Identification of integrin drug targets for 17 solid tumor types.

Integrins are contributors to remodeling of the extracellular matrix and cell migration. Integrins participate in the assembly of the actin cytoskeleton, regulate growth factor signaling pathways, cell proliferation, and control cell motility. In solid tumors, integrins are involved in promoting metastasis to distant sites, and angiogenesis. Integrins are a key target in cancer therapy and imaging. Integrin antagonists have proven successful in halting invasion and migration of tumors. Overexpressed integrins are prime anti-cancer drug targets. To streamline the development of specific integrin cancer therapeutics, we curated data to predict which integrin heterodimers are pausible therapeutic targets against 17 different solid tumors. Computational analysis of The Cancer Genome Atlas (TCGA) gene expression data revealed a set of integrin targets that are differentially expressed in tumors. Filtered by FPKM (Fragments Per Kilobase of transcript per Million mapped reads) expression level, overexpressed subunits were paired into heterodimeric protein targets. By comparing the RNA-seq differential expression results with immunohistochemistry (IHC) data, overexpressed integrin subunits were validated. Biologics and small molecule drug compounds against these identified overexpressed subunits and heterodimeric receptors are potential therapeutics against these cancers. In addition, high-affinity and high-specificity ligands against these integrins can serve as efficient vehicles for delivery of cancer drugs, nanotherapeutics, or imaging probes against cancer

Non-specific binding of antibodies in immunohistochemistry: Fakes and facts

Protocols for blocking non-specific antibody (Ab) binding in immunohistochemistry are based on rather contradictory and outdated reports. This prompted us to prove, whether non-specific Ab binding may really lead to unwanted background staining in routinely processed cell and tissue probes. In this study, the probes were fixed and processed according to routine protocols with and without a blocking step (goat serum or BSA). Surprisingly, all Ab in probes processed without a blocking step did not show any propensity towards non-specific binding that might lead to background staining, thus implying that endogenous Fc receptors do not retain their ability to bind Fc portion of Ab after standard fixation. Likewise in routinely fixed probes, we did not find any non-specific Ab binding ascribed to a combination of ionic and hydrophobic interactions. The traditionally used protein blocking step is useless in immunostaining of routinely fixed tissues

Is the biology of breast cancer changing? A study of hormone receptor status 1984-1986 and 1996-1997

Using archived tumours, those from 1984-1986 and 1996-1997 underwent immunohistochemistry for hormone receptors and grade analysis. A significant shift towards more ER-positive and low-grade disease was found; this appears to reflect screening practices, but could still influence survival

HAGE, a cancer/testis antigen expressed at the protein level in a variety of cancers

The search for novel tumour antigens that are either uniquely expressed or over-expressed in a wide variety of tumours is still ongoing. Because of their expression in a broad spectrum of cancers and limited expression in normal tissues, cancer/testis antigens are considered to be potentially reliable targets for immunotherapy of cancer in general. The helicase antigen HAGE has been identified as a cancer/testis antigen. However, little is known about its expression in normal and cancer tissues. Using a newly developed antibody against HAGE, specific staining of its expression by immunohistochemistry was validated and optimised on murine tumours transfected to express the HAGE protein. The antibody was subsequently used to determine HAGE expression in normal human and cancer tissue microarrays. HAGE protein expression was confirmed in 75% (12/16) of carcinomas as compared to normal tissues, which either did not express HAGE at all or expressed HAGE at very low levels with the exception of testis. Interestingly, discrepancies were also found between mRNA analysis by real time quantitative PCR (RT-qPCR) and protein analysis by immunohistochemistry, emphasising the need to validate the expression of cancer/testis antigens at the protein level prior to the development of new vaccine strategies. HAGE is therefore proposed to be a valid candidate for designing a broad spectrum vaccine against cancer

Automated Segmentation of Cells with IHC Membrane Staining

This study presents a fully automated membrane segmentation technique for immunohistochemical tissue images with membrane staining, which is a critical task in computerized immunohistochemistry (IHC). Membrane segmentation is particularly tricky in immunohistochemical tissue images because the cellular membranes are visible only in the stained tracts of the cell, while the unstained tracts are not visible. Our automated method provides accurate segmentation of the cellular membranes in the stained tracts and reconstructs the approximate location of the unstained tracts using nuclear membranes as a spatial reference. Accurate cell-by-cell membrane segmentation allows per cell morphological analysis and quantification of the target membrane proteins that is fundamental in several medical applications such as cancer characterization and classification, personalized therapy design, and for any other applications requiring cell morphology characterization. Experimental results on real datasets from different anatomical locations demonstrate the wide applicability and high accuracy of our approach in the context of IHC analysi

Immunostaining of skeletal tissues

Peer reviewedPreprin

Felis catus papillomavirus type 2 infection and skin cancer in domestic cats : a thesis presented in partial fulfilment of the requirements for the degree of Doctor of Philosophy in Veterinary Science at Massey University, Manawatū, New Zealand

Felis catus papillomavirus type 2 (FcaPV-2) is a virus which commonly infects the skin of domestic cats. While most infections are asymptomatic, there is growing evidence that FcaPV-2 may play a role in the development of a subset of feline cutaneous squamous cell carcinomas (SCCs). In the first part of this thesis, the natural history of FcaPV-2 infection was investigated with the aim of determining when cats become infected with the virus. A real-time PCR assay was developed to quantify FcaPV-2 DNA in feline skin swabs. This assay was then used to measure the FcaPV-2 DNA load in serial samples from two populations of cats. Results from these studies showed that most kittens are exposed to FcaPV-2 in the first few days of life. Additionally, the primary source of exposure is likely to be direct contact with other cats in the household, particularly their queen, as some of the queens appeared to be shedding large amounts of virus. FcaPV-2 mRNA was also detected in some of the kittens, confirming that they had become infected with FcaPV-2 soon after birth. The aim of the second part of this thesis was to determine the quantity and transcriptional activity of the FcaPV-2 DNA present in feline cutaneous SCCs in order to determine if the virus was involved in cancer development or just present as an innocent bystander. Real-time PCR assays were developed to measure FcaPV-2 gene expression in SCCs and the results clearly distinguished two subsets of feline cutaneous SCCs. The majority of the SCCs had low copy numbers of FcaPV-2 DNA and no FcaPV-2 gene expression, suggesting the virus was an incidental finding. In contrast, around a third of the SCCs had detectable FcaPV-2 gene expression and high copy numbers of FcaPV-2 DNA, similar to that found in the FcaPV-2-induced premalignant lesions. There was also a significant association between FcaPV-2 gene expression and alterations in a host cell cycle regulatory protein (p16). Taken together, these results strongly suggest that FcaPV-2 played a role in the development of around a third of the feline cutaneous SCCs. The results from the studies reported in this thesis support a causative role of FcaPV-2 in a proportion of feline cutaneous SCCs. However, as infection of cats is common and appears to occur early in life, there may be little opportunity to prevent SCC development by preventing FcaPV-2 infection

A33 shows similar sensitivity to but is more specific than CDX2 as an immunomarker of colorectal carcinoma

Aims: CDX2 is widely used as a sensitive and specific immunomarker for colorectal carcinoma (CRC) but neither this sensitivity nor specificity is absolute. This study is the first known comparison of CDX1 and A33 against CDX2 as immunomarkers for CRC. Methods and Results: As a pilot study, whole sections of 51 cases of liver metastatic carcinoma of different origins - colorectum (n=32), breast (n=3), oesophagogastric tract (n=4), lung (n=3), pancreas (n=8), and prostate (n=1) - were immunostained with CDX1, CDX2 and A33. Compared with CDX1, A33 showed higher sensitivity as a CRC immunomarker, greater interobserver reproducibility for assessment of expression, and less background cross-reactivity. Therefore, only A33 was compared with CDX2 for a tissue microarray-based study of primary adenocarcinomas of different origin: CRC (n=55), liver deposits of metastatic CRC (n=60), breast (n=101), lung (n=40), oesophagogastric tract (n=134), ovary (n= 67), pancreas (n= 77), and prostate (n= 56). Combining the whole section and TMA cases of CRC, A33 had a sensitivity of 95.9% and CDX2 a sensitivity of 97.2%. Combining all the whole section and TMA cases of non-colorectal carcinomas, A33 showed 85.4% specificity as a marker of CRC compared to CDX2 which showed a specificity of 64.3%. The higher specificity of A33 as a colorectal carcinoma immunomarker compared with CDX2 was particularly seen amongst pancreatic and ovarian carcinomas. Further, unlike with CDX2, none of the prostatic and lung carcinomas studied showed A33 positivity. Conclusions: A33 shows similar sensitivity to but is more specific than CDX2 as an immunomarker of CRC

A Review on the Applications of Crowdsourcing in Human Pathology

The advent of the digital pathology has introduced new avenues of diagnostic medicine. Among them, crowdsourcing has attracted researchers' attention in the recent years, allowing them to engage thousands of untrained individuals in research and diagnosis. While there exist several articles in this regard, prior works have not collectively documented them. We, therefore, aim to review the applications of crowdsourcing in human pathology in a semi-systematic manner. We firstly, introduce a novel method to do a systematic search of the literature. Utilizing this method, we, then, collect hundreds of articles and screen them against a pre-defined set of criteria. Furthermore, we crowdsource part of the screening process, to examine another potential application of crowdsourcing. Finally, we review the selected articles and characterize the prior uses of crowdsourcing in pathology