Use Of Tissue Microarrays Research Articles

Quality Assurance and Cost Reduction in Histopathology Laboratories Using Tissue Microarrays

In the context of cost increases of both labor and consumables, cheaper and faster histopathology methods are needed. We implemented in our research laboratory the use of tissue microarrays (TMA) for the parallel processing and analysis of tissue samples. In this study, we used seven pre-processed, paraffinated biomimetic sectionable support matrices serving as "recipient" paraffin blocks to embed a total of 196 tissue cores from formalin-fixed paraffin-embedded tissue samples (serving as "donor" paraffin blocks) from seven different rabbit organs. These tissue samples were obtained using four different processing protocols: two 6 h protocols with xylene as the transition solvent, and two using butanol instead (one 10 h in duration and the other 72 h long). While the samples from protocols 1 and 2 (with xylene) quite regularly generated peeling of some of the cores from the slides (most likely because of substandard paraffin infiltration), butanol processing performed flawlessly for both processing protocols. Our proposed technique of using TMAs in the research laboratory brings with it a significant reduction in time and consumable costs (up to 77 and 64%, respectively), but also new challenges for all the upstream processes.

Tissue Microarray Lipidomic Imaging Mass Spectrometry Method: Application to the Study of Alcohol-Related White Matter Neurodegeneration.

Central nervous system (CNS) white matter pathologies accompany many diseases across the lifespan, yet their biochemical bases, mechanisms, and consequences have remained poorly understood due to the complexity of myelin lipid-based research. However, recent advances in matrix-assisted laser desorption/ionization-imaging mass spectrometry (MALDI-IMS) have minimized or eliminated many technical challenges that previously limited progress in CNS disease-based lipidomic research. MALDI-IMS can be used for lipid identification, semi-quantification, and the refined interpretation of histopathology. The present work illustrates the use of tissue micro-arrays (TMAs) for MALDI-IMS analysis of frontal lobe white matter biochemical lipidomic pathology in an experimental rat model of chronic ethanol feeding. The use of TMAs combines workload efficiency with the robustness and uniformity of data acquisition. The methods described for generating TMAs enable simultaneous comparisons of lipid profiles across multiple samples under identical conditions. With the methods described, we demonstrate significant reductions in phosphatidylinositol and increases in phosphatidylcholine in the frontal white matter of chronic ethanol-fed rats. Together with the use of a novel rapid peak alignment protocol, this approach facilitates reliable inter- and intra-group comparisons of MALDI-IMS data from experimental models and could be extended to human disease states, including using archival specimens.

Measurement of Ovarian Tumor Immune Profiles by Multiplex Immunohistochemistry: Implications for Epidemiologic Studies.

Despite the immunogenic nature of many ovarian tumors, treatment with immune checkpoint therapies has not led to substantial improvements in ovarian cancer survival. To advance population-level research on the ovarian tumor immune microenvironment, it is critical to understand methodologic issues related to measurement of immune cells on tissue microarrays (TMA) using multiplex immunofluorescence (mIF) assays. In two prospective cohorts, we collected formalin-fixed, paraffin-embedded ovarian tumors from 486 cases and created seven TMAs. We measured T cells, including several sub-populations, and immune checkpoint markers on the TMAs using two mIF panels. We used Spearman correlations, Fisher exact tests, and multivariable-adjusted beta-binomial models to evaluate factors related to immune cell measurements in TMA tumor cores. Between-core correlations of intratumoral immune markers ranged from 0.52 to 0.72, with more common markers (e.g., CD3+, CD3+CD8+) having higher correlations. Correlations of immune cell markers between the whole core, tumor area, and stromal area were high (range 0.69-0.97). In multivariable-adjusted models, odds of T-cell positivity were lower in clear cell and mucinous versus type II tumors (ORs, 0.13-0.48) and, for several sub-populations, were lower in older tissue (sample age > 30 versus ≤ 10 years; OR, 0.11-0.32). Overall, high correlations between cores for immune markers measured via mIF support the use of TMAs in studying ovarian tumor immune infiltration, although very old samples may have reduced antigenicity. Future epidemiologic studies should evaluate differences in the tumor immune response by histotype and identify modifiable factors that may alter the tumor immune microenvironment.

Expression of selected cytokeratins in human placenta - a preliminary observational study

Abstract Every human body is made up of billions of cells, and every cell consists of thousands of microscopic structures. Thanks to the presence of the cytoskeleton, which is built by microfilaments, microtubules, and intermediate filaments, cells are able to fulfill their main function. Dozens of genes encode a large family of cytoskeletal proteins, which form 10-nanometer-long microfilaments, called cytokeratins. The study was carried out on seven mature human placentas without significant pathology - all eligible mothers were healthy. The collection of basic anthropometric data preceded the dissection of the placentas. Paraffin blocks were made in the usual manner, and hematoxylin and eosin-stained slides were made afterward. Immunohistochemical reactions were performed and the expression of the studied markers was evaluated independently by two observers. Evaluation of microscopic material revealed the absence of expression of antibodies for cytokeratin 5/6 in placental tissues. Moreover, strong expression of cytokeratin 7 was demonstrated in the villi trophoblast in all types of villi. Immunohistochemical reactions were observed in the mesenchyme within the blood vessel wall, as well as in the extravascular tissue. The human placenta is an organ that only exists during the intrauterine period of human development and undergoes rapid changes and dynamic growth during pregnancy. These types of processes define selected placental cells as ‘pseudo-tumorigenic tissue’ because of the numerous similarities trophoblast cells have to tumor cells. The use of tissue microarray (TMA) in combination with immunohistochemistry (IHC) may be a valuable approach to validate the predictive and diagnostic utility of various biomarkers in non-cancerous tissues like placental tissue.

Immune Contexture and Differentiation Features Predict Outcome in Bladder Cancer

BackgroundAn improved risk assessment of patients with bladder cancer (BC) is important to optimize clinical management. ObjectiveTo identify whether immune cell subpopulations and cancer cell-intrinsic features are associated with outcome and response to first-line chemotherapy in BC. Design, setting, and participantsPrimary tumor tissue from 785 patients with BC (stage Ta-T4b) were stained using multiplex immunofluorescence (CD3, CD8, FOXP3, CD20, CD68, CD163, and MHC-I) and immunohistochemistry (pancytokeratin, CK5/6, GATA3, programmed death 1 [PD-1], and programmed death ligand 1 [PD-L1]). A digital image analysis quantified staining results within the carcinoma cell and stromal part of the tumor. Outcome measurements and statistical analysisPrimary endpoints were progression-free survival, recurrence-free survival, and response to first-line chemotherapy. Optimal cutoff values for investigated markers were estimated using maximally selected rank statistics and receiver operating characteristic for each primary endpoint. Time-to-event analyses were performed using Cox regression analyses. Results and limitationsSeveral immune subpopulations were independently associated with clinical outcomes. Especially, high PD-1 and PD-L1 expression was independently associated with an increased risk of recurrence and progression in non–muscle-invasive tumors, but with a lower risk of recurrence in muscle-invasive tumors. Furthermore, we observed a lower likelihood of response to first-line chemotherapy in patients with basal differentiation features. Finally, a model combining clinical risk factors with our most evident prognosticator improved prediction accuracy compared with clinical risk factors alone for progression in non–muscle-invasive BC and recurrence in muscle-invasive BC. The use of tissue microarrays and a long inclusion period are limitations to this study. ConclusionsImmune cell subpopulations and cancer cell-intrinsic features are associated with different clinical outcomes in BC. Patient summaryImmune cells play an important role in cancer development and treatment outcomes. Infiltration with specific immune cells and the presence of markers associated with immune evasion in the tumor predict clinical outcomes in bladder cancer.

Tumor-associated immune cell infiltrate density in penile squamous cell carcinomas

Penile squamous cell carcinomas are rare tumor entities throughout Europe. Early lymphonodal spread urges for aggressive therapeutic approaches in advanced tumor stages. Therefore, understanding tumor biology and its microenvironment and correlation with known survival data is of substantial interest in order to establish treatment strategies adapted to the individual patient. Fifty-five therapy naïve squamous cell carcinomas, age range between 41 and 85 years with known clinicopathological data, were investigated with the use of tissue microarrays (TMA) regarding the tumor-associated immune cell infiltrate density (ICID). Slides were stained with antibodies against CD3, CD8 and CD20. An image analysis software was applied for evaluation. Data were correlated with clinicopathological characteristics and overall survival. There was a significant increase of ICID in squamous cell carcinomas of the penis in relation to tumor adjacent physiological tissue. Higher CD3-positive ICID was significantly associated with lower tumor stage in our cohort. The ICID was not associated with overall survival. Our data sharpens the view on tumor-associated immune cell infiltrate in penile squamous cell carcinomas with an unbiased digital and automated cell count. Further investigations on the immune cell infiltrate and its prognostic and possible therapeutic impact are needed.

NTRK fusions are extremely rare in bone tumours.

AimsBecause of the efficacy of tropomyosin receptor kinase (Trk) inhibitor therapy in tumours with rearrangements of the neurotrophic tyrosine kinase receptor genes (NRTK genes), there has been a surge in demand for NTRK fusion screening. To date, most studies involving mesenchymal tumours have focused on soft tissue tumours, and data on bone tumours are sparse. Hence, we aimed to explore the frequency of NTRK fusions in a large series of primary bone tumours.Methods and resultsImmunohistochemical expression of pan‐Trk was successfully assessed in 354 primary bone tumours by the use of tissue microarrays. In a selection of positive cases, additional molecular analysis for NTRK fusions was performed with anchored multiplex polymerase chain reaction‐based targeted next‐generation sequencing. Positivity was found in 19 cases (5%), which comprised Ewing sarcoma (n = 6, 33%), osteosarcoma (n = 11, 13%), and giant‐cell tumour of bone (n = 2, 3%). In all except one case, cytoplasmic staining was observed. Weak staining was most often observed (n = 13), although five cases showed moderate staining and one case showed focal strong staining. Molecular analysis was successful in six cases, all of which were negative for NTRK fusions.ConclusionThe likelihood of finding an NTRK fusion in bone tumours in clinical practice is extremely low. This may imply that, if more comprehensive large‐scale molecular studies confirm this, routine predictive NTRK testing in bone tumour patients with advanced disease may be reconsidered.

Infiltration of M2 Macrophages and Regulatory T Cells Plays a Role in Recurrence of Renal Cell Carcinoma

BackgroundIt has been hypothesized that M2 macrophages and regulatory T cells (Tregs) may contribute to tumor progression by suppression of antitumor immunity. ObjectiveTo investigate the association between infiltration of CD163+ M2 macrophages and CD4+FOXP3+ Tregs with clinical outcomes in renal cell carcinoma patients. Design, setting, and participantsA cohort of 346 patients diagnosed with renal cell carcinoma at Örebro University Hospital between 1986 and 2011 was evaluated for CD163+ M2 macrophage and CD4+FOXP3+ Treg infiltration by immunohistochemistry. Outcome measurements and statistical analysisAssociations between clinicopathological features and infiltration of CD163+ M2 macrophages and/or CD4+FOXP3+ Tregs were estimated with chi-square or Fisher's exact tests. For survival analyses, Kaplan-Meier curves with log-rank tests and multivariate Cox proportional hazards regression models were used. Results and limitationsWe found that infiltration of CD163+ M2 macrophages and CD4+FOXP3+ Tregs were associated with adverse clinical outcomes. Our data further demonstrate that CD163+ M2 macrophages and CD4+FOXP3+ Tregs colocalize in tumor and normal tissue, and that this colocalization may have synergistic effects on tumor aggressiveness. The use of tissue microarrays rather than whole sections may be viewed as a limitation. ConclusionsInfiltration of CD163+ M2 macrophages and CD4+FOXP3+ Tregs is associated with recurrence of renal cell carcinoma, and colocalization of these cell types may have an association with clinical outcome. Patient summaryThe aim of this study was to investigate the association between infiltration of M2 macrophages and regulatory T cells with clinical outcomes in renal cell carcinoma. We demonstrated that renal cell carcinoma patients with high infiltration of both these cell types are at an increased risk of poor clinical outcomes.

Use of Tissue Microarray in Esophageal Squamous Cell Carcinoma.

Tissue microarray (TMA) is widely used for identifying the expression of markers in many tissues from patients with esophageal squamous cell carcinoma. The technology is mostly used in immunohistochemical studies to test the expression of markers and oncoproteins in signalling pathway as well as targeting proteins involved in therapies for esophageal squamous cell carcinoma. Appropriate use of TMA sections needs consideration of labor, planning, and expertise involved. For the best performance, it is important to design the layout of the TMA as well as use whole-slide scanning for interpretation of the TMA sections.

The adequacy of tissue microarrays in the assessment of inter- and intra-tumoural heterogeneity of infiltrating lymphocyte burden in leiomyosarcoma

The characterisation and clinical relevance of tumour-infiltrating lymphocytes (TILs) in leiomyosarcoma (LMS), a subtype of soft tissue sarcoma that exhibits histological heterogeneity, is not established. The use of tissue microarrays (TMA) in studies that profile TIL burden is attractive but given the potential for intra-tumoural heterogeneity to introduce sampling errors, the adequacy of this approach is undetermined. In this study, we assessed the histological inter- and intra-tumoural heterogeneity in TIL burden within a retrospective cohort of primary LMS specimens. Using a virtual TMA approach, we also analysed the optimal number of TMA cores required to provide an accurate representation of TIL burden in a full tissue section. We establish that LMS have generally low and spatially homogenous TIL burdens, although a small proportion exhibit higher levels and more heterogeneous distribution of TILs. We show that a conventional and practical number (e.g. ≤3) of TMA cores is adequate for correct ordinal categorisation of tumours with high or low TIL burden, but that many more cores (≥11) are required to accurately estimate absolute TIL numbers. Our findings provide a benchmark for the design of future studies aiming to define the clinical relevance of the immune microenvironments of LMS and other sarcoma subtypes.

LACE-Bio: Validation of Predictive and/or Prognostic Immunohistochemistry/Histochemistry-based Biomarkers in Resected Non–small-cell Lung Cancer

BackgroundComplete resection of non–small-cell lung cancer (NSCLC) offers the potential for cure after surgery and adjuvant chemotherapy. Patients may not benefit and may experience severe toxicity. There are no validated molecular tools to allow better patient selection. Materials and MethodsThe LACE-Bio (LACE [Lung Adjuvant Cisplatin Evaluation]) project includes 4 trials (International Adjuvant Lung Cancer Trial [IALT], Adjuvant Navelbine International Trialist Association [ANITA], JBR10, and Cancer and Leukemia Group B (CALGB)-9633). Immunohistochemistry biomarkers shown in one trial to have a prognostic/predictive effect on overall survival were tested. ResultsThe majority of the promising biomarkers could not be validated; the prognostic effect of tumor infiltrating lymphocytes and β-tubulin was confirmed. Potential causes include tissue fixation, storage, the use of tissue microarrays, and varying reagent/antibody batches. ConclusionsImmunohistochemistry assays from single trials may be misleading and require validation before being used for patient selection. LACE-Bio-2 is evaluating potential genomic biomarkers that may allow more precise selection of patients with NSCLC for adjuvant chemotherapy in NSCLC.

In MALDI-Mass Spectrometry Imaging on Formalin-Fixed Paraffin-Embedded Tissue Specimen Section Thickness Significantly Influences m/z Peak Intensity.

In matrix-assisted laser desorption/ionization-mass spectrometry imaging (MALDI-MSI) standardized sample preparation is important to obtain reliable results. Herein, the impact of section thickness in formalin-fixed paraffin embedded (FFPE) tissue microarrays (TMA) on spectral intensities is investigated. TMAs consisting of ten different tissues represented by duplicates of ten patients (n=200 cores) are cut at 1, 3, and 5μm. MSI analysis is performed and mean intensities of all evaluable cores are extracted. Measurements are merged and mean m/z intensities are compared. Visual inspection of spectral intensities between 1, 3, and 5μm reveals generally higher intensities in thinner tissue sections. Specifically, higher intensities are observed in the vast majority of peaks (98.6%, p<0.01) in 1μm compared with 5μm sections. Note that 28.4% and 2.1% of m/z values exhibit a at least two- and threefold intensity difference (p<0.01) in 1μm compared to 5μm sections, respectively. A section thickness of 1μm results in higher spectral intensities compared with 5μm. The results highlight the importance of standardized protocols in light of recent efforts to identify clinically relevant biomarkers using MSI. The use of TMAs for comparative analysis seems advantageous, as section thickness displays less variability.

PD-L1 Expression in Men with Penile Cancer and its Association with Clinical Outcomes

BackgroundIt has been hypothesized that PD-L1 expression in tumor cells and tumor-infiltrating immune (TII) cells may contribute to tumor progression by inhibiting antitumor immunity. ObjectiveTo investigate the association between PD-L1 expression in tumor cells and TII cells and clinical outcomes in penile cancer. Design, setting, and participantsA cohort of 222 men treated for penile squamous cell carcinoma (SqCC) at Örebro University Hospital between 1984 and 2008 with long-term follow-up (median 34 mo) was evaluated for PD-L1 expression in tumor cells and TII cells via immunohistochemistry. Outcome measurements and statistical analysisAssociation between clinicopathological features and PD-L1 expression was estimated using χ2 and Fisher's exact tests. For survival analyses, Kaplan-Meier curves with log-rank tests and multivariate Cox proportional hazards regression models were used. Results and limitationsWe found that 32.1% of the tumors and 64.2% of the TII cells expressed PD-L1. Our data demonstrate that penile SqCC patients with PD-L1–positive tumor cells or TII cells are at significant risk of lower cancer-specific survival and that the prognostic value of PD-L1 expression was strongest for tumor cell positivity. The use of tissue microarrays rather than whole sections may be viewed as a limitation. ConclusionsTumor PD-L1 expression independently identifies penile SqCC patients at risk of poor clinical outcomes. Patient summaryWe investigated how many patients with penile cancer had tumors that manufactured PD-L1, a protein that decreases the ability of the immune system to fight cancer. We found that up to one-third of penile tumors make this protein. Patients whose tumors make PD-L1 have more aggressive penile cancer and worse clinical outcomes.

Tissue microarray is suitable for scientific biomarkers studies in endometrial cancer

The aim of this study was to define the concordance between tissue microarrays (TMAs) of different sizes and whole slide for 15 different antibodies in endometrial cancer and study the use of TMAs in preoperative endometrial samples. Cores of preoperative and hysterectomy specimens of 14 endometrial cancer and three atypical hyperplasia cases were collected in TMA blocks. Two 0.6-mm and two 2.0-mm cores were used from each sample. Different antibodies were tested in TMAs and compared with results of whole slides of hysterectomy. Tested antibodies were as follows: ER, PR, p53, Ki-67, MLH1, PMS2, MSH2, MSH6, ARID1A, stathmin, IMP3, L1CAM, PTEN, β-catenin, and p16. Seventeen cases with four cores per paraffin block (both 0.6 and 2.0 mm in duplicate) and 15 different antibodies resulted in a total of 1020 cores for both preoperative and hysterectomy specimen. Overall, 2.0-mm cores were more assessable for evaluation than 0.6-mm cores (96.0 versus 79.5%, p < 0.01). For most antibodies, a substantial to good agreement between hysterectomy TMA and whole slide was present, with lowest agreement for p16 and stathmin and perfect agreement for mismatch repair proteins. Preoperative TMAs showed for most antibodies moderate to perfect agreement with hysterectomy TMAs. In conclusion, 2.0-mm cores are the preferred size for immunohistochemical studies in endometrial cancer. For all tested antibodies, TMAs are a good alternative for whole slide analysis in scientific studies with large patient cohorts, even in preoperative endometrial samples. However, caution is required for interpretation of TMA results of p16 and stathmin.

Abstract B28: The accuracy of tissue microarrays in the study of the sarcoma immune microenvironment is dependent on the number of sampled cores

Abstract Introduction: The immune tumor microenvironment (iTME) of soft-tissue sarcomas (STS) is not well characterized. The use of tissue microarray (TMA) methods is an attractive means of high-throughput immunohistochemistry-based immune profiling, Typically, between 1-3 tumor cores per case are included on conventional TMAs that are well validated for many protein markers across multiple tumor types. However, the reproducibility/representativeness of TMA for sarcomas is not fully established, and a significant factor is the marked intratumoral morphologic heterogeneity in STS. Here, we examine the variability of lymphoid infiltrate between different regions of the same tumor and how this might be optimally represented on a TMA. Methods: Multiple FFPE blocks, representing spatially discrete tumor areas, were selected from primary surgical specimen of 46 cases of localized leiomyosarcoma (LMS) and 9 cases of localized synovial sarcoma (SS) identified from institutional diagnostic archive. Following sectioning and immunohistochemistry for lymphocyte markers, average tumor-infiltrating lymphocytes (TIL) number per section was manually assessed and scored as a numerical variable. Variability of TIL numbers within and between cases was then statistically assessed through two-way ANOVA and intraclass correlation To determine the optimal number of 1-mm TMA cores that need to be sampled to provide representative overview of immune microenvironment, we performed iterative sampling of 1 to 20 virtual cores from digital microscopy images taken from representative blocks from 46 LMS cases. TILs within each virtual core area were digitally counted, with the average TIL number from the combination of increasing number of cores compared to the tumor overall TIL value. Results: We found a dynamic range in TIL numbers between individual cases of LMS but little variability in low TIL numbers seen between SS cases. Focusing on infiltrating T-lymphocytes in LMS, we found marked variation of TIL numbers between discrete areas of highly infiltrated tumors. However, 2-way ANOVA showed that intratumoral variance only accounted for 0.2% of variation in TILs, whilst intertumoral variation accounted for 54.1% of variance, suggesting greater intertumor than intratumor heterogeneity in lymphocyte infiltration in the LMS cohort. In our virtual core experiment, many more TMA cores (median = 11) than the conventional 1-3 were required in a sample to provide an accurate picture of the degree of an individual tumor's lymphocyte infiltration in an individual tumor (within +/-20% of mean TIL number). However, when the 46-case cohort was divided into high or low lymphocyte-infiltrated tumors around the cohort median TIL value, a single core was sufficient to correctly identify a tumor as being TIL high or TIL low in the majority of cases. Conclusion: The variability in TIL number between LMS cases (intertumor variation) in our cohort was much greater than the variability between different tissue blocks from the same individual tumor (intratumor variation), indicating that choice of tumor-containing block from those available for a given specimen may not be an important source of variation when studying the immune microenvironment of STS cohorts. We found that using a conventional number of TMA cores was sufficient to distinguish between tumors with high and low degrees of lymphocyte infiltration. However, in many cases, far more than 3 cores were required to accurately recapitulate the precise degree of infiltration in a tumor. Researchers investigating the iTME of STS should be wary of the apparent limitations of conventional TMA approaches in this field. The extent and biologic significance in the variability of lymphocyte distribution in STS lesions requires further investigation. Citation Format: Alex Lee, Khin Thway, Seth Pollack, Ian Judson, Paul Huang, Robin Jones. The accuracy of tissue microarrays in the study of the sarcoma immune microenvironment is dependent on the number of sampled cores [abstract]. In: Proceedings of the AACR Conference on Advances in Sarcomas: From Basic Science to Clinical Translation; May 16-19, 2017; Philadelphia, PA. Philadelphia (PA): AACR; Clin Cancer Res 2018;24(2_Suppl):Abstract nr B28.

Tissue microarray methodology identifies complement pathway activation and dysregulation in progressive multiple sclerosis.

The complement pathway has potential contributions to both white (WM) and grey matter (GM) pathology in Multiple Sclerosis (MS). A quantitative assessment of complement involvement is lacking. Here we describe the use of Tissue MicroArray (TMA) methodology in conjunction with immunohistochemistry to investigate the localization of complement pathway proteins in progressive MS cortical GM and subcortical WM. Antibodies targeting complement proteins C1q, C3b, regulatory proteins C1 inhibitor (C1INH, complement receptor 1 (CR1), clusterin, factor H (FH) and the C5a anaphylatoxin receptor (C5aR) were utilised alongside standard markers of tissue pathology. All stained slides were digitised for quantitative analysis. We found that numbers of cells immunolabelled for HLA-DR, GFAP, C5aR, C1q and C3b were increased in WM lesions (WML) and GM lesions (GML) compared to normal appearing WM (NAWM) and GM (NAGM), respectively. The complement regulators C1INH, CR1, FH and clusterin were more abundant in WM lesions, while the number of C1q+ neurons were increased and the number of C1INH+, clusterin+, FH+ and CR1+ neurons decreased in GM lesions. The number of complement component positive cells (C1q, C3b) correlated with complement regulator expression in WM, but there was no statistical association between complement activation and regulator expression in the GM. We conclude that TMA methodology and quantitative analysis provides evidence of complement dysregulation in MS GML, including an association of the numerical density of C1q+ cells with tissue lesions. Our work confirms that complement activation and dysregulation occur in all cases of progressive MS and suggest that complement may provide potential biomarkers of the disease.

Uso de la técnica de micromatrices de tejido en histología veterinaria

Las micromatrices de tejido son plataformas de alto rendimiento que permiten el análisis de decenas a cientos de muestras de tejidos en forma simultánea. Estas se han utilizado, en especial, para analizar tejidos neoplásicos. Sin embargo, los beneficios de su aplicación pueden ser aprovechados en otras áreas de investigación, como la embriología y la histología. El presente trabajo explora el uso de la técnica de micromatrices de tejido para el análisis morfológico y molecular de tejidos animales normales. Un total de 82 muestras de piel canina normal prenatal y posnatal fueron utilizadas para construir la matriz. A partir de ésta, se obtuvo una gran cantidad de secciones en las que se aplicaron técnicas de histología convencional y de inmunohistoquímica. Los resultados nos muestran la eficacia de la técnica para realizar el análisis morfológico y molecular de decenas de muestras de tejido normal en forma simultánea. Esto permite la evaluación más estandarizada de los tejidos, reduciendo así la variabilidad que puede ocurrir cuando se realizan ensayos sobre muestras individuales.

Ki67 expression in invasive breast cancer: the use of tissue microarrays compared with whole tissue sections

BackgroundAlthough the prognostic value of Ki67 in breast cancer is well documented, using optimal cut-points for patient stratification, reproducibility of the scoring and interpretation of the results remains a matter of debate particularly when using tissue microarrays (TMAs). This study aims to assess Ki67 expression assessed on TMAs and their matched whole tissue sections (WTS). Moreover, whether the cut-off used for WTS is reproducible on TMA in BC molecular classes and the association between Ki67 expression cut-off, assessed on TMAs and WTS, and clinicopathological parameters and patient outcome were tested.MethodA large series (n = 707) of primary invasive breast tumours were immunostained for Ki67 using both TMA and WTS and assessed as percentage staining and correlated with each other, clinicopathological parameters and patient outcome. In addition, MKI67 mRNA expression was correlated with Ki67 protein levels on WTS and TMAs in a subset of cases included in the METABRIC study.ResultsThere was moderate concordance in Ki67 expression between WTS and TMA when analysed as a continuous variable (Intraclass correlation coefficient = 0.61) and low concordance when dichotomised (kappa value = 0.3). TMA showed low levels of Ki67 with mean percentage of expression of 35 and 22% on WTS and TMA, respectively. MKI67 mRNA expression was significantly correlated with protein expression determined on WTS (Spearman Correlation, r = 0.52) and to a lesser extent on TMA (r = 0.34) (p < 0.001). Regarding prediction of patient outcome, statistically significant differences were detected upon stratification of patients with tumours expressing Ki67 at 10, 15, 20, 25 or 30% in TMA. Using TMA, ≥20% Ki67 provided the best prognostic cut-off particularly in triple-negative and HER2-positive classes.ConclusionKi67 expression in breast cancer can be evaluated using TMA although different cut-points are required to emulate results from WTS. A cut-off of ≥20% for Ki67 expression in BC provides the best prognostic correlations when TMAs are used.

Constructing Tissue Microarrays: Protocols and Methods Considering Potential Advantages and Disadvantages for Downstream Use.

Tissue microarrays were first constructed in the 1980s but were used by only a limited number of researchers for a considerable period of time. In the last 10 years there has been a dramatic increase in the number of publications describing the successful use of tissue microarrays in studies aimed at discovering and validating biomarkers. This, along with the increased availability of both manual and automated microarray builders on the market, has encouraged even greater use of this novel and powerful tool. This chapter describes the basic techniques required to build a tissue microarray using a manual method in order that the theory behind the practical steps can be fully explained. Guidance is given to ensure potential disadvantages of the technique are fully considered.

Tissue Microarray Is a Reliable Tool for the Evaluation of HER2 Amplification in Breast Cancer.

We examined an economical method for evaluating the amplification of the human epidermal growth factor receptor 2 (HER2) gene in breast cancer specimens. We compared HER2 amplification determined by fluorescence in situ hybridization (FISH) on whole-tissue (WT) blocks used for diagnostic and on tissue microarray (TMA) sections for a cohort of 521 consecutive patients with breast cancer. In a subset of 116 patients, we examined HER2 concordance from the WT section and a TMA section from a randomly chosen additional block (a proxy of the core biopsy). Overall concordance for HER2 amplification between WT and TMA sections was 98.2%, and between sections from WT and from the additional block was 99.0%. The high concordance rates support the use of TMA for the evaluation of HER2 amplification in breast cancer and suggest that FISH can be used to assess HER2 using core biopsies.