PDF file - 54K, Antibodies used for Array and Western Blot Immunostaining

PDF file - 103K, Validation of the reproducibility of the RPMA for HER2 and pHER2 analysis from FFPE-tissues

The aim of our study was to evaluate the quality of histo- and cytomorphological features of PAXgene-fixed specimens and their suitability for histomorphological classification in comparison to standard formalin fixation. Fifteen colon cancer tissues were collected, divided into two mirrored samples and either formalin fixed (FFPE) or PAXgene fixed (PFPE) before paraffin embedding. HE- and PAS-stained sections were scanned and evaluated in a blinded, randomised ring trial by 20 pathologists from Europe and the USA using virtual microscopy. The pathologists evaluated histological grading, histological subtype, presence of adenoma, presence of lymphovascular invasion, quality of histomorphology and quality of nuclear features. Statistical analysis revealed that the reproducibility with regard to grading between both fixation methods was rather satisfactory (weighted kappa statistic ( k w ) = 0.73 (95 % confidence interval (CI), 0.41–0.94)), with a higher agreement between the reference evaluation and the PFPE samples ( k w = 0.86 (95 % CI, 0.67–1.00)). Independent from preservation method, inter-observer reproducibility was not completely satisfactory ( k w = 0.60). Histomorphological quality parameters were scored equal or better for PFPE than for FFPE samples. For example, overall quality and nuclear features, especially the detection of mitosis, were judged significantly better for PFPE cases. By contrast, significant retraction artefacts were observed more frequently in PFPE samples. In conclusion, our findings suggest that the PAXgene Tissue System leads to excellent preservation of histomorphology and nuclear features of colon cancer tissue and allows routine morphological diagnosis.

Abstract Purpose: Targeting of the HER2 protein in human breast cancer represents a major advance in oncology but relies on measurements of total HER2 protein and not HER2 signaling network activation. We used reverse-phase protein microarrays (RPMA) to measure total and phosphorylated HER2 in the context of HER family signaling to understand correlations between phosphorylated and total levels of HER2 and downstream signaling activity. Experimental Design: Three independent study sets, comprising a total of 415 individual patient samples from flash-frozen core biopsy samples and formalin-fixed and paraffin-embedded (FFPE) surgical and core samples, were analyzed via RPMA. The phosphorylation and total levels of the HER receptor family proteins and downstream signaling molecules were measured in laser capture microdissected (LCM) enriched tumor epithelium from 127 frozen pretreatment core biopsy samples and whole-tissue lysates from 288 FFPE samples and these results were compared with FISH and immunohistochemistry (IHC). Results: RPMA measurements of total HER2 were highly concordant (>90% all sets) with FISH and/or IHC data, as was phosphorylation of HER2 in the FISH/IHC+ population. Phosphorylation analysis of HER family signaling identified HER2 activation in some FISH/IHC− tumors and, identical to that seen with FISH/IHC+ tumors, the HER2 activation was concordant with EGF receptor (EGFR) and HER3 phosphorylation and downstream signaling endpoint activation. Conclusions: Molecular profiling of HER2 signaling of a large cohort of human breast cancer specimens using a quantitative and sensitive functional pathway activation mapping technique reveals IHC−/FISH−/pHER2+ tumors with HER2 pathway activation independent of total HER2 levels and functional signaling through HER3 and EGFR. Clin Cancer Res; 18(23); 6426–35. ©2012 AACR.

Formalin fixed and paraffin embedded (FFPE) tissues are the basis for histopathological diagnosis of many diseases around the world. For translational research and routine diagnostics, protein analysis from FFPE tissues is very important. We evaluated the potential influence of six histological stains, including hematoxylin (Mayer and Gill), fast red, light green, methyl blue and toluidine blue, for yield, electrophoretic mobility in 1-D gels, and immunoreactivity of proteins isolated from formalin-fixed breast cancer tissues. Proteins extracted from stained FFPE tissues using a recently established technique were compared with proteins obtained from the same tissues but without prior histological staining. Western blot and quantitative protein lysate microarray analysis demonstrated that histological staining can result in decreased protein yield but may not have much influence on immunoreactivity and electrophoretic mobility. Interestingly, not all staining protocols tested are compatible with subsequent protein analysis. The commonly used hematoxylin staining was found to be suitable for multiplexed quantitative protein measurement technologies although protein extraction was less efficient. For best results we suggest a guided protein extraction method, in which an adjacent hematoxylin/eosin-stained tissue section is used to control dissection of an unstained specimen for subsequent protein extraction and quantification for research and diagnosis.

Epithelial–mesenchymal transition (EMT) involving down‐regulation of E‐cadherin is known to play an important role in tumour progression. The aim of our study was to investigate the mRNA expression of two EMT regulators— Slug and E12/E47 —in primary human gastric carcinomas and to compare this with the expression of E‐cadherin and other EMT regulators ( Snail, Twist , and SIP1 ). We studied a series of 59 gastric carcinomas by real‐time quantitative RT‐PCR in formalin‐fixed and paraffin‐embedded tissues. Thirty‐four cases (58%) showed Slug up‐regulation in the tumour; reduced or negative expression of E‐cadherin was present in 24 of these (71%, p < 0.0001). Twenty‐one cases (36%) showed E12/E47 up‐regulation that was not significantly associated with E‐cadherin down‐regulation ( p = 0.5734). Slug up‐regulation accompanied by E‐cadherin down‐regulation correlated with the presence of distant metastases ( p = 0.0029) and with advanced pTNM stages ( p = 0.0424). A statistically significant association was found between Slug up‐regulation and the expression of SIP1 in intestinal ( p = 0.0014) and Snail in diffuse ( p = 0.0067) carcinomas. We present the first study integrating the analysis of several EMT regulators in primary gastric carcinomas and conclude that Slug up‐regulation is associated with E‐cadherin down‐regulation in diffuse and intestinal‐type gastric carcinoma, and that this effect could be complemented by the presence of other EMT regulators. Copyright © 2007 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.

BACKGROUND:Vascular endothelial growth factor C (VEGF-C) is the only factor known to cause lymphangiogenesis. In esophageal cancer the histologic tumor type and lymph node metastasis are independent predictors of recurrence and poor outcome. To evaluate the rule of VEGF-C expression in esophageal cancer, we investigated 113 specimens, 59 squamous cell and 54 adenocarcinomas of the esophagus.METHODS:The expression of VEGF-C was evaluated using immunohistochemistry (IHC) on 59 paraffin-embedded archival specimens from patients with squamous cell esophageal carcinomas and 54 paraffin-embedded archival specimens of patients with esophageal adenocarcinomas arising in Barrett's mucosa. All patients had a complete tumor resection. A complete and updated follow-up was available for all patients.RESULTS:The expression of VEGF-C was significantly different between the two histological types of esophageal tumors. Patients with squamous cell carcinoma and lymph node metastases had a significantly higher VEGF-C expression (P < 0.01). In patients with adenocarcinoma of the esophagus there was no correlation between VEGF-C expression and clinicopathological parameters. High VEGF-C expression tended to be correlated with poor survival in squamous cell cancer but not in adenocarcinoma of the esophagus.CONCLUSIONS:The present study indicates that VEGF-C may play a role in tumor progression via lymphangiogenesis in squamous cell carcinoma of the esophagus. This seems not to be true for the adenocarcinoma of the esophagus. These data could help with the understanding of the different onset and characteristics of lymph node metastasis in squamous cell carcinoma and adenocarcinoma of the esophagus.

Telepathology (TP) is the practice of evaluating pathology cases by the digital transmission of diagnostic slides as either static pictures (static TP) or by a continuous flow of pictures from a robotic microscopy (dynamic TP). The diagnostic efficacy of dynamic TP-based consultation services has not been widely tested. Dysplasia arising in association with chronic ulcerative colitis (CUC) is, at present, the most important marker for an increased risk of malignancy in patients with this disease. However, the diagnosis of dysplasia suffers from a significant degree of intra- and interobserver variability which usually necessitates a second opinion prior to definitive treatment. Thus, it is often necessary to obtain expert consultation of potential dysplasia cases by dedicated gastrointestinal pathologists. The aim of this study was to evaluate the utility and interobserver variability of diagnosing dysplasia in CUC with the use of dynamic TP. Dynamic TP was used to evaluate digitalized images of 38 CUC cases with areas considered negative, indefinite, or positive for dysplasia (low or high grade) independently by seven pathologists. Subsequently, all cases were graded by each of the pathologists by light microscopic examination of the H&E-stained glass slides. The degree of intra- and interobserver variability was determined by Kappa statistics. Overall, there was a poor degree of interobserver agreement (K=0.32) among the seven pathologists after analysis of the cases by dynamic TP. The poorest level of agreement was in the indefinite and low-grade dysplasia categories, whereas the highest level was in the negative and high-grade dysplasia categories. Grouping together several diagnostic categories (for instance: Indefinite and low, or low- and high-grade dysplasia) had no significant effect on the level of agreement. The degree of variability in interpretation of cases by microscopic slide analysis was similar (K=0.35). After reviewing all the cases by microscopic analysis of the glass slides, the diagnosis was changed in 51% of the observations; in the majority of these (61%), the grade of dysplasia was decreased. In summary, the use of dynamic TP for consultation in CUC-associated dysplasia has a poor level of interobserver agreement, but does not differ significantly from that obtained by the evaluation of the cases by microscopic slide analysis. Diagnoses rendered by dynamic TP tend to be of a higher grade compared to that obtained by microscopic slide analysis. Thus, although dynamic TP may be used for the consultation of CUC dysplasia cases, more specific criteria are needed in the general categorization of dysplasia in CUC.

Aberrant expression and mutation of E-cadherin is frequent in gastric carcinoma (GC) especially of the diffuse type. The frequency of CDH1 (gene encoding E-cadherin) mutation in populations with high incidence of diffuse GC and its prognostic significance is unknown. One hundred seventy-seven gastrectomies from Mexican mestizo patients with intestinal (53), mixed (55), or diffuse (69) GC were included. In addition, 101 endoscopic biopsies from patients with GC not subjected to surgery were analyzed. Immunohistochemistry against wild-type E-cadherin (clone 36) and against 2 mutation-specific antibodies (MSA) recognizing mutant CDH1 lacking exon-8 (del 8) or exon-9 (del 9) were performed. Staining was correlated with histotype, tumor node metastasis stage, and follow-up. Abnormal or absent E-cadherin expression (clone 36) was identified in 84% GC, predominantly in diffuse or mixed tumors (P = 0.004) in advanced stages (P = 0.003). No survival differences at 1 and 2 years were observed among patients showing normal, abnormal, or absent wild type E-cadherin expression. Overall reactivity with the MSA was observed in 10 (5.6%) patients who were treated with surgery. In 140 patients, dead from the disease or alive with the disease, the survival at 1 and 2 years was 37% versus 17% and 14% versus 0 for patients without and with del 8/9 positivity, respectively (log rank P = 0.01). Biopsies from patients with inoperable-GC (101) rendered 5 (4.95%) with del 8 or 9 immunoreactivity. Abnormal E-cadherin expression is frequent in GC. However, exon 8 or 9 deletions were observed in only 5.3% tumors in this series from Mexico, at a lower rate than previously published, but associated with a worse prognosis.

Alterations of the cell adhesion molecule E-cadherin have been demonstrated in sporadic and hereditary gastric carcinomas. A cell adhesion molecule with functional similarity to E-cadherin is desmoglein 2 (Dsg2), a major component of the desmosomes. In this study, we investigated whether alterations of Dsg2 are involved in gastric carcinogenesis and whether germline mutations contribute to a genetic predisposition in familial gastric cancer patients with no germline mutations in the E-cadherin gene. Seventy-five formalin-fixed, paraffin-embedded tissues from 37 familial and 38 sporadic gastric carcinomas were analysed for Dsg2 expression by immunohistochemistry. DNA from 31 familial gastric cancer patients was analysed for germline mutations and five sporadic tumours were analysed for somatic mutations by DHPLC. Of the 75 tumours, 25 (33%) demonstrated abnormal (reduced and/or non-membrane-associated) Dsg2 expression. There was a trend towards more frequent abnormal expression in diffuse type (42%) than in intestinal type tumours (18%) (p = 0.066). One germline missense variant leading to a non-conservative amino acid change (c. 2810 C > A, Thr 937 Asn) was found in a familial gastric cancer patient with a diffuse type tumour. No somatic mutations were identified. The observed abnormal expression of Dsg2 protein suggests that this molecule is involved in the carcinogenesis of a subset of gastric carcinomas, in particular of the diffuse type. Somatic mutations in the gene do not seem to be a very frequent inactivation event and the finding of no clear pathogenic germline mutation rules out Dsg2 as a major gastric cancer predisposition gene.

Background: Hereditary non-polyposis colorectal cancer (HNPCC) is clinically defined by familial clustering of colorectal cancer and other associated tumours.Methods: By thorough molecular and clinical evaluation of 41 families, two different groups were characterised: group 1, 25 families with truncating mutations in MLH1 or MSH2 (12 novel mutations); and group 2, 16 Amsterdam positive families without mutations in these genes and without microsatellite instability in their corresponding tumours.Results: Significant clinical differences between these two groups were found. Firstly, earlier age of onset for all colorectal cancers (median 41 v 55 years; p<0.001) and all tumours (median 43 v 56 years; p = 0.022) was observed, comparing groups 1 and 2. Secondly, 68% of the index colorectal cancers were localised proximally of the splenic flexure in group 1 compared with 14% in group 2 (p<0.010). Thirdly, more synchronous and metachronous colorectal (p = 0.017) and extracolorectal tumours (p<0.001) were found in group 1. Fourthly, a higher colorectal adenoma/carcinoma ratio (p = 0.030) and a tendency towards more synchronous or metachronous adenomas in group 2 (p = 0.084) was observed, indicating a slower progression of adenomas to carcinomas. As three mutation negative tumours revealed chromosomal instability after comparative genomic hybridisation, these tumours may be caused by one or more highly penetrant disease alleles from the chromosomal instability pathway.Conclusion: These data show that HNPCC includes at least two entities with clinical and molecular differences. This will have implications for surveillance programmes and for cancer research.

Germline mutations of the cell adhesion molecule E-cadherin have been shown for the first time to underlie a hereditary diffuse type gastric cancer syndrome (HDGC) in Maori families,1 and subsequently have been reported in HDGC patients of various ethnic origins.2–9 Gastric cancer may also be associated with other hereditary tumour syndromes, which are mainly characterised by carcinomas of other organs. One of these syndromes is the HNPCC syndrome (hereditary nonpolyposis colorectal cancer syndrome), which is caused by germline mutations in DNA mismatch repair genes.10 Furthermore, gastric cancer has been observed in the context of the Li Fraumeni syndrome, a rare cancer syndrome due to germline mutations of the TP53 tumour suppressor gene,11–14 as well as in association with the FAP and Peutz–Jeghers syndrome.15Despite known molecular genetic causes, contributing to a genetic predisposition to gastric cancer, a considerable number of familial cases have been reported that were not attributable to one of this hereditary syndromes, suggesting that unknown susceptibility genes for gastric cancer might exist. Putative tumour suppressor genes that are commonly inactivated in sporadic gastric cancers, such as the RUNX3 or HPP1 genes, are potential candidate susceptibility genes.16,17In a previous study, we identified one E-cadherin germline mutation among seven diffuse type familial gastric cancer patients, indicating that in addition to E-cadherin , other genes might be involved in genetic predisposition to the disease in this patient group.4There were three goals of the present study. First, we aimed to extend our analysis of E-cadherin germline mutations to a higher number of patients, to evaluate the contribution of germline mutations in this gene to German familial and early onset gastric cancer patients and to characterise identified missense mutations for their functional relevance. Secondly, we wished to analyse the role …

Not many models of mammalian cell cycle system exist due to its complexity. Some models are too complex and hard to understand, while some others are too simple and not comprehensive enough. Moreover, some essential aspects, such as the response of G1-S and G2-M checkpoints to DNA damage as well as the growth factor signalling, have not been investigated from a systems point of view in current mammalian cell cycle models. To address these issues, we bring a holistic perspective to cell cycle by mathematically modelling it as a complex system consisting of important sub-systems that interact with each other. This retains the functionality of the system and provides a clearer interpretation to the processes within it while reducing the complexity in comprehending these processes. To achieve this, we first update a published ODE mathematical model of cell cycle with current knowledge. Then the part of the mathematical model relevant to each sub-system is shown separately in conjunction with a diagram of the sub-system as part of this representation. The model sub-systems are Growth Factor, DNA damage, G1-S, and G2-M checkpoint signalling. To further simplify the model and better explore the function of sub-systems, they are further divided into modules. Here we also add important new modules of: chk-related rapid cell cycle arrest, p53 modules expanded to seamlessly integrate with the rapid arrest module, Tyrosine phosphatase modules that activate Cyc_Cdk complexes and play a crucial role in rapid and delay arrest at both G1-S and G2-M, Tyrosine Kinase module that is important for inactivating nuclear transport of CycB_cdk1 through Wee1 to resist M phase entry, Plk1-Related module that is crucial in activating Tyrosine phosphatases and inactivating Tyrosine kinase, and APC-Related module to show steps in CycB degradation. This multi-level systems approach incorporating all known aspects of cell cycle allowed us to (i) study, through dynamic simulation of an ODE model, comprehensive details of cell cycle dynamics under normal and DNA damage conditions revealing the role and value of the added new modules and elements, (ii) assess, through a global sensitivity analysis, the most influential sub-systems, modules and parameters on system response, such as G1-S and G2-M transitions, and (iii) probe deeply into the relationship between DNA damage and cell cycle progression and test the biological evidence that G1-S is relatively inefficient in arresting damaged cells compared to G2-M checkpoint. To perform sensitivity analysis, Self-Organizing Map with Correlation Coefficient Analysis (SOMCCA) is developed which shows that Growth Factor and G1-S Checkpoint sub-systems and 13 parameters in the modules within them are crucial for G1-S and G2-M transitions. To study the relative efficiency of DNA damage checkpoints, a Checkpoint Efficiency Evaluator (CEE) is developed based on perturbation studies and statistical Type II error. Accordingly, cell cycle is about 96% efficient in arresting damaged cells with G2-M checkpoint being more efficient than G1-S. Further, both checkpoint systems are near perfect (98.6%) in passing healthy cells. Thus this study has shown the efficacy of the proposed systems approach to gain a better understanding of different aspects of mammalian cell cycle system separately and as an integrated system that will also be useful in investigating targeted therapy in future cancer treatments.

In our study, we aimed to investigate the expression of N‐cadherin and E‐cadherin and their dependency on epithelial‐mesenchymal transition regulators SNAI1, SIP1 and TWIST in human colon cancer. Expression of E‐cadherin and N‐cadherin was examined by immunohistochemistry in 80 colon carcinomas by using paraffin embedded and formalin fixed tissues. Those cases were partly analyzed for mRNA expression of N‐cadherin (42 cases), TWIST (18 cases), SNAI1 (25 cases) and SIP1 (25 cases) by real‐time quantitative RT‐PCR. Additionally, colon carcinomas that showed amplification of 20q13, the localization of the human SNAI1 gene, were examined. We found cytoplasmic and/or membrane‐associated immunoreactivity of N‐cadherin in 35/80 (44%) of the cases. However, there was no correlation to upregulated TWIST mRNA levels, as we have shown previously for diffuse‐type gastric cancers with abnormal N‐cadherin expression. Reduced and/or cytoplasmic E‐cadherin immunoreactivity was detected in 19% (15/80) of the cases. Expression of SNAI1 or SIP1 mRNA was not seen in any of the 25 cases analyzed. There was no correlation between amplification of 20q13 and SNAI1 mRNA expression. Remarkably, N‐cadherin was almost exclusively expressed in those cases showing normal E‐cadherin immunoreactivity, suggesting a mutual exclusion between abnormal E‐cadherin reduction and upregulation of N‐cadherin. For the first time, we postulate a role for N‐cadherin in primary colon cancer progression, which may be similar to the effect discovered by others in breast cancer cell lines, where coexpressed N‐cadherin can exert a dominant function over E‐cadherin's adhesive function and thus promote tumor invasiveness. © 2004 Wiley‐Liss, Inc.

The aim of the study was to determine epidermal growth factor receptor (EGFR) expression in gastric adenocarcinoma by standardized immunohistochemistry and to correlate EGFR expression with clinical features and patient survival. EGFR expression was investigated in paraffin sections of resection specimens of 89 gastric carcinomas from Mexican Mestizo patients using standardized immunohistochemistry with antigen retrieval (Dako EGFRpharmDxTM assay detection system). Membrane staining of EGFR was evaluated in the neoplastic cells and graded using a semiquantitative score (0–3+). Of the 89 carcinomas examined, staining of neoplastic cells was weak in 17 (19.1%, score 1+), moderate in 16 (18.0%, score 2+), and strong in nine cases (10.1%, score 3+). EGFR reactivity was heterogeneous, frequently showing completely negative up to 3+ positive areas within an individual tumor. EGFR reactivity score correlated with distant metastases (P=0.002) and clinical stage (P=0.033). EGFR score 0/1+ was significantly associated with an increase in patient survival when compared to score 2+/3+ (P=0.0006). In a multivariate analysis, EGFR positive cells in muscularis or subserosa (P=0.004), distant metastases (P=0.016) and residual disease (P=0.039) were significantly correlated with decreased survival. The prognosis was associated with the EGFR reactivity score (P=0.003), distant metastases (P=0.0001) and residual disease (P=0.012) in a univariate analysis. EGFR reactivity in neoplastic cells is an independent prognostic factor in gastric adenocarcinoma. The relevance of the heterogeneity in EGFR expression with regard to tumor progression, metastasis and anti-EGFR therapy needs to be studied.

To characterize cytogenetic alterations found in Barrett's adenocarcinoma (BA) and, more importantly, its premalignant stages, we studied chromosomal imbalances in various lesions in the histologically proposed metaplasia-dysplasia-carcinoma sequence using comparative genomic hybridization (CGH). Using 30 esophageal adenocarcinoma resection specimens, we were able to study 30 areas of Barrett's adenocarcinoma and 8 lymph node metastases (LN). In addition, we investigated 25 premalignant lesions adjacent to BA derived from a subset of 14 resection specimens including 11 areas of high grade dysplasia (HGD), 8 areas of low grade dysplasia (LGD), and 6 areas of intestinal metaplasia (IM), which were laser-microdissected and studied with CGH. To validate the CGH findings, fluorescence in situ hybridization analysis on 13 BA with probes specific for HER-2/neu and 20q13.2 were performed. The chromosomal alterations most often identified in BA were: gains on 8q (80%), 20q (60%), 2p, 7p and 10q (47% each), 6p (37%), 15q (33%) and 17q (30%). Losses were observed predominantly on the Y-chromosome (76%), 4q (50%), 5q and 9p (43. each), 18q (40%), 7q (33%) and 14q (30%). High-level amplifications were observed on 8q23-qter, 8p12-pter, 7p11-p14, 7q21–31, 17q11-q23. Recurrent chromosomal changes were also identified in metaplastic (gains on 8q, 6p, 10q, losses on 13q, Y, 9p) and dysplastic epithelium (gains on 8q, 20q, 2p, 10q, 15q, losses on Y, 5q, 9p, 13q, 18q). Novel amplified chromosomal regions on chromosomes 2p and 10q were detected in both Barrett's adenocarcinoma and premalignant lesions. An increase of the average number of detected chromosomal imbalances from IM (7.0 ± 1.7), to LGD (10.8 ± 2.2), HGD (13.4 ± 1.1), BA (13.3 ± 1.4), and LN (22 ± 1.2) was seen. Although the detection of common chromosomal alterations in premalignant lesions and adjacent carcinomas suggest a process of clonal expansion, the occurrence of several chromosomal changes in an apparently random order relative to one another is striking evidence that clonal evolution is more complex than would be predicted by linear models. This is probably a reflection of the existence of many divergent neoplastic subpopulations and highlights one of the main problems associated with surveillance of Barrett's patients, namely sampling error.