The utility of cMet as a diagnostic tissue biomarker in primary colorectal cancer

The transmembrane protein, cMet, is thought to be overexpressed and activated in colorectal cancer (CRC). This study explored its potential as a diagnostic tissue biomarker for CRC in a large human CRC tissue collection obtained from a randomized clinical trial.Tissue microarrays of matched normal colorectal epithelium and primary cancer were prepared from specimens obtained from 280 patients recruited to the MRC CLASICC trial (ISRCTN 74883561) and interrogated using immunohistochemistry for cMet expression. The distribution and intensity of immunopositivity was graded using a validated, semiquantifiable score, and differences in median scores analysed using the Wilcoxon signedrank test. A receiver operating characteristic (ROC) curve was plotted to measure the diagnostic accuracy of cMet as a biomarker in CRC.Epithelial cell membrane expression of cMet differed significantly between CRC and normal colorectal tissue: median 12.00 (Interquartile range (IQR) 615) versus median 6.00 (IQR 2.7012.00) respectively (P=<.0001). ROCAUC analysis of cMet expression yielded a CRC diagnostic probability of 0.66 (95% CI: 0.61 to 0.70; P<.0001). A score of 14.50 showed high specificity at 85.32% (95% CI 80.33%89.45%) but sensitivity of only 30.92% (CI 25.37%36.90%).Thus cMet is consistently overexpressed in human CRC as compared to normal colorectal epithelium tissue. cMet expression may have a role in diagnosis and prognostication if combined with other biomarkers.     

Aberrant promoter methylation of beta‐1,4 galactosyltransferase 1 as potential cancer‐specific biomarker of colorectal tumors

Epigenetic alterations, such as CpG islands methylation and histone modifications, are recognized key characteristics of cancer. Glycogenes are a group of genes which epigenetic status was found to be changed in several tumors. In this study, we determined promoter methylation status of the glycogene beta‐1,4‐galactosyltransferase 1 (B4GALT1) in colorectal cancer patients. Methylation status of B4GALT1 was assessed in 130 colorectal adenocarcinomas, 13 adenomas, and in paired normal tissue using quantitative methylation specific PCR (QMSP). B4GALT1 mRNA expression was evaluated in methylated/unmethylated tumor and normal specimens. We also investigated microsatellite stability and microsatellite instability status and KRAS/BRAF mutations. Discriminatory power of QMSP was assessed by receiving operating curve (ROC) analysis on a training set of 24 colorectal cancers and paired mucosa. The area under the ROC curve (AUC) was 0.737 (95% confidence interval [CI]:0.591–0.881, P = 0.005) with an optimal cutoff value of 2.07 yielding a 54% sensitivity (95% CI: 35.1%–72.1%) and a specificity of 91.7% (95% CI: 74.1%–97.7%). These results were confirmed in an independent validation set where B4GALT1 methylation was detected in 52/106 patients. An inverse correlation was observed between methylation and B4GALT1 mRNA expression levels (r = ?0.482, P = 0.037). Significant differences in methylation levels and frequencies was demonstrated in invasive lesions as compared with normal mucosa (P = 0.0001) and in carcinoma samples as compared with adenoma (P = 0.009). B4GALT1 methylation is a frequent and specific event in colorectal cancer and correlates with downregulation of mRNA expression. These results suggest that the glycogene B4GALT1 represent a valuable candidate biomarker of invasive phenotype of colorectal cancer. © 2012 Wiley Periodicals, Inc.     

Simultaneous assessment of DNA ploidy and biomarker expression in paraffin‐embedded tissue sections

Fleskens S J H M, Takes R P, Otte‐Höller I, van Doesburg L, Smeets A, Speel E‐J M, Slootweg P J & van der Laak J A W M
(2010) Histopathology 57, 14–26
Simultaneous assessment of DNA ploidy and biomarker expression in paraffin‐embedded tissue sections Aims: Aneuploidy is a potential biomarker for predicting progression of premalignancies. Ploidy assessment is mostly performed on nuclei isolated from tissue sections. Ploidy assessment in situ in tissue sections may be a large improvement, enabling selective sampling of nuclei, thus allowing the correlation between ploidy and histology. Existing ploidy analysis methods in sections suffer from limited sensitivity. The aim was to reliably assess ploidy in sections, combined with simultaneous assessment of other markers at the individual cell level. Methods and results: Ploidy was measured in 22 paraffin‐embedded oral premalignancies. The DNA stoichiometric Feulgen procedure was used on isolated nuclei, as well as fluoresence in situ hybridization analysis. In tissue sections, Feulgen was combined with immunohistochemistry for Ki67 proliferation marker, enabling distinction between cycling euploid and aneuploid cells. Aneuploidy was reliably detected in tissue sections (sensitivity 100%, specificity 92%). One section in which aneuploidy was detected was misclassified in isolated nuclei analysis. Sections were also successfully analysed using our model combined with DNA double strand break marker γ‐H2AX in fluorescence microscopy, underlining the power of biomarker evaluation on single cells in tissue sections. Conclusions: The analysis model proposed in this study enables the combined analysis of histology, genotypic and phenotypic information.     

E‐cadherin immunohistochemistry in breast pathology: uses and pitfalls

E‐cadherin immunohistochemistry is used commonly in surgical pathology practice to help distinguish lobular carcinoma in situ from ductal carcinoma in situ and invasive lobular carcinoma from invasive ductal carcinoma in histologically problematic or indeterminate cases. However, the interpretation of E‐cadherin immunostains is not always straightforward. Failure to recognize the pitfalls and limitations of E‐cadherin immunostains can lead to an erroneous diagnosis which may result in inappropriate patient management, particularly for patients with in‐situ lesions. In this paper we review the uses and, particularly, the pitfalls in the interpretation of E‐cadherin immunostains in distinguishing lobular from ductal lesions of the breast.     

Tissue microarray technology: validation in colorectal carcinoma and analysis of p53, hMLH1, and hMSH2 immunohistochemical expression

Tissue microarray technology enables the analysis of hundreds of specimens by arranging numerous 0.6-mm tissue core biopsy specimens into a single paraffin block. Validation studies are necessary to evaluate the representativeness of small disks taken from the original tissue. We validated the tissue microarray technology in colorectal carcinoma by analyzing the immunohistochemical expression of proteins involved in the two main pathways of colorectal carcinogenesis: p53 protein for loss of heterozygosity tumors, hMLH1 and hMSH2 proteins for microsatellite instability (MSI) tumors. We compared in 30 colorectal carcinomas (15 MSI^– and 15 MSI⁺), 8 microarrays disks, and the whole section of the block from which they were derived. Tumoral tissue was present in 95.7% of the microarray disks. The analysis of three disks per case was comparable to the analysis of the whole section in 99.6% (p53), 98.8% (hMLH1), and 99.2% (hMSH2) of cases. In the second part we applied the tissue microarray technology to 263 consecutive cases of colorectal carcinoma, sampled by three cores. We showed that 48.5% overexpressed p53 and 8.7% lost hMLH1 or hMSH2. Tissue microarray technology, validated in colorectal carcinoma, appears as a useful research tool for rapid analysis of the clinical interest of molecular alterations.     

Keratin 7 expression in colorectal cancer – freak of nature or significant finding?

Harbaum L, Pollheimer M J, Kornprat P, Lindtner R A, Schlemmer A, Rehak P & Langner C
(2011) Histopathology 59 , 225–234 Keratin 7 expression in colorectal cancer – freak of nature or significant finding? Aims: To assess the prevalence of keratin 7 (K7) expression in colorectal cancer and to correlate findings with clinicopathological parameters and patients' outcome. Method and results: A total of 370 patients were evaluated for K7 expression by immunohistochemistry using a tissue microarray technique. K7 expression was scored semiquantitatively as either focal (<10%), moderate (10–50%) or extensive (>50%). In all, 32 (9%) tumours were immunoreactive for K7, with five cases showing extensive, four moderate and 23 focal expression, respectively. K7 expression was associated with poor tumour differentiation (P = 0.005) and the extent of tumour budding (P = 0.02). In whole sections, K7 immunoreactivity prevailed in single cells and small clusters of cells at the invasion front. Analysis of serial sections showed that K7‐positive cells colocalized with keratin 20, whereas they lacked immunoreactivity for E‐cadherin, MUC2 and MIB‐1. Disease progression occurred in 52% of patients with K7‐positive tumours and 41% with K7‐negative tumours (P = 0.19); 48% of patients with K7‐positive tumours but only 33% with K7‐negative tumours died of disease (P = 0.06). Conclusions: Aberrant expression of K7 in budding cancer cells represents a modification of the epithelial phenotype (‘epithelial–epithelial transition’: EET) which may be linked to gains in motility and invasive potential.     

Tpl-2表达与结直肠癌发生发展的关系及意义

目的: 研究Tpl-2在结直肠癌旁正常黏膜、结直肠腺瘤以及结直肠腺癌中的蛋白表达情况,及其与结直肠癌临床病理参数的关系,探讨Tpl-2在结直肠癌发生发展中的意义。方法: 使用组织芯片及免疫组化方法检测Tpl-2在结直肠正常黏膜、腺瘤、癌组织中的表达情况,分析Tpl-2表达与结直肠癌的临床病理参数的相关性。结果: 24例癌旁正常黏膜中,Tpl-2低表达20例(83.3%)、高表达为4例(16.7%);24例腺瘤组织中Tpl-2低表达17例(70.8%)、高表达7例(29.2%);96例癌组织中Tpl-2低表达31例(32.3%)、高表达65例(67.7%)。与正常和腺瘤比较,癌组织Tpl-2表达均明显增高(P<0.01)。腺瘤和正常黏膜中Tpl-2表达无显著差异(P>0.05)。在96例结直肠癌组织中,Tpl-2表达与N分期、TNM分期相关(P<0.05),与性别、年龄、身高体重指数(BMI)、肿瘤大小、分化程度、T分期、M分期以及K-ras基因突变状况无明显相关(P>0.05)。结论: Tpl-2与结直肠癌发生及疾病进展具有较密切的关系,可能是一个促癌因子。     

HER2/neu (c‐erbB‐2) gene amplification and protein expression are rare in uterine cervical neoplasia: a tissue microarray study of 814 archival specimens

Published studies have reported widely variable incidence of HER2/neu (c‐erbB‐2) protein expression and HER2/neu (c‐erbB‐2) gene amplification in cervical carcinoma. We examined tissue microarrays (TMAs) constructed from 814 formaldehyde‐fixed paraffin‐embedded archival specimens of cervical intraepithelial neoplasia (CIN)1 (n = 262), CIN2 (n = 230), CIN3 (n = 186) and invasive carcinoma (n = 136), for HER2/neu protein expression by immunohistochemistry (IHC) and for HER2/neu gene amplification by chromogenic in situ hybridization (CISH). We found moderate or strong immunohistochemical positivity for HER2/neu in 64 of 814 specimens (7.9%). Using CISH, polysomy of the HER2/neu gene was detected in 87 cases (10.7%), low/borderline amplification in five cases (0.6%) and true amplification in four cases (0.5%). The correlation between IHC and CISH was statistically significant in CIN2, CIN3 and invasive cervical carcinoma specimens. When present, Her‐2/neu positivity is more commonly seen in higher grades of cervical dysplasia and in carcinoma. However, this large TMA study shows that HER2/neu oncoprotein expression and HER2/neu gene amplification overall are uncommon events in cervical neoplasia. This provides compelling evidence that HER2/neu plays no major role in the development and progression of cervical neoplasia.     

Development and validation of a novel protein extraction methodology for quantitation of protein expression in formalin‐fixed paraffin‐embedded tissues using western blotting

The development of efficient formaldehyde cross‐link reversal strategies will make the vast diagnostic tissue archives of pathology departments amenable to prospective and retrospective translational research, particularly in biomarker‐driven proteomic investigations. Heat‐induced antigen retrieval strategies (HIARs) have achieved varying degrees of cross‐link reversal, potentially enabling archival tissue usage for proteomic applications outside its current remit of immunohistochemistry (IHC). While most successes achieved so far have been based on retrieving tryptic peptide fragments using shot‐gun proteomic approaches, attempts at extracting full‐length, non‐degraded, immunoreactive proteins from archival tissue have proved challenging. We have developed a novel heat‐induced antigen retrieval strategy using SDS‐containing Laemmli buffer for efficient intact protein recovery from formalin‐fixed tissues for subsequent analysis by western blotting. Protocol optimization and comparison of extraction efficacies with frozen tissues and current leader methodology is presented. Quantitative validation of methodology was carried out in a cohort of matched tumour/normal, frozen/FFPE renal tissue samples from 10 patients, probed by western blotting for a selected panel of seven proteins known to be differentially expressed in renal cancer. Our data show that the protocol enables efficient extraction of non‐degraded, full‐length, immunoreactive protein, with tumour versus normal differential expression profiles for a majority of the panel of proteins tested being comparable to matched frozen tissue controls (rank correlation, r = 0.7292, p < 1.825e‐09). However, the variability observed in extraction efficacies for some membrane proteins emphasizes the need for cautious interpretation of quantitative data from this subset of proteins. The method provides a viable, cost‐effective quantitative option for the validation of potential biomarker panels through a range of clinical samples from existing diagnostic archives, provided that validation of the method is first carried out for the specific proteins under study. Copyright © 2008 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.     

Whole‐genome methylation analysis of benign and malignant colorectal tumours

Changes in DNA methylation, whether hypo‐ or hypermethylation, have been shown to be associated with the progression of colorectal cancer. Methylation changes substantially in the progression from normal mucosa to adenoma and to carcinoma. This phenomenon has not been studied extensively and studies have been restricted to individual CpG islands, rather than taking a whole‐genome approach. We aimed to study genome‐wide methylation changes in colorectal cancer. We obtained 10 fresh‐frozen normal tissue–cancer sample pairs, and five fresh‐frozen adenoma samples. These were run on the lllumina HumanMethylation27 whole‐genome methylation analysis system. Differential methylation between normal tissue, adenoma and carcinoma was analysed using Bayesian regression modelling, gene set enrichment analysis (GSEA) and hierarchical clustering (HC). The highest‐rated individual gene for differential methylation in carcinomas versus normal tissue and adenomas versus normal tissue was GRASP (p_adjusted = 1.59 × 10^–5, BF = 12.62, p_adjusted = 1.68 × 10^–6, BF = 14.53). The highest‐rated gene when comparing carcinomas versus adenomas was ATM (p_adjusted = 2.0 × 10^–4, BF = 10.17). Hierarchical clustering demonstrated poor clustering by the CIMP criteria for methylation. GSEA demonstrated methylation changes in the Netrin–DCC and SLIT–ROBO pathways. Widespread changes in DNA methylation are seen in the transition from adenoma to carcinoma. The finding that GRASP, which encodes the general receptor for phosphoinositide 1‐associated scaffold protein, was differentially methylated in colorectal cancer is interesting. This may be a potential biomarker for colorectal cancer. Copyright © 2012 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.     

Colorectal signet ring cell carcinoma: Influence of EGFR,E-cadherin and MMP-13 expression on clinicopathological features and prognosis

Signet ring cell carcinoma (SRCC) is unique rare subtype of mucin-producing colorectal adenocarcinoma characterized by presence of signet ring cells, in > 50% of the tumor tissue. This study aims to investigate expression of EGFR, E-cadherin and MMP-13 expression on clinicopathological features of signet ring cell type and its prognostic effect using manual tissue microarray technique. In this work, we studied tumor tissue specimens from 150 patients with colorectal cancer cases among which 19 cases of SRCC. High density manual tissue microarrays were constructed using modified mechanical pencil tips technique and immunohistochemistry for EGFR, E-cadherin and MMP-13 expression was done. We found that SRCC was significantly associated with younger age and more frequency of LN metastasis than all other groups. SRCC was also significantly associated with annular gross picture, more depth of invasion, advanced stage, more lymphovascular emboli, more perineural invasion and less arousal from an overlying adenoma. In conclusion, colorectal SRCC has distinctive clinicopathological and histological features with different unique mechanisms of carcinogenesis and more aggressive biologic behavior than other colorectal carcinoma subtypes. Negative/low expressions of EGFR and E-cadherin and MMP-13 were found in SRCC with no effect on the prognosis.     

Distinct expression pattern of claudin‐6, a primitive phenotypic tight junction molecule,in germ cell tumours and visceral carcinomas

Ushiku T, Shinozaki‐Ushiku A, Maeda D, Morita S & Fukayama M
(2012) Histopathology
Distinct expression pattern of claudin‐6, a primitive phenotypic tight junction molecule, in germ cell tumours and visceral carcinomas Aims: This study aimed to clarify claudin‐6 expression patterns in cancers. Methods and results: We surveyed claudin‐6 expression by immunohistochemistry using tissue microarray in 860 tumours, including germ cell tumours and major carcinomas. Claudin‐6 was expressed consistently in germ cell tumours (28 of 28, 100%), whereas only 64 (8%) of 832 non‐germ cell tumours demonstrated claudin‐6 expression. Further immunohistochemical study in full tissue sections demonstrated diffuse claudin‐6 staining in all seminomas (n = 14), embryonal carcinomas (n = 10), yolk sac tumours (n = 12) and mononuclear trophoblastic cells of choriocarcinomas (n = 3), and focal staining in immature epithelial components of immature teratomas (n = 6). Additionally, because alpha‐fetoprotein (AFP)‐producing gastric adenocarcinomas and pulmonary high‐grade fetal adenocarcinomas were among the claudin‐6 expressing non‐germ cell tumours in the microarray studies, we predicted that claudin‐6 may be a biomarker for them and studied additional tumours in full sections, which showed claudin‐6 expression in AFP‐producing gastric adenocarcinomas (18 of 20, 90%) and pulmonary high‐grade fetal adenocarcinomas (four of five, 80%). Only one of 11 hepatoblastomas demonstrated focal claudin‐6 staining. Conclusions: This study demonstrated that claudin‐6 is a novel diagnostic marker for primitive germ cell tumours and is also expressed frequently in some cancers with a primitive phenotype.     

Tissue microarrays: what will they bring to molecular and anatomic pathology?

The analysis of a large number of tumor tissues with conventional techniques of molecular pathology is tedious and slow. The authors recently developed the tissue microarray technology that makes it possible to sample up to 1,000 tumors on one glass slide, which then can be analyzed by fluorescence in situ hybridization, RNA in situ hybridization, or immunohistochemistry. The tissue microarray technology has the potential to significantly accelerate molecular studies that seek associations between molecular changes and clinicopathologic features of the cancer. Examples of potential applications for tissue microarrays include testing and optimization of probes and antibodies, the organization of large tissue repositories, and the facilitation of multicenter studies. Further, tissue microarrays can be used for educational purposes as well as to improve quality control and standardization of staining methods and interpretation. Tissue microarrays have become one of the most promising tools for the molecular and anatomic pathologist and will have many applications in cancer research, as well as in other fields of pathology. This review article gives an overview of current applications of tissue microarrays as well as possible future development of the technology.     

Tumor–microenvironment interactions studied by zonal transcriptional profiling of squamous cell lung carcinoma

Invasion is a critical step in lung tumor progression. The interaction between tumor cells and their surroundings may play an important role in tumor invasion and metastasis. To better understand the mechanisms of tumor invasion and tumor–microenvironment interactions in lung tumors, total RNA was isolated from the inner tumor, tumor invasion front, adjacent lung, and distant normal lung tissue from 17 patients with primary squamous cell lung carcinoma using punch‐aided laser capture microdissection. Messenger RNA expression profiles were obtained by microarray analysis, and microRNA profiles were generated from eight of these samples using TaqMan Low Density Arrays. Statistical analysis of the expression data showed extensive changes in gene expression in the inner tumor and tumor front compared with the normal lung and adjacent lung tissue. Only a few genes were differentially expressed between tumor front and the inner tumor. Several genes were validated by immunohistochemistry. Evaluation of the microRNA data revealed zonal expression differences in nearly a fourth of the microRNAs analyzed. Validation of selected microRNAs by in situ hybridization demonstrated strong expression of hsa‐miR‐196a in the inner tumor; moderate expression of hsa‐miR‐224 in the inner tumor and tumor front, and strong expression of hsa‐miR‐650 in the adjacent lung tissue. Pathway analysis placed the majority of genes differentially expressed between tumor and nontumor cells in intrinsic processes associated with inflammation and extrinsic processes related to lymphocyte physiology. Genes differentially expressed between the inner tumor and the adjacent lung/normal lung tissue affected pathways of arachidonic acid metabolism and eicosanoid signaling. © 2012 Wiley Periodicals, Inc.     

Prognostic relevance of tumour cell‐associated uPAR expression in invasive ductal breast carcinoma

Kotzsch M, Bernt K, Friedrich K, Luther E, Albrecht S, Gatzweiler A, Magdolen V, Baretton G, Zietz C & Luther T
(2010) Histopathology 57 , 461–471
Prognostic relevance of tumour cell‐associated uPAR expression in invasive ductal breast carcinoma Aims: The urokinase‐type plasminogen activator receptor (uPAR) is a key molecule for pericellular proteolysis in tumour cell invasion and metastasis. The aim was to evaluate the prognostic impact of uPAR in invasive breast cancer dependent on which cell types within the tumour express uPAR. Methods and results: uPAR expression was analysed by immunohistochemistry in 270 tumour tissue specimens of invasive ductal breast carcinomas using tissue microarrays. For evaluation of uPAR immunoexpression we used the epitope‐mapped, uPAR domain II‐specific monoclonal antibody IID7. High uPAR score values in both tumour cells (uPAR‐Tc) and stromal cells were significantly related to high tumour grade (G3), and inversely correlated with oestrogen receptor status. On multivariate analysis, high uPAR‐Tc values contributed independent prognostic information for disease‐free survival (hazard ratio 1.93, P = 0.007) when adjusted for prognostically relevant clinicopathological parameters, whereas uPAR expression in stromal cells was not related to prognosis. In addition, elevated uPAR‐Tc values were found to be prognostic indicators in clinically relevant subgroups of patients with invasive breast cancer. Conclusions: In invasive breast cancer uPAR expression in invasive carcinoma cells, but not in stromal cells, has a significant impact on patients’ prognosis, and contributes to a more aggressive tumour phenotype.     

Quantification of immunohistochemistry--issues concerning methods, utility and semiquantitative assessment II

Immunohistochemistry is entering its fourth decade of use on formalin‐fixed paraffin‐embedded tissues. Over this period the method has evolved to become a major part of the practice of diagnostic surgical pathology worldwide. From the beginning immunohistochemistry has been adapted to provide a range of markers of cell lineage and tissue type, with particular application to the diagnosis and classification of tumours. In this modality immunohistochemical methods were employed simply as ‘special stains’, the results of which were evaluated qualitatively by the pathologist, as for any other stain. More recently, attention has shifted to the demonstration of prognostic markers in tumour cells, driven by the advent of molecular biology and the discovery of numerous regulatory molecules, coupled with manufacture of the corresponding specific antibodies. Immunohistochemistry has rapidly adapted to this new use, but in so doing the demand for quantification has become paramount; it is no longer enough that the ‘stain’ is there; rather it is a question of ‘How much is there?’. This review explores the limitations of immunohistochemistry when employed in a semiquantitative mode, and explores the possibility of fulfilling the full potential of immunohistochemistry, as a true quantitative immunoassay applied in a tissue section environment.