James J. Going

Expression of the HER1-4 family of receptor tyrosine kinases in breast cancer.

EGFr/HER1 and c‐erbB‐2/HER2 expression are associated with poor prognosis in breast cancer. The type I receptor tyrosine kinase (RTK) family to which they belong has four members (HER1–4). In this study, expression of HER1–4 and oestrogen receptor (ER) expression were determined by immunohistochemistry in 220 breast carcinomas. Elevated expression of HER1 was observed in 16.4%, HER2 in 22.8%, HER3 in 17.5%, and HER4 in 11.9% of these tumours. Patients whose tumours overexpressed HER1, 2 or 3 had reduced survival (p = <0.001), whereas those whose tumours overexpressed HER4 had increased survival (p = 0.013); 38.6% of cases overexpressed one or more of HER1, 2 or 3. HER4 was rarely overexpressed with other HERs (1.4% of cases). Coxs multiple regression analysis demonstrated that overexpression of HER1/2/3, HER4, and standard prognostic indicators independently affected survival. HER1–3 expression was related to ER negativity (p < 0.0001, χ2). Patients with ER‐positive, HER1–3‐positive tumours had a significantly poorer survival (p < 0.001) than those with ER‐positive/HER‐negative or HER4‐positive tumours. Expression of HER RTKs displays complex interactions between different family members. There is a strong interaction, in terms of survival, between HER expression and ER expression. The development of HER‐targeted agents (eg Herceptin, Iressa), and agents targeted at the downstream signalling pathways, therefore provides new possibilities in the treatment of breast cancer. Copyright

Consensus Statements for Management of Barrett's Dysplasia and Early-Stage Esophageal Adenocarcinoma, Based on a Delphi Process

BACKGROUND & AIMS Esophageal adenocarcinoma (EA) is increasingly common among patients with Barretts esophagus (BE). We aimed to provide consensus recommendations based on the medical literature that clinicians could use to manage patients with BE and low-grade dysplasia, high-grade dysplasia (HGD), or early-stage EA. METHODS We performed an international, multidisciplinary, systematic, evidence-based review of different management strategies for patients with BE and dysplasia or early-stage EA. We used a Delphi process to develop consensus statements. The results of literature searches were screened using a unique, interactive, Web-based data-sifting platform; we used 11,904 papers to inform the choice of statements selected. An a priori threshold of 80% agreement was used to establish consensus for each statement. RESULTS Eighty-one of the 91 statements achieved consensus despite generally low quality of evidence, including 8 clinical statements: (1) specimens from endoscopic resection are better than biopsies for staging lesions, (2) it is important to carefully map the size of the dysplastic areas, (3) patients that receive ablative or surgical therapy require endoscopic follow-up, (4) high-resolution endoscopy is necessary for accurate diagnosis, (5) endoscopic therapy for HGD is preferred to surveillance, (6) endoscopic therapy for HGD is preferred to surgery, (7) the combination of endoscopic resection and radiofrequency ablation is the most effective therapy, and (8) after endoscopic removal of lesions from patients with HGD, all areas of BE should be ablated. CONCLUSIONS We developed a data-sifting platform and used the Delphi process to create evidence-based consensus statements for the management of patients with BE and early-stage EA. This approach identified important clinical features of the diseases and areas for future studies.

Evaluating HER2 amplification and overexpression in breast cancer

The development of Herceptin (Trazumatab) makes testing for HER2 status important for choosing optimal therapy in breast cancer. This study addresses the precision, accuracy, and reproducibility of HER2 assays. HER2 was assessed retrospectively by immunohistochemistry (IHC) with Dako ‘Herceptest’, by IHC with the monoclonal antibody CB11, and by fluorescence in situ hybridization (FISH, PathVysion), in a series of 216 formalin‐fixed breast carcinomas including 191 for which quantitative HER2 data from radioimmunohistochemistry (Q‐IHC) were available. All tests were scored independently by two observers. Positivity rates varied between Herceptest (12.6%), FISH (19.4%), and CB11 IHC (28.5%). Kappa values showed that IHC‐based tests were more susceptible to inter‐observer variation (κ=0.67 and 0.74 for Herceptest and CB11, respectively) than FISH (κ=0.973). Overall test accuracy (see the Materials and methods section) for CB11 IHC (83.8%) was lower than Herceptest (87.4%) or FISH (93.2%). FISH predicted p185 HER2 overexpression (determined by Q‐IHC) better (concordance index C.Ind. 0.90) than CB11 IHC (C.Ind.=0.85) or Herceptest (C.Ind.=0.81). Of 42 cases with gene amplification by FISH, 67% were positive in the Herceptest (2+ or 3+) vs. 83% with CB11. Of 174 cases negative by FISH, 96% were negative in the Herceptest and 68% with CB11. In conclusion, FISH is the most accurate, reproducible, and precise predictor of HER2 overexpression in routine diagnostic laboratories. Copyright

Oral contraceptive use influences resting breast proliferation

The controversy that surrounds oral contraceptive use and breast cancer risk has arisen from epidemiologic studies, yet the direct effect of such use on breast tissue remains undefined. Breast epithelial proliferation was assessed by 3H-thymidine labeling of normal lobular units dissected from benign biopsies of 347 females aged 14 to 48 years. Factors shown to influence this response included cycle phase, time since menarche (breast age), and parity status. Multivariate analysis allowing for these influences was used to compare activity of natural cycles and those artificially regulated by oral contraceptives (OC). The increased activity in nulliparous OC users was highly significant (P less than .005). Comparing the effect of differences in OC type, whether combined, triphasic, progestin only, or according to estrogen or progestin content, showed a heterogeneity in response that was significant (P less than .01). Examined specifically, the formulation of OC according to progestin content did not have a significant influence, although progestin-only OC was most active, while the influence of increasing estrogen content was significant (P less than .05). However, emphasis is given to acknowledging the multiple factors and interactive processes responsible for breast epithelial stimulation when considering strategies of intervention.

Amplification, increased dosage and in situ expression of the telomerase RNA gene in human cancer

Telomere length is maintained by the enzyme, telomerase, which has been linked to cellular immortality and tumour progression. However, the reasons for the high levels of telomerase found in human tumours are unknown. We have mapped the human telomerase RNA gene, (hTR), to chromosome 3q26.3 and show the hTR gene to be amplified in four carcinomas, (2/33 cervix, 1/31 head and neck, 1/9 lung). In addition, increased copy numbers of the hTR locus was also observed in 97% of tumours. By in situ hybridisation, the histological distribution of high levels of hTR expression could be demonstrated in a lung tumour and its metastasis with hTR amplification. These results are the first report of genetic alterations involving a known component of telomerase in human cancer. Indeed, it is also the first report of the amplification of a specific locus within the chromosome 3q region frequently subject to copy number gains in human tumours. In addition, we also show for the first time the histological distribution of the RNA component of telomerase in human tumours.

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 ⩽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.

Positive Mobilization Margins Alone Do Not Influence Survival Following Pancreatico-Duodenectomy for Pancreatic Ductal Adenocarcinoma

Objective:To determine the prognostic influence of residual tumor at or within 1 mm of the mobilization margins (R1Mobilization) compared with transection margins (R1Transection) following pancreaticoduodenectomy for pancreatic ductal adenocarcinoma (PDAC). Background:The prognostic strength of R1 status increases with frequency of margin positivity and is enhanced by protocol driven pathology reporting. Currently, margins are treated uniformly with tumor at or close to any margin considered of equal prognostic significance. The resection involves a mobilization phase freeing the posterior margin and anterior surface then a transection phase requiring lympho-vascular division forming the medial resection and pancreatic transection margin. The comparative assessment of the relative importance of tumor involvement of these different margins has not previously been investigated. Methods:Retrospective analysis of 148 consecutive resections for PDAC from 1996–2007 was performed. The individual (pancreatic transection, medial, posterior, and anterior surface) margins were separately identified and analyzed by a senior pathologist. An R1 resection was defined as microscopic evidence of tumor ≤1 mm from a resection margin. R1Mobilization tumor extension included both R1Anterior and R1Posterior cases; while R1Transection included pancreatic neck/body transection, R1Medial and adjacent transection margins. Results:R1 status was confirmed in 109 patients (74%). The medial (46%) and posterior (44%) margins were most commonly involved. R1 status was found to an independent predictor of poor outcome (P < 0.001). R1Mobilization involvement only (n = 48) was associated with a significantly longer median survival of 18.9 months (95% CI, 13.7–24.8) versus 11.1 months (95% CI, 7.1–15.0) for those with R1Transection tumor involvement (n = 61) (P < 0.001). There was no significant difference in the survival of the R1Mobilization compared with R0 group (P = 0.52). Conclusions:Following pancreaticoduodenectomy for PDAC, involvement of the transection margins in contrast to mobilization margins defines a group whose outcome is significantly worse. This may impact upon the allocation of adjuvant therapy within the setting of randomized controlled trials.

Escaping from Flatland: clinical and biological aspects of human mammary duct anatomy in three dimensions

Accurate knowledge of breast duct anatomy in three dimensions is needed to understand normal breast development, how intraepithelial neoplasia may spread through a breast, and the potential for diagnostic and therapeutic access to breast parenchyma via the nipple. This paper reports three related exploratory studies. In study 1, the median number of milk‐collecting ducts in the nipple was determined in 72 breasts excised for cancer; in study 2, the volumes of all 20 complete duct systems (‘lobes’) in an autopsy breast were measured from 2 mm serial ‘subgross’ sections; and in study 3, a 3D digital model of all collecting ducts in a mastectomy nipple was made from 68 100 µm serial sections. The mastectomy nipples contained 11–48 central ducts (median 27, inter‐quartile range 21–30). In the autopsy breast, the largest ‘lobe’ drained 23% of breast volume; half of the breast was drained by three ducts and 75% by the largest six. Conversely, eight small duct systems together accounted for only 1.6% of breast volume. The 3D model of the nipple revealed three distinct nipple duct populations. Seven ducts maintained a wide lumen up to the skin surface (population A); 20 ducts tapered to a minute lumen at their origin in the vicinity of skin appendages (population B) on the apex of the nipple; and a minor duct population (C) arose around the base of the papilla. Major variations in duct morphology and extent define highly variable territories in which intraepithelial neoplasia could grow. While population A ducts appear accessible to duct endoscopy or lavage, population B and population C ducts may be less accessible. Copyright

Expression of gap junction proteins connexin 26 and connexin 43 in normal human breast and in breast tumours

Gap junctional intercellular communication (GJIC) has been proposed as a cellular mechanism for tumour suppression and there is experimental evidence in support of this. If aberrant GJIC contributes to the formation of human breast tumours, one might expect that the connexins (gap junction proteins) expressed by epithelial cells in normal human breast would be down‐regulated in tumour epithelial cells, or that tumour cells might show aberrant expression of other connexin family members. This study examines the immunocytochemical expression of connexins 26 (Cx26) and 43 (Cx43) in normal human breast, 11 benign breast lesions, two special‐type carcinomas, and 27 invasive carcinomas of no special histological type (NST). Cx26 generally was not expressed at detectable levels in normal human breast, but punctate Cx43 immunostaining of the myoepithelial cells was found. Cx43 staining of the myoepithelium was also a feature of the benign lesions and ductal carcinoma in situ (DCIS). In general, the epithelial cells of benign lesions failed to stain for either connexin. Similarly, a lobular carcinoma did not express Cx26 or Cx43, but there was punctate Cx43 in the epithelial cells of a mucoid carcinoma. Cx26 was up‐regulated in the carcinoma cells of 15 of the 27 invasive NST carcinomas, although the staining was usually cytoplasmic and heterogeneous. Cx43 was expressed by stromal cells, possibly myofibroblasts, in all NST carcinomas. Furthermore, there was heterogeneous Cx43 expression in the carcinoma cells of 14 of the 27 NST carcinomas and the staining was often intercellular and punctate, characteristic of functional connexins. Up‐regulation of Cx26 and/or Cx43 in the carcinoma cells of over two‐thirds of invasive lesions of NST is not necessarily inconsistent with a tumour suppressor role for GJIC. However, the role of gap junctions in the formation and progression of solid human tumours is likely to be more complex than indicated from experimental systems.

Aberrant expression of minichromosome maintenance proteins 2 and 5, and Ki-67 in dysplastic squamous oesophageal epithelium and Barrett's mucosa

Background: Minichromosome maintenance (Mcm) proteins are essential for eukaryotic DNA replication, and their expression implies potential for cell proliferation. Expression is dysregulated in dysplastic states but data for oesophageal squamous mucosa and Barretts mucosa have not been published. Aim: To test the hypothesis that Mcm proteins are downregulated together with the proliferation marker Ki-67 in differentiating epithelial compartments of non-dysplastic squamous and Barretts epithelium, and that this process does not occur in dysplastic mucosae. Methods and cases: Forty five patients with Barretts oesophagus included 20 with glandular dysplasia (10 low grade, eight high grade, two both, and four with invasive adenocarcinoma). Twenty five other patients included 12 with oesophageal squamous dysplasia (three low grade, six high grade, three both, and four with invasive squamous carcinoma). Formalin fixed paraffin embedded tissue sections from biopsy series and resections were immunostained using antibodies to Mcm2, Mcm5, and Ki-67. Percentage of nuclei positive for Mcm2, Mcm5, and Ki-67 was estimated and scored from 0 to 6 as: 0, none +; 1, <10%+; 2, 10–30%+; 3, 30–70%+; 4, 70–90%+; 5, >90%+; 6, all+. Four separate epithelial strata were scored: in squamous epithelium the basal layer and thirds to the surface, in Barretts mucosa the luminal surface, upper and lower crypt, and deep glands. Results: In non-dysplastic squamous epithelium and Barretts mucosa, high level expression of Mcm2, Mcm5, and Ki-67 proteins was largely confined to the proliferative compartments and downregulated in differentiated compartments. Expression persisted up to the mucosal surface in dysplastic squamous epithelium and Barretts mucosa. Conclusions: Persistent expression of Mcm2, Mcm5, and Ki-67 proteins in luminal compartments of dysplastic oesophageal squamous epithelium and dysplastic Barretts mucosa may be diagnostic markers and imply disruption of cell cycle control and differentiation in these dysplastic epithelia.