBE356 antibody is a more accurate predictor of Her2 gene amplification by FISH than HercepTest in breast carcinoma.

BACKGROUND: Her2 (c-erbB-2/neu) overexpression in breast carcinoma predicts response to the anti-Her2 monoclonal antibody, trastuzumab, and is associated with a poor prognosis. When considering patients for trastuzumab treatment, Her2 protein expression is measured by imunohistochemistry (IHC) and, where staining is equivocal, by fluorescence in situ hybridisation (FISH) detection of Her2 gene amplification. AIMS: To compare IHC using CBE356 with IHC using the Food and Drug Administration approved HercepTesttrade mark. METHODS: CBE356 and HercepTest were analysed using 167 FISH characterised breast carcinomas. Immunohistochemical expression of Her2 was measured semiquantitatively. Sensitivity, specificity, predictive values, and overall accuracy were calculated for both IHC methods using gene amplification by FISH as the end point, and IHC and FISH assays were tested in Kaplan-Meier survival analysis. RESULTS: The accuracy, sensitivity, specificity, positive predictive value (PPV), and negative predictive value (NPV) of CBE356 positive (2+ and 3+) cases were 94%, 89%, 95%, 84%, and 97%, respectively, and of HercepTest positive (2+ and 3+) cases were 91%, 66%, 98%, 92%, and 91%, respectively. A positive result with CBE356, HercepTest, or FISH was associated with significantly decreased overall survival (log rank p = 0.005, p = 0.0017, and p = 0.0005, respectively). CONCLUSIONS: Positive IHC staining for Her2 using CBE356 is 3% more accurate and 23% more sensitive at predicting Her2 gene amplification by FISH than positive staining with HercepTest. Negative IHC using CBE356 antibody is 6% more likely to represent a truly negative result than negative staining with HercepTest. Overall, CBE356 was a more accurate predictor of Her2 gene amplification by FISH than HercepTest.

Diagnosis of oesophageal cancer by detection of minichromosome maintenance 5 protein in gastric aspirates.

Symptomatic oesophageal cancer is usually advanced and the prognosis poor. Lethality of symptomatic oesophageal cancer has motivated screening for these diseases earlier in their evolution, but reliable methods for early diagnosis remain elusive. We have demonstrated that dysregulated expression of minichromosome maintenance (MCM) proteins 2-7 is characteristic of early epithelial carcinogenesis, and that these key DNA replication initiation factors can be used as diagnostic markers for cervical and genito-urinary tract cancer. In this study, we investigated whether minichromosome maintenance protein 5 (Mcm5) can be used to detect oesophageal cancer cells in gastric aspirates. Two monoclonal antibodies raised against His-tagged human Mcm5 were used in a time-resolved immunofluorometric assay to measure Mcm5 levels in cells isolated from gastric aspirates of 40 patients undergoing gastroscopy for suspected or known oesophageal carcinoma or symptoms of dyspepsia. The test discriminated with high specificity and sensitivity between patients with and without oesophageal cancer (85% sensitivity (95% confidence interval (CI)=62-97%), 85% specificity (CI=66-96%)), as demonstrated by the large area under the receiver operating characteristics curve (0.93 (95% CI=0.85-0.99)). Elevated levels of Mcm5 in gastric aspirates are highly predictive of oesophageal cancer. This simple test for oesophageal cancer is readily automated with potential applications in primary diagnosis, surveillance and screening.

A role for BRCA1 in sporadic breast cancer.

To test the hypothesis that altered expression of BRCA1 protein may play an important role in sporadic breast cancer development, 50 randomly selected primary breast cancers (frozen sections, 5 years' median follow-up) were immunolabelled with two monoclonal BRCA1 antibodies (MS110 and MS13). MS110 labelling was exclusively nuclear showing no relation to outcome or tumour pathology. Western blotting demonstrated crossreactivity, suggesting antibody nonspecificity. MS13 labelling was predominantly cytoplasmic. Intense labelling predicted decreased overall survival (P=0.012), disease-free survival (P=0.029), oestrogen receptor negativity (P=0.0004) and c-erbB-2 overexpression (P=0.006). Western blotting detected a 110 kDa molecule consistent with BRCA1 delta11b splice variant. BRCA1 protein is postulated to function as a tumour suppressor. We demonstrate cytoplasmic localisation in sporadic breast cancer suggesting excess delta11b splice variant production, reduced production of full-length BRCA1 and thus postulate reduced tumour suppressor activity. BRCA1 protein appears to have a significant role in both sporadic and hereditary breast cancers.

Human squamous cell carcinomas lose a mortality gene from chromosome 6q14.3 to q15.

Normal human keratinocytes possess a finite replicative lifespan. Most advanced squamous cell carcinomas (SCCs), however, are immortal, a phenotype that is associated with p53 and INK4A dysfunction, high levels of telomerase and loss of heterozygosity (LOH) at several genetic loci, suggestive of the dysfunction of other mortality genes. We show here that human chromosome 6 specifically reduces the proliferation or viability of a human SCC line, BICR31, possessing LOH across the chromosome. This was determined by an 88% reduction in colony yield (P<0.001), following the reintroduction of an intact normal chromosome 6 by monochromosome transfer. Deletion analysis of immortal segregants using polymorphic markers revealed the loss of a 2.9 Mbp interval, centred on marker D6S1045 at 6q14.3-q15, in 6/19 segregants. Crucially, allelic losses of this region were not identified in control hybrids constructed between chromosome 6 and the BICR6 SCC cell line that is heterozygous for chromosome 6 and which showed no reduction in colony formation relative to the control chromosome transfers. This indicates that the minimally deleted region at D6S1045 is not the result of fragile sites, a recombination hot spot, or a feature of the monochromosome transfer technique. LOH of D6S1045 was found in 2/9 immortal SCC lines and was part of a minimally deleted region of line BICR19. Furthermore, allelic imbalance, consistent with LOH, was detected in 3/17 advanced SCCs of the tongue. These results suggest the existence of a suppressor of SCC immortality and tumour development at chromosome 6q14.3-q15, which is important to a subset of human SCCs.