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ObjectiveOvarian cancer (OC) is the second most lethal gynecological cancer among women throughout the world. Protocadherin-8 (PCDH8) could function as a candidate tumor suppressor. However, the link between PCDH8 and OC development is poorly understood.Materials and MethodsA total of 68 OC patients were retrospectively enrolled. Clinical information was collected and cancer tissues were used for tissue microarray. The PCDH8 expression was determined on tissue microarray by immunohistochemical staining, and PCDH8 protein was detected in cancer tissues and adjacent tissue by western blotting. Human OC cell lines (SKOV-3 and OVCAR-3) were used to assess the effects of PCDH8 overexpression by western blot and real-time PCR analysis. 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide cell proliferation assay, wound healing migration assay, colony formation assay and invasion assays were performed to assess the influence of PCDH8 on cell function. Cells with Luc-nonspecific Lentiviral or Luc-Lentiviral with PCDH8 gene were subcutaneously injected into nude mice to observe the effect of PCDH8 gene on tumor growth. Bioluminescence imaging was used to observe tumor volume.ResultsWe found a low expression of PCDH8 in OC tissues versus the corresponding adjacent tissue. The PCDH8 expression, International Federation of Gynecology and Obstetrics stage, metastasis and recurrence were the independent prognostic factors for over-all survival by multivariate analyses. Furthermore, the patients with recurrence presented a low level of PCDH8 in OC tissues, and patients with advanced tumor stage also had a low PCDH8 expression. Importantly, the low expression of PCDH8 in OC tissues had a poor prognosis with a low overall survival rate. Overexpression of PCDH8 could inhibit OC cell growth/proliferation, migration, invasion, and colony formation in vitro. In vivo experiments also proved that overexpression of PCDH8 could inhibit OC cell growth/proliferation.ConclusionsProtocadherin-8 might be considered as a candidate tumor suppressor and play a crucial role in the progression of OC.

The proliferation marker Ki-67 has been used as a prognostic marker to separate low- and high-risk breast cancer subtypes and guide treatment decisions for adjuvant chemotherapy. The association of Ki-67 with response to tamoxifen therapy is unclear. High-throughput automated scoring of Ki-67 might enable standardization of quantification and definition of clinical cut-off values. We hypothesized that digital image analysis (DIA) of Ki-67 can be used to evaluate proliferation in breast cancer tumors, and that Ki-67 may be associated with tamoxifen resistance in early-stage breast cancer. Here, we apply DIA technology from Visiopharm using a custom designed algorithm for quantifying the expression of Ki-67, in a case-control study nested in the Danish Breast Cancer Group clinical database, consisting of stages I, II, or III breast cancer patients of 35-69 years of age, diagnosed during 1985-2001, in the Jutland peninsula, Denmark. We assessed DIA-Ki-67 score on tissue microarrays (TMAs) from breast cancer patients in a case-control study including 541 ER-positive and 300 ER-negative recurrent cases and their non-recurrent controls, matched on ER-status, cancer stage, menopausal status, year of diagnosis, and county of residence. We used logistic regression to estimate odds ratios and associated 95% confidence intervals to determine the association of Ki-67 expression with recurrence risk, adjusting for matching factors, chemotherapy, type of surgery, receipt of radiation therapy, age category, and comorbidity. Ki-67 was not associated with increased risk of recurrence in tamoxifen-treated patients (ORadj =0.72, 95% CI 0.54, 0.96) or ER-negative patients (ORadj =0.85, 95% CI 0.54, 1.34). Our findings suggest that Ki-67 digital image analysis in TMAs is not associated with increased risk of recurrence among tamoxifen-treated ER-positive breast cancer or ER-negative breast cancer patients. Overall, our findings do not support an increased risk of recurrence associated with Ki-67 expression.

Activated leukocyte cell adhesion molecule (ALCAM) is a membranous cell adhesion protein that is often expressed in breast cancer. Data on the prognostic impact of ALCAM expression is highly controversial in this cancer. To evaluate the clinical impact of ALCAM expression in a sufficiently large patient cohort, we utilized a tissue microarray (TMA) containing more than 2,100 primary breast cancers with clinical follow-up data by immunohistochemistry. TMA spots containing normal breast epithelium showed moderate to strong membranous ALCAM staining. ALCAM staining was strong in 66.2%, moderate in 10.9%, weak in 11.1% and absent in 11.8% of 1,778 (80.9%) interpretable breast cancer tissue spots. Decreased ALCAM expression was significantly associated with advanced tumor size (p=0.0017), unfavorable tumor grade (p<0.0001), negative ER and PR status (p<0.0001 each) as well as high Ki67 labeling index (p<0.0001). Cancers with ACLAM expression loss had a significantly poorer overall (p<0.0001) and disease-specific survival (p=0.0088). This association also held true in the subset of nodal positive cancers (p<0.0001). In conclusion, these data demonstrate that ALCAM is generally expressed in normal and cancerous breast epithelium and that a marked reduction of ALCAM expression characterizes a subset of breast cancer patients with adverse tumor characteristics and unfavorable clinical outcome.

Background and Objective: Tissue microarray (TMA) is a method of harvesting small disks of tissue from a range of standard paraffin tissue blocks and placing them in an array on a recipient paraffin block such that hundreds of cases can be analyzed simultaneously by using only a few microliters of antibody in immunohistochemistry as a single experiment. The TMA construction done with the help of automated tissue arrayer or commercially available rubber molds are expensive. This study involved the fabrication of TMA using rubber-based additional silicone mold constructed in the department and comparison of this method with two other methods of fabricating TMA. Materials and Methods: The TMA mold was fabricated using silicone material in the department. The recipient blocks were prepared. The tissue core prepared from donor blocks were inserted into the recipient blocks. The sections taken from this were compared with the TMA using double-sided adhesive tape technique and TMA by punching out holes in prefabricated dummy paraffin recipient block for insertion of tissue core. Results: The TMA using a mold made of silicone showed more advantages than other two methods. Conclusion: Fabricating TMA mold using silicone in the department is inexpensive and yet efficient.

The limited clinical success of anti‐HGF/MET drugs can be attributed to the lack of predictive biomarkers that adequately select patients for treatment. We demonstrate here that quantitative digital imaging of formalin fixed paraffin embedded tissues stained by immunohistochemistry can be used to measure signals from weakly staining antibodies and provides new opportunities to develop assays for detection of MET receptor activity. To establish a biomarker panel of MET activation, we employed seven antibodies measuring protein expression in the HGF/MET pathway in 20 cases and up to 80 cores from 18 human cancer types. The antibodies bind to epitopes in the extra (EC)‐ and intracellular (IC) domains of MET (MET4EC, SP44_METIC, D1C2_METIC), to MET‐pY1234/pY1235, a marker of MET kinase activation, as well as to HGF, pSFK or pMAPK. Expression of HGF was determined in tumour cells (T_HGF) as well as in stroma surrounding cancer (St_HGF). Remarkably, MET4EC correlated more strongly with pMET (r = 0.47) than SP44_METIC (r = 0.21) or D1C2_METIC (r = 0.08) across 18 cancer types. In addition, correlation coefficients of pMET and T_HGF (r = 0.38) and pMET and pSFK (r = 0.56) were high. Prediction models of MET activation reveal cancer‐type specific differences in performance of MET4EC, SP44_METIC and anti‐HGF antibodies. Thus, we conclude that assays to predict the response to HGF/MET inhibitors require a cancer‐type specific antibody selection and should be developed in those cancer types in which they are employed clinically.

Abstract Introduction: Trophoblast cell surface antigen 2 (TROP2) is the target of sacituzumab govitecan, an antibody-drug conjugate approved for treatment of triple negative breast cancer and urothelial carcinoma. Methods: A tissue microarray containing 18,563 samples from 150 different tumor types and subtypes as well as 608 samples of 76 different normal tissue types was analyzed by TROP2 immunohistochemistry. Results: TROP2 positivity was found in 109 tumor categories, including squamous cell carcinomas of various origins, urothelial, breast, prostate, pancreatic, and ovarian cancers (>95% positive). High TROP2 expression was linked to advanced stage (p = 0.0069) and nodal metastasis (p < 0.0001) in colorectal cancer as well as to nodal metastasis in gastric adenocarcinoma (p = 0.0246) and papillary thyroid cancer (p = 0.0013). Low TROP2 expression was linked to advanced stage in urothelial carcinoma (p < 0.0001), high pT (p = 0.0024), and high grade (p < 0.0001) in breast cancer, as well as with high Fuhrmann grade (p < 0.0001) and pT stage (p = 0.0009) in papillary renal cell carcinomas. Conclusion: TROP2 is expressed in many epithelial neoplasms. TROP2 deregulation can be associated with cancer progression in a tumor-type dependent manner. Since anti-TROP2 cancer drugs have demonstrated efficiency, they may be applicable to a broad range of tumor entities in the future.

Thyroid transcription factor 1 (TTF-1) immunohistochemistry (IHC) is routinely used for the distinction of primary pulmonary adenocarcinomas. However, TTF-1 can also occur in other malignancies. A tissue microarray containing 17,772 samples from 152 different tumor types was analyzed. Napsin-A, CK20, SATB2, FABP1, and Villin-1 IHC data were available from previous studies. TTF-1 staining was seen in 82 of 152 tumor categories including thyroidal cancers (19–100%), adenocarcinomas (94%), neuroendocrine tumors (67%) of the lung, small cell neuroendocrine carcinomas (71–80%), mesenchymal tumors (up to 42%), and thymomas (39%). Comparative analysis of TTF-1 and Napsin-A revealed a sensitivity/specificity of 94%/86% (TTF-1), 87%/98% (Napsin-A), and 85%/99.1% (TTF-1 and Napsin-A) for the distinction of pulmonary adenocarcinomas. Combined analysis of TTF-1 and enteric markers revealed a positivity for TTF-1 and at least one enteric marker in 22% of pulmonary adenocarcinomas but also a TTF-1 positivity in 6% of colorectal, 2% of pancreatic, and 3% of gastric adenocarcinomas. TTF-1 is a marker of high sensitivity but insufficient specificity for pulmonary adenocarcinomas. A small fraction of TTF-1-positive gastrointestinal adenocarcinomas represents a pitfall mimicking enteric-type pulmonary adenocarcinoma. Combined analysis of TTF-1 and Napsin-A improves the specificity of pulmonary adenocarcinoma diagnosis.

Summary A novel metabolomics analysis technique, termed matrix‐assisted laser desorption/ionization mass spectrometry imaging‐based plant tissue microarray (MALDI‐MSI‐PTMA), was successfully developed for high‐throughput metabolite detection and imaging from plant tissues. This technique completely overcomes the disadvantage that metabolites cannot be accessible on an intact plant tissue due to the limitations of the special structures of plant cells (e.g. epicuticular wax, cuticle and cell wall) through homogenization of plant tissues, preparation of PTMA moulds and matrix spraying of PTMA sections. Our study shows several properties of MALDI‐MSI‐PTMA, including no need of sample separation and enrichment, high‐throughput metabolite detection and imaging (>1000 samples per day), high‐stability mass spectrometry data acquisition and imaging reconstruction and high reproducibility of data. This novel technique was successfully used to quickly evaluate the effects of two plant growth regulator treatments (i.e. 6‐benzylaminopurine and N‐phenyl‐N′‐1,2,3‐thiadiazol‐5‐ylurea) on endogenous metabolite expression in plant tissue culture specimens of Dracocephalum rupestre Hance (D. rupestre). Intra‐day and inter‐day evaluations indicated that the metabolite data detected on PTMA sections had good reproducibility and stability. A total of 312 metabolite ion signals in leaves tissues of D. rupestre were detected, of which 228 metabolite ion signals were identified, they were composed of 122 primary metabolites, 90 secondary metabolites and 16 identified metabolites of unknown classification. The results demonstrated the advantages of MALDI‐MSI‐PTMA technique for enhancing the overall detection ability of metabolites in plant tissues, indicating that MALDI‐MSI‐PTMA has the potential to become a powerful routine practice for high‐throughput metabolite study in plant science. MALDI‐MSI‐PTMA was reported as a novel high‐throughput analysis technique for the enhancement of metabolite detection in plant science.

Tissue microarray (TMA) technology is a high-throughput research tool, which has greatly facilitated and accelerated tissue analyses by in-situ technologies. TMAs are amenable to every research method that can be applied on the standard whole sections at enhanced speed. It plays a central role in target verification of results from cDNA arrays, expression profiling of tumors and tissues, and is proving to be a powerful platform for proteomic research. In this review article, primarily meant for students of pathology and oncology, we briefly discuss its basic methodology, applications and merits and limitations.