Immunohistochemical Detection of Markers for Translational Studies of Lung Disease in Pigs and Humans.

Genetically engineered pigs are increasingly recognized as valuable models for the study of human disease. Immunohistochemical study of cellular markers of disease is an important tool for the investigation of these novel models so as to evaluate genotype and treatment differences. Even so, there remains a lack of validated markers for pig tissues that can serve as a translational link to human disease in organs such as the lung. Herein, we evaluate markers of cellular inflammation (cluster of differentiation [CD]3, CD79a, B cell lymphoma [BCL] 6, ionized calcium-binding adapter molecule [IBA]1, and myeloperoxidase) and those that may be involved with tissue remodeling (alpha-smooth muscle actin, beta-tubulin-III, lactoferrin, mucin [MUC]5AC, MUC5B, and cystic fibrosis transmembrane conductance regulator [CFTR]) for study of lung tissues. We compare the utility of these markers between pig and human lungs to validate translational relevance of each marker. Our results suggest these markers can be a useful addition in the pathological evaluation of porcine models of human disease.

Prognostic and therapeutic value of the Hippo pathway, RABL6A, and p53-MDM2 axes in sarcomas.

Additional prognostic and therapeutic biomarkers effective across different histological types of sarcoma are needed. Herein we evaluate expression of TAZ and YAP, the p53-MDM2 axis, and RABL6A, a novel oncoprotein with potential ties to both pathways, in sarcomas of different histological types. Immunohistochemical staining of a tissue microarray including 163 sarcomas and correlation with clinical data showed that elevated YAP and TAZ independently predict worse overall and progression-free survival, respectively. In the absence of p53 expression, combined TAZ and YAP expression adversely affect overall, progression free, and metastasis free survival more than TAZ or YAP activation alone. RABL6A independently predicted shorter time to metastasis and was positively correlated with p53, MDM2 and YAP expression, supporting a possible functional relationship between the biomarkers. Network analysis further showed that TAZ is positively correlated with MDM2 expression. The data implicate all five proteins as clinically relevant downstream players in the Hippo pathway. Finally, a novel inhibitor of MDM2 (MA242), effectively suppressed the survival of sarcoma cell lines from different histological types regardless of p53 status. These findings suggest both independent and cooperative roles for all five biomarkers across different histological types of sarcoma in predicting patient outcomes and potentially guiding future therapeutic approaches.

Impact of Nuclear Interleukin-1 Alpha and EGFR Expression on Recurrence and Survival Outcomes in Oral Squamous Cell Carcinomas.

Interleukin-1 alpha (IL-1α) is a pleiotropic cytokine involved in inflammation and immune response and is upregulated in many solid tumors including head and neck squamous cell carcinomas. Although IL-1α expression is generally associated with poor prognosis, the implications of the subcellular localization of IL-1α expression in patient outcomes are poorly understood. This study is aimed at investigating the prognostic value of nuclear and cytoplasmic immunohistochemical IL-1α expression in oral squamous cell carcinomas (OSCCs). Tissue microarrays containing 146 OSCCs were analyzed for IL-1α and epidermal growth factor receptor (EGFR) expression by immunohistochemistry. IL-1α and EGFR expression scores were correlated with clinicopathological parameters and survival outcomes. IL-1α expression was observed in the nuclear and/or cytoplasmic compartments in 98% of evaluable tumors and 78% of tumors expressed IL-1α in both compartments. There were no differences observed in overall survival or progression-free survival between high, moderate, or negative IL-1α nuclear/cytoplasmic expression scores. When IL-1α nuclear/cytoplasmic expression scores were stratified by positive or negative EGFR expression, tumors with a combined EGFR-positive and high nuclear IL-1α expression profile were significantly more likely to possess perineural invasion and were significantly associated with a high risk of tumor recurrence and worse progression-free survival compared to all other EGFR and combined IL-1α/EGFR expression profiles. Altogether, nuclear IL-1α expression may enhance the prognostic value of EGFR in OSCC and warrants further study as a prognostic biomarker for recurrence.

Glycogen depletion can increase the specificity of mucin detection in airway tissues.

OBJECTIVE: Mucin is an important parameter for detection and assessment in studies of airway disease including asthma and cystic fibrosis. Histochemical techniques are often used to evaluate mucin in tissues sections. Periodic acid Schiff (PAS) is a common technique to detect neutral mucins in tissue, but this technique also detects other tissue components including cellular glycogen. We tested whether depletion of glycogen, a common cellular constituent, could impact the detection of mucin in the surface epithelium of the trachea. RESULTS: Normal tissues stained by PAS had significantly more staining than serial sections of glycogen-depleted tissue with PAS staining (i.e. dPAS technique) based on both quantitative analysis and semiquantitative scores. Most of the excess stain by the PAS technique was detected in ciliated cells adjacent to goblet cells. We also compared normal tissues using the Alcian blue technique, which does not have reported glycogen staining, with the dPAS technique. These groups had similar amounts of staining consistent with a high degree of mucin specificity. Our results suggest that when using PAS techniques to stain airways, the dPAS approach is preferred as it enhances the specificity for airway mucin.

Immunohistochemical Markers for Prospective Studies in Neurofibromatosis-1 Porcine Models.

Neurofibromatosis type 1 (NF1) is a common, cancer-predisposing disease caused by mutations in the NF1 tumor gene. Patients with NF1 have an increased risk for benign and malignant tumors of the nervous system (e.g., neurofibromas, malignant peripheral nerve sheath tumors, gliomas) and other tissues (e.g., leukemias, rhabdomyosarcoma, etc.) as well as increased susceptibility to learning disabilities, chronic pain/migraines, hypertension, pigmentary changes, and developmental lesions (e.g., tibial pseudoarthrosis). Pigs are an attractive and upcoming animal model for future NF1 studies, but a potential limitation to porcine model research has been the lack of validated reagents for direct translational study to humans. To address that issue, we used formalin-fixed tissues (human and pigs) to evaluate select immunohistochemical markers (activated caspase-3, allograft inflammatory factor-1, beta-tubulin III, calbindin D, CD13, CD20, desmin, epithelial membrane antigen, glial fibrillary acidic protein, glucose transporter-1, laminin, myelin basic protein, myoglobin, proliferating cell nuclear antigen, S100, vimentin, and von Willebrand factor). The markers were validated by comparing known expression and localization in human and pig tissues. Validation of these markers on fixed tissues will facilitate prospective immunohistochemical studies of NF1 pigs, as well as other pig models, in a more efficient, reproducible, and translationally relevant manner.

A method for histopathological study of the multifocal nature of spinal cord lesions in murine experimental autoimmune encephalomyelitis.

Experimental autoimmune encephalomyelitis (EAE) is a well-established mouse model for multiple sclerosis and is characterized by infiltration of mononuclear cells and demyelination within the central nervous system along with the clinical symptoms of paralysis. EAE is a multifocal and random disease, which sometimes makes histopathologic analysis of lesions difficult as it may not be possible to predict where lesions will occur, especially when evaluating cross sections of spinal cord. Consequently, lesions may be easily missed due to limited sampling in traditional approaches. To evaluate the entire length of the spinal cord while maintaining anatomic integrity, we have developed a method to section the cord within the decalcified spinal column, which allows for the study of the multifocal nature of this disease and also minimizes handling artifact. HE and Luxol fast blue staining of these spinal cord sections revealed a paucity of lesions in some areas, while others showed marked inflammation and demyelination. The percentage of spinal cord affected by EAE was evaluated at four separate areas of longitudinally sectioned cord and it varied greatly within each animal. Immunohistochemical staining of in situ spinal cords which had undergone decalcification was successful for key immuno-markers used in EAE research including CD3 for T cells, B220 for B cells and F4/80 for murine macrophages. This method will allow investigators to look at the entire spinal cord on a single slide and evaluate the spinal cord with and without classic EAE lesions.

TAZ and YAP are frequently activated oncoproteins in sarcomas.

TAZ (WWTR1) and YAP are transcriptional coactivators and oncoproteins inhibited by the Hippo pathway. Herein we evaluate 159 sarcomas representing the most prevalent sarcoma types by immunohistochemistry for expression and activation (nuclear localization) of TAZ and YAP. We show that 50% of sarcomas demonstrate activation of YAP while 66% of sarcomas demonstrate activated TAZ. Differential activation of TAZ and YAP are identified in various sarcoma types. At an RNA level, expression of WWTR1 or YAP1 predicts overall survival in undifferentiated pleomorphic sarcoma and dedifferentiated liposarcoma. Immunohistochemistry demonstrates that TAZ and YAP expression and activation are positively correlated with grade in the well-differentiated liposarcoma to dedifferentiated liposarcoma tumor progression sequence as well as conventional chondrosarcomas. TAZ and YAP are constitutively activated oncoproteins in sarcoma cell lines. Knock-down of TAZ and YAP demonstrate differential activity for the two proteins. Verteporfin decreases colony formation in soft agar as well as CTGF expression in sarcoma cell lines harboring activated TAZ and YAP.