Validation of tissue microarray technology in breast carcinoma

The recent development of tissue microarray technology has potentiated large-scale retrospective cohort studies using archival formalin-fixed, paraffin-embedded tissues. A major obstacle to broad acceptance of microarrays is that they reduce the amount of tissue analyzed from a whole tissue section to a disk, 0.6 mm in diameter, that may not be representative of the protein expression patterns of the entire tumor. In this study, we examine the number to disks required to adequately represent the expression of three common antigens in invasive breast carcinoma--estrogen receptor, progesterone receptor, and the Her2/neu oncogene--in 38 cases of invasive breast carcinoma. We compared the staining of 2 to 10 microarray disks and the whole tissue sections from which they were derived and determined that analysis of two disks is comparable to analysis of a whole tissue section in more than 95% of cases. To evaluate the potential for using archival tissue in such arrays, we created a breast cancer microarray of 8 to 11 cases from each decade beginning in 1932 to the present day and evaluated the antigenicity of these markers and others. This array demonstrates that many proteins retain their antigenicity for more than 60 years, thus validating their study on archival tissues. We conclude that the tissue microarray technique, with 2-fold redundancy, is a valuable and accurate method for analysis of protein expression in large archival cohorts.     

Validation of tissue microarray technology in ovarian carcinoma.

High-throughput tissue microarray allows many clinical specimens to be analyzed simultaneously on a single slide. One potential limitation of tissue microarray is the correct representation of each tumor with the small tissue core. Because tumors from different organs have different levels of heterogeneity, it requires a validation study for each one of them. We compared immunostaining of Ki-67, estrogen receptors, and p53 in whole sections of 45 cases of high-grade serous ovarian carcinoma with six core samples from those sections with regard to the number of tissue cores needed to reliably represent a whole section. Staining for Ki-67 was graded high or low by automated image analysis of 10 high-power fields; staining for estrogen receptor and p53 was scored on a 0-to-3 scale. Correlation coefficients for whole-section vs core stains were 0.86 for Ki-67, 0.93 for estrogen receptors, and 0.82 for p53. A total of 54 (6.6%) of the cores were inadequate for scoring. The probability that results from one core would correctly represent all three markers in the whole section was 91%; that for two cores was 96%; and that for three cores was 98%. Our results show that analysis of a single readable core matched the staining pattern of a whole section more than 90% of the time, and analysis of two cores increased that value to more than 95%, demonstrating that ovarian carcinoma tissue microarray is a reliable technique to analyze the expression of markers.     

Validation of tissue microarray technology in squamous cell carcinoma of the esophagus

Tissue microarray (TMA) technology has been developed to facilitate high-throughput immunohistochemical and in situ hybridization analysis of tissues by inserting small tissue biopsy cores into a single paraffin block. Several studies have revealed novel prognostic biomarkers in esophageal squamous cell carcinoma (ESCC) by means of TMA technology, although this technique has not yet been validated for these tumors. Because representativeness of the donor tissue cores may be a disadvantage compared to full sections, the aim of this study was to assess if TMA technology provides representative immunohistochemical results in ESCC. A TMA was constructed containing triplicate cores of 108 formalin-fixed, paraffin-embedded squamous cell carcinomas of the esophagus. The agreement in the differentiation grade and immunohistochemical staining scores of CK5/6, CK14, E-cadherin, Ki-67, and p53 between TMA cores and a subset of 64 randomly selected donor paraffin blocks was determined using kappa statistics. The concurrence between TMA cores and donor blocks was moderate for Ki-67 (κ = 0.42) and E-cadherin (κ = 0.47), substantial for differentiation grade (κ = 0.65) and CK14 (κ = 0.71), and almost perfect for p53 (κ = 0.86) and CK5/6 (κ = 0.93). TMA technology appears to be a valid method for immunohistochemical analysis of molecular markers in ESCC provided that the staining pattern in the tumor is homogeneous.     

A sertoliform endometrioid adenocarcinoma of the endometrium

Sertoliform endometrioid adenocarcinomas of the ovary are well recognized but, curiously, a sertoliform pattern has not previously been noted in endometrioid adenocarcinomas of the endometrium. An endometrial tumour is described which showed in some areas the typical appearances of an endometrioid adenocarcinoma and in others a pattern closely resembling that of a Sertoli cell tumour.     

Clinical validation of breast cancer biomarkers using tissue microarray technology.

The results of previous studies done in our laboratory on breast cancer gene expression profile, using DNA microarrays, led to the discovery of several genes associated with breast cancer progression. Further evaluation of these genes and their involvement at various stages of cancer progression required performance of immunohistochemistry on thousands of different tissue blocks. Tissue microarray (TMA) technology facilitates rapid translation of DNA microarrays results to clinical specimens by using immunohistochemical analysis of protein expression. DNA microarray analysis done in our laboratory showed a significantly higher expression of prostatic-specific antigen (PSA) in invasive ductal carcinomas as compared to ductal carcinoma in situ, a finding contrary to the previously published data for PSA immunoreactivity in breast carcinomas. To find out whether TMA strategy could be used to explore the expression of the candidate genes involved in the breast cancer progression, we constructed a breast cancer progression TMA. It consisted of 2 normal ductal epithelium, 8 ductal carcinoma in situ, 19 invasive ductal carcinomas, and 3 metastatic ductal carcinomas of breast in triplets. Two prostatic adenocarcinomas and 2 normal colons were used as positive and negative controls, respectively. We first used well-documented and well-tested markers, such as antibodies to estrogen receptor, progesterone receptor, and p53. Results of these 3 antibodies were according to the previously published data. To validate our result, we then used antibody to PSA and looked for the expression of this protein on breast cancer progression TMA. Except for the 2 positive controls all 98 cores were found to be negative for PSA expression highlighting the importance of validation studies for DNA microarray results.     

Validation of usefulness of tissue microarray technology in primary tumours of the canine and feline central nervous system

High-throughput tissue microarray (TMA) technology allows analysis of many specimens of tumours simultaneously on a single slide. One potential limitation of TMAs is the correct representation of each tumour with the small tissue core. The aim of this study was to validate TMA technology for 10 primary tumours of the canine?and feline central nervous system (CNS) by comparing histology and immunohistochemical labelling of duplicate core biopsies on TMA with the results of full-section analysis. Concordance between results was not rejected by using paired student's t-test. The accuracy of the TMA technology of sampling a representative tumour area was 95% without significant differences in various histological parameters. The loss of ～0.9% of tissue cores during histological and immunohistochemical processing was very low. There were no significant differences in immunohistochemical labelling between the two tissue cores, between the mean score of both tissue?cores and the conventional tissue section and between each tissue core alone compared with the full tissue section. This investigation confirms the applicability of the TMA technology for primary CNS tumours of dogs and cats.     

Expression analysis of URI/RMP gene in endometrioid adenocarcinoma by tissue microarray immunohistochemistry

Multiple studies have recently demonstrated the oncogenic property of URI (or RMP, a member of the prefoldin family of molecular chaperones) during progression of hepatocellular carcinoma, ovarian cancer, and possibly prostate cancer. Most recently, we have shown that URI/RMP is up-regulated in cervical cancer, another reproductive system tumor beside ovarian and prostate cancers. To investigate if URI/RMP also plays a role in other reproductive system tumors, especially in endometrioid adenocarcinoma, we analyzed URI/RMP expression in a TMA (tissue microarray) containing tissues from 30 cases of endometrioid adenocarcinoma (which covers tumor tissues from Grade I through Grade III) and adjacent endometrium by immunohistochemistry (IHC) and densitometry analysis using image-pro plus 6.0 software. Our results showed that the mean density of URI/RMP expression in cancerous tissue is slightly higher than that of the adjacent endometrial tissue, though not statistically significant (p>0.05). There is no significant difference either between the mean density of Grade III cancerous tissue and that of Grade I and II cancers. Notably, we detected significantly higher signal intensity in cancerous tissue of all 7 Grade III cases than that of their adjacent endometrial tissue (p<0.05), suggesting a correlation of URI/RMP expression with the differentiation and pathological classification of endometrioid adenocarcinoma. Together, our results demonstrate the heterogeneous expression of URI/RMP in endometrioid adenocarcinoma. The higher level of URI/RMP expression in high-grade endometrioid adenocarcinomas compared to tissues of adjacent endometrium or gland suggests a diagnostic and possibly, a prognostic value of URI/RMP in endometrioid adenocarcinoma.     

Demystified...tissue microarray technology.

Several "high throughput methods" have been introduced into research and routine laboratories during the past decade. Providing a new approach to the analysis of genomic alterations and RNA or protein expression patterns, these new techniques generate a plethora of new data in a relatively short time, and promise to deliver clues to the diagnosis and treatment of human cancer. Along with these revolutionary developments, new tools for the interpretation of these large sets of data became necessary and are now widely available. Tissue microarray (TMA) technology is one of these new tools. It is based on the idea of applying miniaturisation and a high throughput approach to the analysis of intact tissues. The potential and the scientific value of TMAs in modern research have been demonstrated in a logarithmically increasing number of studies. The spectrum for additional applications is widening rapidly, and comprises quality control in histotechnology, longterm tissue banking, and the continuing education of pathologists. This review covers the basic technical aspects of TMA production and discusses the current and potential future applications of TMA technology.     

E-cadherin expression in prostate cancer: a broad survey using high-density tissue microarray technology

E-cadherin is a calcium 2+-dependent cell-adhesion molecule that determines epithelial development in the embryo and maintains adult differentiated epithelium and homeostasis. Aberrant or decreased expression has been reported to be associated with prostate carcinoma progression. The degree of E-cadherin expression in prostate cancer remains controversial. Some studies have reported decreased expression of E-cadherin as tumors advance and metastasize. Other studies have not demonstrated this relationship. To address these variations, we undertook a study to systematically evaluate E-cadherin expression in a broad range of prostate tissue. Benign prostate, clinically localized prostate cancer, and hormone-refractory metastatic prostate cancer were analyzed under uniform conditions using high-density tissue microarrays (TMA). Formalin-fixed, paraffin-embedded prostate carcinoma from men with clinically localized prostate carcinoma and autopsy material from men who died of widely metastatic, hormone-refractory prostate carcinoma were arrayed into 6 high-density TMA blocks. Benign and atrophic prostate tissue and high-grade prostatic intraepithelial neoplasia (PIN) were also included from the clinically localized cases. Immunohistochemistry was performed using the immunoglobulin G1 mouse monoclonal antibody (HECD-1; Zymed, San Francisco, CA). Membranous staining was recorded as low (aberrant) or high (normal). E-cadherin expression was considered aberrant if less than 70% of the cells had strong membranous staining. A total of 1,220 prostate TMA samples were analyzed. High (normal) E-cadherin expression was seen in 87% of 757 benign, 80% of 41 high-grade PIN, 82% of 325 prostate carcinoma and 90% of 97 hormone-refractory prostate carcinoma TMA samples. Mean E-cadherin expression was determined for each of the 128 clinically localized prostate cancer cases. Aberrant E-cadherin expression showed a statistical trend toward an association with positive surgical margins (P =.012), higher Gleason score (P =.18), and prostate-specific antigen (PSA) failure (Kaplan-Meier analysis, log-rank P =.09). There was a statistically significant association between aberrant E-cadherin expression and larger tumor size (P =.01). No significant associations were seen with extraprostatic extension and seminal vesicle invasion. The current study shows a broad-spectrum approach to evaluating E-cadherin protein expression in prostate carcinoma. Clinically localized prostate tumors, treated with surgery alone, show a high level of E-cadherin expression. Aberrant expression was identified in tumors with positive surgical margins, higher Gleason score, and a higher rate of PSA failure. However, these trends were not statistically significant. A statically significant association between aberrant E-cadherin expression and larger tumor size was identified. In the metastatic hormone-refractory prostate tumors, E-cadherin expression was vastly expressed, and only rare cases had aberrant expression. Therefore, the findings of this study are most consistent with a transient down-regulation of E-cadherin in localized prostate cancer. Metastatic prostate cancer shows strong E-cadherin expression as determined by anti-E-cadherin antibody HECD-1.     

在组织芯片上利用量子点技术进行抗原的检测

在组织培养和组织切片上,可利用放射性和非放射性方法检测抗原的定位.而在传统的免疫荧光分析技术中,有机荧光基团存在激发波长范围很窄、发光时间短,容易猝灭等缺点,限制了它们在蛋白定量上的使用.荧光半导体纳米颗粒量子点(quantum dots)提供了一种潜在的方法去克服有机荧光基团的这些缺陷,开始在免疫荧光分析中初步应用,并取得了令人满意的效果.     

Prognostic significance of the infiltrative pattern invasion in endometrioid adenocarcinoma of the endometrium

The prognostic significance of the invasive type of carcinoma cells in endometrial carcinoma is not defined. We evaluated the prognostic significance of the invasive type, as well as the immunostains of p53, c-erbB-2, Ki-67 antigen and MDM2 in endometrial endometrioid adenocarcinoma. This prospective analysis comprised 112 patients with endometrioid adenocarcinoma of the uterine corpus who had undergone surgery and were traced for more than 5 years after the operation. They were divided into recurrence (16 patients) and non-recurrence (96 patients) groups. The invasive type of carcinoma cells was divided into expansile, mixed (expansile and infiltrative) and infiltrative pattern. The difference in the invasive type (P < 0.001) and p53 expression (P = 0.004) between the recurrence and non-recurrence groups was significant in the univariate analysis. Moreover, the invasive type was significant in the multivariate analysis (P = 0.004). In contrast, the difference in MDM2 expression, c-erbB-2 expression and the Ki-67 labeling index in both groups was not significant in the univariate analysis. The infiltrative pattern of the invasive type (P < 0.001) and p53 expression (P = 0.043) were significantly related to a poor prognosis in the Kaplan-Meier method using the log-rank test. In conclusion, the current study indicated that the infiltrative pattern of the carcinoma cells is a predictor for poor prognosis in endometrioid adenocarcinoma in the uterine corpus. It was also indicated that p53 immunostains are useful as a predictor, but Ki-67 antigen, c-erbB-2 and MDM2 stains are not.     

Body mass index as a prognostic factor in endometrioid adenocarcinoma of the endometrium

OBJECTIVE: To determine if body mass index (BMI) influences tumor expression of HER-2/neu, estrogen and progesterone receptors (ER/PR), and survival in women with endometrial adenocarcinoma. METHODS: Patients diagnosed between January 1992 and December 2001 with endometrioid adenocarcinoma of the uterus were identified. Clinical and pathologic data were retrospectively collected. HER-2/neu, estrogen and progesterone receptor expression were determined by immunohistochemistry. Differences in these variables and other prognostic factors were analyzed and correlated with effect on survival. RESULTS: One-hundred-sixty-five patients were included in this analysis. Lower BMI was associated with high stage (p=0.04) and HER-2/neu expression (p=0.04). Black race, high grade, high stage and lack of ER/PR expression were all associated with decreased survival. Despite having better prognostic factors, women with a BMI >25 had a lower survival than women with a BMI <25 (p=0.36). When five-year survival rates were calculated for BMI category and stratified by prognostic factors, for almost every high risk factor, survival was lower in overweight patients. CONCLUSION: In patients with endometrioid adenocarcinoma, low BMI is associated with high stage and tumor expression of HER-2/neu. Despite better prognostic factors, overweight women experience poorer survival.     

cDNA microarray analysis and immunohistochemistry reveal a distinct molecular phenotype in serous endometrial cancer compared to endometrioid endometrial cancer

Purpose

The main objective of this study was to refine more precisely the gene expression patterns used to distinguish serous from endometrioid endometrial carcinoma.

Methods

A low-density cDNA microarray containing 492 genes was designed and constructed. The gene expression profiles of 32 endometrioid and 5 serous endometrial cancer tissue samples were compared. The expression of 5 differentially expressed genes: NDC80, BUB1, FUT8, ANXA4 and BBC3 in endometrioid and serous adenocarcinoma samples was further evaluated by quantitative real-time PCR and immunohistochemistry.

Results

Unsupervised cluster analysis revealed that the 5 serous adenocarcinomas clustered together. These were separated from the endometrioid adenocarcinomas which were further sorted into 3 additional clusters. A comparative analysis indicated that there was a significant difference in FIGO stage with no significant difference in depth of myometrial invasion among the 4 clusters. The FIGO ternary grading system could not distinctly separate the 3 clusters of endometrioid adenocarcinomas, but a binary grading system was able to do so. Using a supervised analysis, we have identified 46 genes exhibiting > 2-fold differences that can be used to statistically differentiate serous adenocarcinomas from endometrioid adenocarcinomas. The directions of gene and protein expression change of five differentially expressed genes estimated by real-time PCR and immunohistochemistry are consistent with those estimated from microarray.

Conclusions

Serous adenocarcinoma exhibits distinct gene expression profiles, compared with those of endometrioid adenocarcinoma. These differences make it feasible to validate microarray data by immunohistochemistry, and they will ultimately allow us to identify tumors according to their immunohistochemical phenotype. The accuracy of classifying endometrial tumors using a system based on their gene expression patterns is much higher than the accuracy of the FIGO grading system. Thus, this gene expression pattern-based system may prove to be crucial in developing novel treatment strategies for endometrial cancers at the molecular level in future.     

Applications of tissue microarray technology in immunohistochemistry: a study on c-kit expression in small cell lung cancer

Tissue microarray technology allows the immediate evaluation of molecular profiles of numerous different tissues, with savings of money and time. It was created for rapid, large-scale molecular studies, and the main concern regarding its possible broad acceptance is that the analysis of tissue microarrays instead of whole tissue sections may lead to false negative or positive results because of tissue heterogeneity. In the present study, we analyzed in 54 small cell lung cancers, by immunohistochemistry, the expression of the antigen c-kit, which seems to be important in these neoplasms' tumorigenesis, and compared the staining obtained on whole sections with that of the corresponding tissue microarrays. Although c-kit expression of the whole sections agreed with that of the corresponding biopsies in many cases, the correlation between whole sections and all the companion nonlost single cores or their mean value turned out to be highly significant only if the 36 double negatives (ie, both whole sections and companion tissue microarrays negative) were included (P <0.0001). In fact, if only cases positive to at least 1 of the tests (i.e. whole sections or corresponding tissue microarrays positive) were considered, the correlation was not significant (P=0.055). Tissue microarrays showed a good specificity (94.2% for all single cores and 92.3% for their mean value) but a rather poor sensitivity (respectively, 69.4% and 71.4%). Moreover, a high percentage (13.4%) of cores was lost, and this loss was not random. To sum up, in our experience, tissue microarray technology cannot be a substitute for whole sections in clinical diagnosis of individual cases.     

Strategies for distinguishing low-grade endometrioid and serous carcinomas of endometrium

Distinction between endometrioid and serous carcinomas of the endometrium has important prognostic and therapeutic implications. Misdiagnosing a serous carcinoma as endometrioid can have significant consequences for the patient and pathologist. Although many cases are straightforward and easy to classify, there are occasional problematic cases. This review focuses on strategies that help differentiate between low-grade endometrioid carcinoma and serous carcinoma of the endometrium. We will discuss clinical, morphologic, and immunohistochemical differences between the 2 entities and provide practical tips for practicing pathologists when confronted with this differential diagnosis.     

Evaluation of the tissue microarray technique for immunohistochemical analysis in rectal cancer

BACKGROUND: Immunohistochemical staining for tumor-associated proteins is widely used for the identification of novel prognostic markers. Recently, a tissue-conserving, high-throughput technique, tissue microarray, has been introduced. This technique uses 0.6-mm tissue core biopsy specimens, 500 to 1000 of which are brought into a new paraffin array block, which can be sectioned up to 100 times. METHODS: We evaluated the tissue microarray technique for immunohistochemical analysis in 20 rectal cancers. Immunohistochemical staining was performed for the proliferation marker Ki-67 and the tumor suppressor protein p53 in whole tissue sections and in tissue core biopsy specimens. RESULTS: The whole tissue sections were assessed by counting all cells in 10 high-power fields (x40), which resulted in a mean fraction of Ki-67-expressing tumor cells of 0.81 (range, 0.54-1.0). p53 expression assessed in whole tissue sections showed nuclear staining in 15 (75%) of 20 rectal carcinomas. For the tissue microarray technique, a median of 3 (range, 3-5) 0.6-mm tissue core biopsy specimens were studied from each of the 20 tumor specimens. The tissue microarray method gave a mean Ki-67 expression of 0.85 (range, 0.50-1.0) in tumor cell nuclei and showed p53 protein expression in the same 15 of 20 tumors as in the whole tissue sections. CONCLUSION: We conclude that the tissue microarray technique for immunohistochemical staining in rectal cancer yields staining of good quality and expression data for Ki-67 and p53 comparable to those obtained with whole tissue staining. The feasibility of tissue microarray thus enables time- and tissue-preserving studies of multiple markers in large tumor series.