Standardization of concentrated antibodies for use in automated immunohistochemistry

Background: Immunohistochemistry (IHC) is a method of identifying proteins in cells or tissues that is useful for diagnosis and research. In modern pathology, it has assumed an important supporting role in the molecular diagnosis of certain neoplasia, with an exponential contribution in personalized medicine. Automation in immunohistochemistry contributes to reduced test variability through standardization. The transition to automation is a process, and for a laboratory that already has a collection of stockconcentrated primary antibodies, ideally, these antibodies will be compatible with the chosen automated method, as antibodies are of high value to be discarded as a consequence of migration to automated immunohistochemistry. Methods: 78 concentrated antibodies were tested for use in Ventana Medical Systems’ Benchmark XT automation platform. Thirty-one human tissue samples containing the antigens of interest were used as positive control. Results: All antibodies tested showed good performance, indicating the feasibility of using these antibodies concentrated in the automation platform in question. The protocol most frequently used was the one with antigenic retrieval with Cell Conditioning 1 for 60 minutes and incubation in the primary antibody for 32 minutes at 42ºC. The dilutions of the primary antibodies in automation ranged from 1:20 to 1:4000. Conclusion: Under the aforementioned conditions, it was possible to take advantage of the portfolio of concentrated antibodies present in the laboratory at the time of transition from manual to automated immunohistochemistry

Nuclear morphometry and chromatin texture changes in hepatocellular carcinoma samples may predict outcomes of liver transplanted patients

BACKGROUND: Nuclear changes are typical in the carcinogenesis of hepatocellular carcinoma (HCC). Morphometry and chromatin texture analysis are quantitative methods for their quantification. In this study, we analyzed nuclear morphometry and chromatin texture parameters in samples of hepatocellular carcinoma from liver transplant patients and their associations with clinicopathologic variables. METHODS: Samples of HCC and adjacent tissue from 34 individuals were collected in tissue microarray blocks. Stained slides were microphotographed using an optical microscope and nuclear parameters analyzed in ImageJ (FracLac plug-in). ROC curve analysis was used to find accurate cut-offs for differentiation of neoplastic and non-neoplastic cells. The inter-rater agreement was also evaluated. RESULTS: Nuclear morphometric and textural differences were observed between the samples of HCC and adjacent tissue of liver transplant patients. Lower mean gray value (p = 0.034) and Feret diameter (p = 0.024) were associated with higher Model for End-Stage Liver Disease (MELD) scores. Nuclei with larger area (p = 0.014) and larger Feret diameter (p = 0.035) were associated with lower survival. Lower aspect ratio was associated with HCC recurrence after the transplant (p = 0.048). The cut-off of 1.13 μm (p =  \u3c 0.001) for aspect ratio and cut-off of 21.15 μm (p = 0.038) for perimeter were established for the differentiation of neoplastic and non-neoplastic cells. The morphometric analysis was reproducible to area, circularity, Feret diameter, mean gray value and aspect ratio between observers (p =  \u3c 0.001). CONCLUSIONS: Nuclear morphometric differences between the HCC and the adjacent tissue samples were associated with prognostic variables (MELD scores, recurrence and survival) and may predict liver transplant patients\u27 outcomes

Overexpression of PIK3CA impacts global survival of patients with HER2 subtype breast carcinoma

Purpose: To investigate the expression of proteins fosfatidilinositol-4,5-bifosfate 3-quinase (PIK3CA) and phosphatase and tensin homolog (PTEN) in HER2-positive breast cancer and verify their associations with clinical and pathological variables. Methods: We assessed PTEN and PIK3CA status using immunohistochemistry (IHC), which was performed in formalin-fixed paraffin-embedded biopsies from 50 patients with HER2-positive breast cancer. Medical records were studied for collection of clinical-pathological information, including overall survival (OS). The HIC markers PTEN and PIK3CA were analyzed semi-quantitatively by two blinded independent researchers. The relationship between the variables were evaluated using the chi-square test and Kaplan-Meier curves plus log-rank test for survival. Results: In IHC, the expression level of PIK3CA was 86%, and loss of PTEN expression was observed in 46% of the cases. The expression of the markers showed no significant correlation with each other or with the clinical and pathological parameters studied: tumor grade, staging, ER, PR, Ki67 and recurrence. The highest expression of PIK3CA was associated with lower number of deaths (p=0.016) and longer OS of patients (p=0.001). The PTEN marker showed no significant effect on OS. Conclusions: The PIK3CA expression showed a protective effect in relation to the OS of patients with HER2-positive breast cancer

Nuclear morphometry and chromatin texture changes in hepatocellular carcinoma samples may predict outcomes of liver transplanted patients

Background: Nuclear changes are typical in the carcinogenesis of hepatocellular carcinoma (HCC). Morphometry and chromatin texture analysis are quantitative methods for their quantification. In this study, we analyzed nuclear morphometry and chromatin texture parameters in samples of hepatocellular carcinoma from liver transplant patients and their associations with clinicopathologic variables. Methods: Samples of HCC and adjacent tissue from 34 individuals were collected in tissue microarray blocks. Stained slides were microphotographed using an optical microscope and nuclear parameters analyzed in ImageJ (FracLac plug-in). ROC curve analysis was used to find accurate cut-offs for differentiation of neoplastic and non-neoplastic cells. The inter-rater agreement was also evaluated. Results: Nuclear morphometric and textural differences were observed between the samples of HCC and adjacent tissue of liver transplant patients. Lower mean gray value (p=0.034) and Feret diameter (p=0.024) were associated with higher Model for End-Stage Liver Disease (MELD) scores. Nuclei with larger area (p=0.014) and larger Feret diameter (p=0.035) were associated with lower survival. Lower aspect ratio was associated with HCC recurrence after the transplant (p=0.048). The cut-off of 1.13 μm (p= < 0.001) for aspect ratio and cut-off of 21.15 μm (p=0.038) for perimeter were established for the differentiation of neoplastic and non-neoplastic cells. The morphometric analysis was reproducible to area, circularity, Feret diameter, mean gray value and aspect ratio between observers (p= < 0.001). Conclusions: Nuclear morphometric differences between the HCC and the adjacent tissue samples were associated with prognostic variables (MELD scores, recurrence and survival) and may predict liver transplant patients’ outcomes

Damage-associated molecular patterns (DAMPs) related to immunogenic cell death are differentially triggered by clinically relevant chemotherapeutics in lung adenocarcinoma cells

Background: Chemotherapeutics can stimulate immune antitumor response by inducing immunogenic cell death (ICD), which is activated by Damage-Associated Molecular Patterns (DAMPs) like the exposure of calreticulin (CRT) on the cell surface, the release of ATP and the secretion of High Mobility Group Box 1 (HMGB1). Methods: Here, we investigated the levels of ICD-associated DAMPs induced by chemotherapeutics commonly used in the clinical practice of non-small cell lung cancer (NSCLC) and the association of these DAMPs with apoptosis and autophagy. A549 human lung adenocarcinoma cells were treated with clinically relevant doses of cisplatin, carboplatin, etoposide, paclitaxel and gemcitabine. We assessed ICD-associated DAMPs, cell viability, apoptosis and autophagy in an integrated way. Results: Cisplatin and its combination with etoposide induced the highest levels of apoptosis, while etoposide was the less pro-apoptotic treatment. Cisplatin also induced the highest levels of ICD-associated DAMPs, which was not incremented by co-treatments. Etoposide induced the lower levels of ICD and the highest levels of autophagy, suggesting that the cytoprotective role of autophagy is dominant in relation to its pro-ICD role. High levels of CRT were associated with better prognosis in TCGA databank. In an integrative analysis we found a strong positive correlation between DAMPs and apoptosis, and a negative correlation between cell number and ICD-associated DAMPs as well as between autophagy and apoptosis markers. We also purpose a mathematical integration of ICD-associated DAMPs in an index that may represent with greater biological relevance this process. Cisplatin-treated cells showed the highest IndImmunog, while etoposide was the less immunogenic and the more pro-autophagic treatment. Conclusions: Cisplatin alone induced the highest levels of ICD-associated DAMPs, so that its combination with immunotherapy may be a promising therapeutic strategy in NSCLC