An exploratory study for the discovery of non-invasive hepatocellular carcinoma biomarkers among high-risk hepatitis C virus infected patients

Hepatocellular Carcinoma (HCC) is a major healthcare problem, representing the third most common cause of cancer-related mortality worldwide. Chronic infections with Hepatitis B virus (HBV) and/or Hepatitis C virus (HCV) are the major risk factors for the development of HCC. The incidence of HBV -associated HCC is in decline as a result of an effective HBV vaccine; however, since an equally effective HCV vaccine has not yet been developed, there are 130 million HCV infected patients worldwide who are at a high-risk for developing HCC. Because reliable parameters and/or tools for the early detection of HCC among high-risk individuals are severely lacking, HCC patients are always diagnosed at a late stage where surgical solutions or effective treatment are not possible. Using urine as a non-invasive sample source, two different approaches (proteomic-based and genomic-based approaches) were pursued with the common goal of discovering potential biomarker candidates for the early detection of HCC among high-risk chronic HCV infected patients. Urine was collected from 106 HCV infected Egyptian patients, 32 of whom had already developed HCC and 74 patients who were diagnosed as HCC-free at the time of initial sample collection. In addition to these patients, urine samples were also collected from 12 healthy control individuals. Total urinary proteins, Trans-renal nucleic acid (Tr-NA) and microRNA (miRNA) were isolated from urine using novel methodologies and silicon carbide-loaded spin columns. In the first, "proteomic-based", approach, liquid chromatography coupled with tandem mass spectrometry (LC-MS/MS) was used to identify potential candidates from pooled urine samples. This was followed by validating relative expression levels of proteins present in urine among all the patients using quantitative real time-PCR (qRT-PCR). This approach revealed that significant over-expression of three proteins: DJ-1, Chromatin Assembly Factor-1 (CAF-1) and 11 Moemen Abdalla HCC Biomarkers Heat Shock Protein 60 (HSP60), were characteristic events among HCC-post HCV infected patients. As a single-based HCC biomarker, CAF-1 over-expression identified HCC among HCV infected patients with a specificity of 90%, sensitivity of 66% and with an overall diagnostic accuracy of 78%. Moreover, the CAF-lIHSP60 tandem identified HCC among HCV infected patients with a specificity of 92%, sensitivity of 61 % and with an overall diagnostic accuracy of 77%. In the second genomic-based approach, two different approaches were processed. The first approach was the miRNA-based approach. The expression levels of miRNAs isolated from urine were studied using the Illumina MicroRNA Expression Profiling Assay. This was followed by qRT-PCR-based validation of deregulated expression of identified miRNA candidates among all the patients. This approach shed the light on the deregulated expression of a number of miRNAs, which may have a role in either the development of HCC among HCV infected patients (i.e. miR-640, miR-765, miR-200a, miR-521 and miR-520) or may allow for a better understanding of the viral-host interaction (miR-152, miR-486, miR-219, miR452, miR-425, miR-154 and miR-31). Moreover, the deregulated expression of both miR-618 and miR-650 appeared to be a common event among HCC-post HCV infected patients. The results of the search for putative targets of these two miRNA suggested that miR-618 may be a potent oncogene, as it targets the tumor-suppressor gene Low density lipoprotein-related protein 12 (LPR12), while miR-650 may be a potent tumor-suppressor gene, as it is supposed to downregulate the TNF receptor-associated factor-4 (TRAF4) oncogene. The specificity of miR-618 and miR-650 deregulated expression patterns for the early detection of HCC among HCV infected patients was 68% and 58%, respectively, whereas the sensitivity was 64% and 72%, respectively. When the deregulated expression of both miRNAs was combined as a tandem biomarker, the specificity and the sensitivity were 75% and 58% respectively. 111 Moemen Abdalla HCC Biomarkers In the second, "Trans-renal nucleic acid-based", approach, the urinary apoptotic nucleic acid (uaNA) levels of 70ng/mL or more were found to be a good predictor of HCC among chronic HCV infected patients. The specificity and the sensitivity of this diagnostic approach were 76% and 86%, respectively, with an overall diagnostic value of 81 %. The uaNA levels positively correlated to HCC disease progression as monitored by epigenetic changes of a panel of eight tumor-suppressor genes (TSGs) using methylation-sensitive PCR. Moreover, the pairing of high uaNA levels (:::: 70 ng/mL) and CAF-1 over-expreSSIOn produced a highly specific (l 00%) multiple-based HCC biomarker with an acceptable sensitivity of 64%, and with a diagnostic accuracy of 82%. In comparison to the previous pairing, the uaNA levels (:::: 70 ng/mL) in tandem with HSP60 over-expression was less specific (89%) but highly sensitive (72%), resulting in a diagnostic accuracy of 64%. The specificities of miR-650 deregulated expression in combination with either high uaNA content or HSP 60 over-expression were 82% and 79%, respectively, whereas, the sensitivities of these combinations were 64% and 58%, respectively. The potential biomarkers identified in this study compare favorably with the diagnostic accuracy of the a-fetoprotein levels test, which has a specificity of 75%, sensitivity of 68% and an overall diagnostic accuracy of 70%. Here we present an intriguing study which shows the significance of using urine as a noninvasive sample source for the identification of promising HCC biomarkers. We have also introduced new techniques for the isolation of different urinary macromolecules, especially miRNA, from urine. Furthermore, we strongly recommend the potential biomarkers indentified in this study as focal points of any future research on HCC diagnosis. A larger testing pool will determine if their use is practical for mass population screening. This explorative study identified potential targets that merit further investigation for the development of diagnostically accurate biomarkers isolated from 1-2 mL urine samples that were acquired in a non-invasive manner.

Cabinet Card by C. Arthur, Photographer, St. Catharines [n.d.]

An unidentified young woman is featured in this black and white cabinet card by photographer C. Arthur, of St. Catharines, Ontario. The address of Mr. Arthur's studio is printed along the bottom of the photograph. This photograph was among the family memorabilia owned by Iris Sloman Bell of St. Catharines. The Sloman - Bell families include relatives who were former African American slaves who settled in Canada.Charles A. Arthur is listed as a photographer in St. Catharines from 1880 - 1883 and Charles P. Arthur (possibly a son?) was a photographer in the same city in 1898. Source: Phillips, Glen C. The Ontario photographers list (1851-1900). Sarnia: Iron Gate Publishing Co., 1990.

Photograph of Unidentified Woman by C. Arthur, Photographer, St. Catharines [n.d.]

An unidentified young woman is featured in this undated black and white studio photograph by C. Arthur, Photographer, of St. Catharines, Ontario. This small cabinet card photograph was in the possession of the Iris Sloman Bell family of St. Catharines. Relatives of the Sloman - Bell families include former African American slaves who settled in Canada.Two photographers by the name of Arthur are listed as working in St. Catharines, Ontario in the second half of the 19th century. Charles A. Arthur was a photographer from 1880 - 1883, while Charles P. Arthur (possibly a son?) is listed as a photographer in 1898. Source: Phillips, Glen C. The Ontario photographers list (1851-1900). Sarnia: Iron Gate Publishing Co., 1990.

Cabinet Card of Young Woman Photographed by N. C. Shorey, of Toronto [n.d.]

An unidentified African Canadian young woman is the subject of this cabinet card portrait photograph by N. C. Shorey, of Toronto, Ontario. The photo is undated but believed to be taken in the late 1890s. The photographer's stamp, with his name and address, appears in the gold lettering at the bottom of the card. The young woman in the photograph could possibly be a relative of the Sloman - Bell family, who resided in the London and St. Catharines areas. This cabinet card was in the possession of Iris Sloman Bell, of St. Catharines, Ontario. The Sloman - Bell family relatives are descended from former Black slaves from the United States.N. C. Shorey is listed as a photographer in Toronto, Ont. from 1892 - 1900. Source: Phillips, Glen C. The Ontario photographers list (1851-1900). Sarnia: Iron Gate Publishing Co., 1990. "Cabinet card photographs were first introduced in 1866. They were initially employed for landscapes rather than portraitures. Cabinet cards replaced Carte de visite photographs as the popular mode of photography. Cabinet cards became the standard for photographic portraits in 1870. Cabinet cards experienced their peak in popularity in the 1880's. Cabinet cards were still being produced in the United States until the early 1900's and continued to be produced in Europe even longer. The best way to describe a cabinet card is that it is a thin photograph that is mounted on a card that measures 4 1/4″ by 6 1/2″. Cabinet cards frequently have artistic logos and information on the bottom or the reverse of the card which advertised the photographer or the photography studio's services." Source: http://cabinetcardgallery.wordpress.com/category/cabinet-card-history/