组织芯片技术原位检测石蜡包埋肝细胞癌组织的研究

目的探讨组织芯片技术高通量原位免疫组织化学检测石蜡包埋肝细胞癌组织的价值。方法1991年1月-2002年6月西南肝胆医院实施根治性肝切除,病理证实为肝细胞癌234例石蜡组织块,健康成人肝组织18例(西南医院肝胆外科研究所肝移植供肝组织)作为对照,重新包埋石蜡组织,制备成为2个微阵列,分别为13×10(129点)和14×9(123点),微阵列点直径1.0mm,点间距为0.5mm。Envision法检测Vimentin和E cadherin的表达。结果组织芯片中组织样本的组织学结构完整,可满足病理学实验要求。结论组织芯片技术提供了一种快速、有效的组织学方法,可广泛应用大样本肝细胞癌的免疫组织化学检测。     

Tissue-Microarrays

High-throughput molecular screening tools have created a need for equally rapid means to verify potential biomarkers. Tissue microarray (TMA) technology facilitates effective analysis on the protein level. Hundreds of 0.6-mm-diameter tissue samples are placed on a single standard glass slide. This approach allows analysis with different in situ methods of all tissue samples in one experiment under standardized conditions. TMAs are not only perfectly suited for molecular epidemiologic studies if applied on multitumor arrays composed of a variety of tumor entities, but they are also used for tumor-specific investigations of molecular markers. In this report the technical aspects of TMAs as a miniaturized high-throughput technology are described. We further demonstrate how the TMA technique will help to accelerate the transition from basic research to clinical application in prostate cancer disease.     

Tissue microarrays. High-throughput procedures to verify potential biomarkers

High-throughput molecular screening tools have created a need for equally rapid means to verify potential biomarkers. Tissue microarray (TMA) technology facilitates effective analysis on the protein level. Hundreds of 0.6-mm-diameter tissue samples are placed on a single standard glass slide. This approach allows analysis with different in situ methods of all tissue samples in one experiment under standardized conditions. TMAs are not only perfectly suited for molecular epidemiologic studies if applied on multitumor arrays composed of a variety of tumor entities, but they are also used for tumor-specific investigations of molecular markers. In this report the technical aspects of TMAs as a miniaturized high-throughput technology are described. We further demonstrate how the TMA technique will help to accelerate the transition from basic research to clinical application in prostate cancer disease.     

Prognostic factors in soft tissue sarcoma. Tissue microarray for immunostaining, the importance of whole-tumor sections and time-dependence

In adult soft tissue sarcoma (STS) of the extremities and trunk wall, improved prognostic factors are needed to identify patients at high-risk for metastasis. Various factors are included in the many prognostic systems currently in use and the prognostic value of immunohistochemical (IHC) expression of biological markers is unclear. The tissue-preserving, high throughput tissue microarray (TMA) technique for analysis of immunohistochemical expression of biological markers was validated for Ki-67, and was found to yield results comparable to conventional staining methods. TMA was used to study the IHC expression of multiple markers (Ki-67, p53, cyclin A, bcl-2, beta-catenin, CD44, and Pgp) in 218 malignant fibrous histiocytomas (MFH) and in 140 mixed STS. In the MFH series, tumor size and Ki-67, as the only IHC marker, provided independent prognostic information. In the mixed STS series whole-tumor sections were used and TMA was performed in the peripheral tumor growth zone. Whole-tumor sections facilitated assessment of the strong independent prognostic factors for metastasis vascular invasion, hazard ratio (HR) 3.5, tumor necrosis (HR 2.8), and tumor growth pattern (HR 3.2), and the latter also correlated with local recurrence (LR). In comparison, histological malignancy grade, tumor size, and depth were not of independent prognostic value. When TMA was performed from the peripheral tumor growth zone, the IHC expression of Ki-67 (HR 1.9), beta-catenin (HR 2.7), CD44 (HR 2.1) and Pgp (HR 2.4) were independent prognostic factors. Finally, prognostic factors were found to be time-dependent, and most had lost their prognostic value after 2 years, whereas LR was a strong prognostic factor for metastasis whenever it occurred.     

Application of tissue microarrays for the diagnosis, prognosis and therapeutic decision making in renal cell carcinoma

The tissue microarray technique (TMA) represents a powerful diagnostic "high-throughput" tool to analyse DNA/RNA or protein alterations in large patient cohorts in a time and cost effective way. This review focuses on the clinical application of TMA in renal cell carcinoma in modern "translational" medicine--"from bench to bedside". In particular, the advantages of TMA for diagnosis, prognosis and decisions for therapy will be considered in relation to renal cell cancer.     

Tissue microarrays in clinical urology--technical considerations

The tissue microarray (TMA) technique is a 'high-throughput' tool which enables a rapid and concurrent analysis of molecular targets in large numbers of specimens at the DNA, RNA and protein levels under standardized conditions. In addition, TMA is a cost- and time-efficient method. This review focuses on its technical side, outlining its advantages and weaknesses. It also gives an overview of some major studies of bladder and prostate cancer in which the TMA technique was used.     

Tissue Microarrays

High-throughput tissue microarray (TMA) technology facilitates the assessment of the clinical relevance of molecular markers by enabling the simultaneous analysis of hundreds of tissue specimens. The widespread adoption of TMAs in many laboratories replaces the conventional one-slide-one-section approach, in which individual archival clinical specimens were placed on separate microscope slides, with the ability to assess RNA, DNA, or protein expression in hundreds of individual patient specimens in a single experiment. One of the applications of this technology is to significantly accelerate advances in translational research through more efficient assessment of novel markers of outcome and response, and as a result, a more rapid application of this knowledge to clinical practice.     

Prognostic markers in pT3 bladder cancer: A study from the international bladder cancer tissue microarray project

PurposeWe evaluated the prognostic value of 10 putative tumor markers by immunohistochemistry in a large multi-institutional cohort of patients with locally advanced urothelial cancer of the bladder (UCB) with the aim to validate their clinical value and to harmonize protocols for their evaluation.Materials and MethodsPrimary tumor specimens from 576 patients with pathologic (p)T3 UCB were collected from 24 institutions in North America and Europe. Three replicate 0.6-mm core diameter samples were collected for the construction of a tissue microarray (TMA). Immunohistochemistry (IHC) for 10 previously described tumor markers was performed and scored at 3 laboratories independently according to a standardized protocol. Associations between marker positivity and freedom from recurrence (FFR) or overall survival (OS) were analyzed separately for each individual laboratory using Cox regression analysis.ResultsThe overall agreement of the IHC scoring among laboratories was poor. Correlation among the 3 laboratories varied across the 10 markers. There was generally a lack of association between the individual markers and FFR or OS. The number of altered cell cycle regulators (p53, Rb, and p21) was associated with increased risk of cancer recurrence (P < 0.032). There was no clear pattern in the relationship between the percentage of markers altered in an 8-marker panel and FFR or OS.ConclusionsThis large international TMA of locally advanced (pT3) UCB suggests that altered expression of p53, Rb, and p21 is associated with worse outcome. However this study also highlights limitations in the reproducibility of IHC even in the most expert hands.     

Anwendung von Tissue-Microarrays für die Diagnose, Prognose und Therapieentscheidung beim Nierenzellkarzinom

The tissue microarray technique (TMA) represents a powerful diagnostic “high-throughput” tool to analyse DNA/RNA or protein alterations in large patient cohorts in a time and cost effective way. This review focuses on the clinical application of TMA in renal cell carcinoma in modern “translational” medicine — “from bench to bedside”. In particular, the advantages of TMA for diagnosis, prognosis and decisions for therapy will be considered in relation to renal cell cancer.     

High-throughput tissue microarray analysis of erbB-2 gene amplification in urinary bladder cancer. A study of Bulgarian patients

INTRODUCTION: Overexpression of the receptor Her-2 leads to increased proliferative response to epidermal growth factors which plays a key role in tumor development and growth. Increased oncoprotein level, in the majority of cases, is caused by erbB-2 gene amplification. To define the frequency of amplifications of erbB-2 in bladder cancer, and to determine their association with the tumor phenotype we utilized tissue microarray (TMA) approach. MATERIALS AND METHODS: We analyzed a TMA consisting of 159 transitional cell bladder carcinomas for erbB-2 gene amplification by dual-color FISH. RESULTS: The frequency of erbB-2 amplification was 5.3% of all successfully analyzed samples (4 of 75). It increased from minimally invasive (pT1) to muscle-invasive (pT2-4) tumors, as well as with advanced tumor grade (G1 to G2 to G3). All amplifications were cluster amplifications confirming the fact that erbB-2 amplification occurs intrachromosomally in bladder cancer. CONCLUSIONS: We concluded that despite its low incidence, erbB-2 amplification is of importance for the bladder tumor invasion and progression.     

Feasibility of constructing tissue microarrays from diagnostic prostate biopsies

OBJECTIVES: The ability to construct prostate tissue microarrays (TMAs) using prostate needle biopsies could allow high throughput molecular profiling to search for prostate cancer prognostic biomarkers. MATERIALS AND METHODS: Diagnostic prostate biopsies from 13 patients (diagnosed 1996-2000) were obtained from the University of North Carolina (UNC) to construct one prostate TMA under uniform conditions. A second prostate TMA was attempted using diagnostic prostate biopsies from 45 patients (diagnosed 2004) obtained from the North Carolina-Louisiana Prostate Cancer Project (PCaP). RESULTS: Eleven men had sufficient prostate cancer in their diagnostic prostate biopsy blocks to construct a UNC TMA that yielded six-micron sections that provided an average of 76% of cores (maximum 99%) to a depth of 360 microm. Diagnostic prostate biopsy blocks from 35 PCaP research subjects were unsuitable for TMA construction as a result of no remaining prostate cancer in 4, insufficient prostate cancer in 9, and insufficient prostate tissue in 22. The PCaP TMA constructed from 10 men yielded an average of 37%, and a maximum of 45%, of cores when sectioned to a depth of 360 microm. CONCLUSIONS: TMAs may be constructed from diagnostic prostate biopsies obtained at an academic center under uniform conditions. However, excessive facing of blocks and the large number of re-cuts ordered by many community pathology laboratories deplete diagnostic prostate biopsy tissue. The experience of a population-based study of men with newly diagnosed prostate cancer in Louisiana and North Carolina suggests that TMAs may not be constructed using diagnostic prostate biopsies from men diagnosed with prostate cancer throughout the United States.     

Evaluation of P‐glycoprotein (Pgp) expression in human osteosarcoma by high‐throughput tissue microarray

Survival of osteosarcoma patients is currently limited by the development of metastases and multidrug resistance (MDR). A well‐established cause of MDR involves overexpression of P‐glycoprotein (Pgp) in tumor cells. However, some discrepancies still exist as to the clinical significance of Pgp in osteosarcoma. We sought to elucidate further whether the Pgp expression correlated with clinical behavior in a series of patients with osteosarcoma via high‐throughput tissue microarray (TMA) analysis. Immunohistochemical analysis of Pgp expression in a TMA of 114 specimens with a retrospective review of 70 osteosarcoma patients admitted to the Massachusetts General Hospital (MGH) was performed. High Pgp expression was correlated with metastasis development and poor response to pre‐operative chemotherapy in osteosarcoma patients. Eighteen of the fifty‐seven patients initially admitted with primary osteosarcoma showed high Pgp expression. Among these 18 patients with high Pgp expression, 13 of 18 (72%) patients eventually developed metastases. There was no significant clinical relevance between Pgp expression and osteosarcoma survival. These results support that high expression of Pgp is important, but cannot be assigned as, an individual predictor in the development of human osteosarcoma. © 2016 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 34:1606–1612, 2016.     

Tissue microarrays from renal cell tumors: exclusion criteria and rate of exclusion

OBJECTIVES: Exclusion of tissue microarray (TMA) cores can cause the total loss of a tumor case, and this can have a potentially negative effect on the results of TMA-based studies. The main aim of this study was to evaluate the loss of informative cores having cut a given number of slices from a TMA block. A further objective was to investigate the effect in various subtypes of renal cell tumors and the detailed reasons for the loss of informative cores. MATERIAL AND METHODS: A TMA was constructed from renal tumor specimens (n=461). The cause and rate of exclusion were evaluated in the first slice (FS) and last slice (LS) (i.e. the 40th) cut from the TMA blocks. Furthermore, the overall case loss under the assumptions that only one, two or three cores per case were punched was extrapolated. RESULTS: Sarcomatoid and papillary renal cell carcinomas showed the highest overall exclusion rate. Irrespective of the type of tumor, however, the case loss was approximately tripled from FS to LS. Furthermore, extrapolation showed that a reduction in the number of cores punched per case, for example by one, would further double the number of cases lost. Reasons for exclusion were mainly as follows: core loss; <25% tumorous tissue per core; core folding; and core with necrotic area. CONCLUSION: This study shows that punching at least three to four cores per case is advisable when constructing TMAs from oncocytoma and renal cell carcinoma specimens, and that the type of tumor has an effect on the cause and rate of core exclusion.     

Tissue microarray sampling strategy for prostate cancer biomarker analysis

High-density tissue microarrays (TMA) are useful for profiling protein expression in a large number of samples but their use for clinical biomarker studies may be limited in heterogeneous tumors like prostate cancer. In this study, the optimization and validation of a tumor sampling strategy for a prostate cancer outcomes TMA is performed. Prostate cancer proliferation determined by Ki-67 immunohistochemistry was tested. Ten replicate measurements of proliferation using digital image analysis (CAS200, Bacus Labs, Lombard, IL, USA) were made on 10 regions of prostate cancer from a standard glass slide. Five matching tissue microarray sample cores (0.6 mm diameter) were sampled from each of the 10 regions in the parallel study. A bootstrap resampling analysis was used to statistically simulate all possible permutations of TMA sample number per region or sample. Statistical analysis compared TMA samples with Ki-67 expression in standard pathology immunohistochemistry slides. The optimal sampling for TMA cores was reached at 3 as fewer TMA samples significantly increased Ki-67 variability and a larger number did not significantly improve accuracy. To validate these results, a prostate cancer outcomes tissue microarray containing 10 replicate tumor samples from 88 cases was constructed. Similar to the initial study, 1 to 10 randomly selected cores were used to evaluate the Ki-67 expression for each case, computing the 90th percentile of the expression from all samples used in each model. Using this value, a Cox proportional hazards analysis was performed to determine predictors of time until prostate-specific antigen (PSA) recurrence after radical prostatectomy for clinically localized prostate cancer. Examination of multiple models demonstrated that 4 cores was optimal. Using a model with 4 cores, a Cox regression model demonstrated that Ki-67 expression, preoperative PSA, and surgical margin status predicted time to PSA recurrence with hazard ratios of 1.49 (95% confidence interval [CI] 1.01-2.20, p = 0.047), 2.36 (95% CI 1.15-4.85, p = 0.020), and 9.04 (95% CI 2.42-33.81, p = 0.001), respectively. Models with 3 cores to determine Ki-67 expression were also found to predict outcome. In summary, 3 cores were required to optimally represent Ki-67 expression with respect to the standard tumor slide. Three to 4 cores gave the optimal predictive value in a prostate cancer outcomes array. Sampling strategies with fewer than 3 cores may not accurately represent tumor protein expression. Conversely, more than 4 cores will not add significant information. This prostate cancer outcomes array should be useful in evaluating other putative prostate cancer biomarkers.     

Nonsmall cell lung carcinoma with neuroendocrine differentiation--an entity of no clinical or prognostic significance

The existence of non-small cell lung carcinoma with neuroendocrine differentiation as a distinct entity and its relevance for prognostic and treatment purposes is controversial. This study assesses the frequency and biologic and prognostic significance of neuroendocrine (NE) expression of synaptophysin (SNP), chromogranin (Ch), and neural cell adhesion molecule (N-CAM) using tissue microarray (TMA) and immunohistochemistry. Six hundred nine nonsmall cell lung carcinomas (NSCLCs) were reviewed for subclassification. TMA blocks were made using duplicate 0.6-mm-diameter tissue cores and slides stained with SNP, Ch, and N-CAM. Immunoreactivity was considered if 1% or more of tumor cells were positive. Hematoxylin and eosin-stained sections were subclassified as: 243 adenocarcinoma (ACA), 272 squamous cell carcinoma (SCC), 35 large cell carcinoma, 32 non-small cell carcinoma NOS, and 6 other (carcinosarcoma, giant cell carcinoma). Positivity for either marker was identified in 13.6% of NSCLC (76/558). NSCLC showed reactivity for Ch in 0.4% of cases (2/524), for SNP in 7.5% of cases (39/521) and for N-CAM in 8.6% of cases (44/511), whereas only 0.2% of cases (1/517) showed coexpression of SNP and Ch and none of all 3 markers. The assessment of NE differentiation in NSCLC is unnecessary and expensive and is of no clinical or prognostic significance. SNP or N-CAM stains a small minority of NSCLC, whereas Ch immunoreactivity is less common. Positivity for any 2 NE markers is rare. SNP is more likely to be expressed in adenocarcinoma (P=0.01) and N-CAM in squamous-cell carcinoma (P=0.008). Otherwise there was no correlation between immunoreactivity and tumor morphology. Disease specific and overall survival is not influenced by NE differentiation and therefore non-small cell lung carcinoma with neuroendocrine differentiation should not be a subclass distinct from the other NSCLC.     

Eculizumab Improves Posttransplant Thrombotic Microangiopathy Due to Antiphospholipid Syndrome Recurrence but Fails to Prevent Chronic Vascular Changes

Thrombotic microangiopathy (TMA) is one of the hallmark vascular lesions of antiphospholipid syndrome nephropathy (APSN). These lesions are at high risk of recurrence after kidney transplantation. The complement pathway is thought to be active in this process. We used eculizumab to treat three consecutive kidney transplant recipients with posttransplant TMA due to APSN recurrence that was resistant to plasmapheresis and explored the complement deposition and apoptotic and vascular cell markers on the sequential transplant biopsies. Treatment with eculizumab resulted in a rapid and dramatic improvement of the graft function in all three patients and in improvement of the TMA lesions within the graft. None of these patients had TMA flares after eculizumab was withdrawn. At the time of TMA diagnosis, immunofluorescence studies revealed intense C5b‐9 and C4d depositions at the endothelial cell surface of the injured vessels. Moreover, C5b‐9 colocalized with vessels exhibiting a high rate of apoptotic cells. Examination of sequential biopsies during eculizumab therapy showed that TMA lesions, C4d and apoptotic markers were rapidly cleared but the C5b‐9 deposits persisted for several months as a footprint of the TMA. Finally, we noticed that complement inhibition did not prevent the development of the chronic vascular changes associated with APSN. Eculizumab seems to be an efficient method for treating severe forms of posttransplant TMA due to APSN recurrence. Terminal complement inhibition does not prevent the development of chronic APSN.     

Human kidney injury molecule-1 (hKIM-1): a useful immunohistochemical marker for diagnosing renal cell carcinoma and ovarian clear cell carcinoma

Human kidney injury molecule-1 (hKIM-1), a type I transmembrane glycoprotein expressed in injured renal proximal tubules, was also found in renal cell carcinoma (RCC). The current study attempts to evaluate the diagnostic utility of hKIM-1 in a large series of 480 neoplasms including defined subtypes of renal cell tumors, metastatic RCCs, and nonrenal tumors. Tissue microarray (TMA) sections containing 179 renal cell tumors (73 clear cell RCC, 30 papillary RCC, 16 chromophobe RCC, 15 oncocytoma, and 45 metastatic RCC) were included in this study. In addition, 80 cases of renal cell neoplasm and 221 nonrenal tumors in routine tissue sections were also included. Both TMA and routine sections were incubated with anti-hKIM-1 monoclonal antibody using an EnVision-HRP kit. The results demonstrated that a membranous/cytoplasmic staining pattern for hKIM-1 was observed in 54 of 73 (74%) clear cell RCCs and 28 of 30 (93%) papillary RCCs on TMA sections. Zero of 54 chromophobe RCCs and 4 of 41 (9.75%) oncocytomas were positive for hKIM-1 when combining TMA and routine sections. Similar staining results were observed in 35 of 45 (78%) metastatic RCCs. Data from cDNA microarray expression and Western blot demonstrated similar findings. Fifteen of 16 cases (93.8%) of clear cell carcinoma of the ovary demonstrated positive reactivity for hKIM-1. These data indicate that hKIM-1: (1) is a relatively sensitive and specific marker for papillary, clear cell, and metastatic RCCs, (2) can be used to distinguish clear cell from chromophobe RCC, and (3) may serve as a diagnostic marker for clear cell carcinoma of the ovary.     

Predictive Value of Tumor Proliferative Indices in Periampullary Cancers: Ki-67, Mitotic Activity Index (MI) and Volume Corrected Mitotic Index (M/V) Using Tissue Microarrays

Background  

Morphometry [nuclear Ki-67 labelling, mitotic activity index (MI), and volume-corrected mitotic index (M/V)] for periampullary cancers using tissue microarrays has not been performed previously. The purpose of the study was to assess these indices on tissue microarray (TMA) sections constructed from patients with periampullary cancers and study their association with clinicopathological variables.