**2.1.3 Circulating tumor cells**

274 Biomarker

Several studies have reported optimized isolation protocols to enhance the recovery of microRNAs in the stabilized samples. For example it was shown that microRNAs could be isolated from PAXgene-stabilized blood of sufficient quantity and quality that is suitable for

Another problem hampering the analysis of microarray gene expression data in whole blood is the presence of globin. Globin mRNA in red blood cells accounts for over 70% of all mRNA in whole blood and interferes with the accurate assessment of other genes (Field et al., 2007; Wright et al., 2008). Several approaches have been developed to mitigate this effect and tested in microarray experiments (Liu et al., 2006; Vartanian et al., 2009; Wright et al., 2008). Globin reduction techniques based on biotinylated DNA capture oligos (Ambion GLOBINclear processing protocol) produced sensitive results but was least reproducible among all the methods tested (Vartanian et al., 2009). An alternative protocol with globin PNAs (peptide nucleic acid inhibitory oligos) proved to be the best in sensitivity and reproducibility, but was the most time-consuming and required the highest amount of total RNA input (Liu et al., 2006; Vartanian et al., 2009). An alternative approach was suggested by Eklund and colleagues (Eklund et al., 2006). NuGEN's Ovation WB sample preparation protocol, based on single primer isothermal amplification (SPIA), generates cDNA target. The hybridization kinetics of the cDNA target are less affected than cRNA targets by the abundant globin RNA present in whole blood extract. The high specificity and sensitivity of cDNA targets, and the highly reproducible SPIA protocol have been shown to be as good or better for mitigating the interference of globin transcripts compared to other protocols (Fricano et al., 2011; Li et al., 2008; Parrish et al., 2010). The strong performance of this technique, and the relatively low input requirements (50ng of total RNA) have made the NuGEN Ovation WB protocol the method of choice for gene expression profiling in the

Both plasma and serum are widely used specimen types for molecular diagnostics. Nucleic acids that can be found in small amounts in cell-free preparations of whole blood are frequently called "circulating nucleic acids". To date, a number of studies show that plasma and serum nucleic acids can serve as both tumor- and fetal-specific markers for cancer detection and prenatal diagnosis, respectively. For example, several studies reported increased concentrations of DNA in the plasma or serum of cancer patients sharing some characteristics with DNA of tumor cells (Leon et al., 1977; Stroun et al., 1989). Interestingly, DNA levels decreased by up to 90% after radiotherapy, while persistently high or increasing DNA concentrations were associated with a lack of response to treatment (Anker et al., 2001). RNA has also been found circulating in the plasma or serum of normal subjects and cancer patients (Feng et al., 2008; Tsui et al., 2002, 2006). The recent discovery that serum and plasma contain a large amount of stable miRNAs derived from various tissues/organs has lead to multiple studies on circulating miRNA expression as well (Mitchell et al., 2008;

Analysis of circulating nucleic acids, however, requires modified extraction methods to utilize plasma or serum as the source material. First, plasma and serum are biospecimens that have a very high concentration of protein that can interfere with sample preparation and detection techniques. Second, the yield of circulating nucleic acids from small volume

downstream applications (Kruhøffer et al., 2007).

microarray community.

**2.1.2 Serum and plasma** 

Chen et al., 2008; Zhu et al., 2009).

Circulating tumor cells (CTCs) are cells that have been sloughed off of primary tumors and circulate in the bloodstream. Their numbers can be very small (1-10 cells per mL of whole blood) and these cells are not easily detected. Even though CTCs were first observed by Thomas Ashworth back in 1869, the technology with the requisite sensitivity and reproducibility to detect CTC in patients with metastatic disease was developed only recently (Sleijfer et al., 2007). While the presence of circulating tumor cells themselves can serve as a marker of poor clinical outcome, there is an opportunity to develop new biomarkers by studying the gene or protein expression in these cells. Changes in the phenotype of tumor cells can occur after the original diagnosis and resistance to a treatment can only be inferred after the treatment has failed. CTCs offer a tool to understand the complex biology of tumor cells, without the need of invasive biopsies.

Recently, CTCs have been the target of multiple molecular profiling studies (Bosma et al., 2002; Punnoose et al., 2010; Smirnov et al., 2005; Tewes et al., 2009). mRNA expression and DNA mutations can be measured from captured CTCs. RT-PCR using a multi-marker panel of cancer-associated genes was found to be the most sensitive technique for the detection of CTC in blood of breast cancer patients (Bosma et al., 2002; Tewes et al., 2009). Another approach involves the analysis of CTC-enriched samples by microarray gene expression profiling, where numerous genes like S100A14 and S100A16 have been detected (Smirnov et al., 2005).

### **2.1.4 Dried blood spots**

The method of collecting capillary blood on filter paper was introduced in Scotland by Robert Guthrie in 1963 and since then has become a mainstream approach for blood sample collection from newborns in more than 20 countries (Consultant Paediatricians and Medical Officers of Health of the SE Scotland Hospital Region, 1968; Scriver, 1998). These samples were found invaluable for screening for congenital metabolic disorders. Dried blood spots (DBS) are easily acquired through a simple needle stick and transfer to paper cards that are stored and handled at room temperature in ambient atmospheric conditions. This approach eliminates many costly, time-consuming, and unpleasant aspects of sample collection, and can also significantly reduce the cost for shipping samples. The collection of DBS samples requires very little infrastructure and can be done in resource-limiting locations. Vidal-Taboada and colleagues even showed that both patients and investigators prefer this as a method of DNA collection and storage (Vidal-Taboada et al., 2006).

The limitation of small sample volume has restricted the usage of dried blood spots for the development of molecular diagnostics until recently. Advances in technology have

Novel Tissue Types for the Development of Genomic Biomarkers 277

collection kits (Oragene or Norgen products). This ease of collection results in higher compliance by the patients. As is often the case in biological samples, the difference in yield is usually a donor dependent value (van Schie & Wilson, 1997). It is possible that saliva samples could replace blood samples for DNA studies. A study in Australia and New Zealand compared 10 matched pairs of blood and saliva, as well as nearly 2000 samples of either blood (Australia) or saliva (New Zealand; Oragene collection system) for genotyping. This study was larger than the van Schie & Wilson study, but corroborated that there is a donor dependency to DNA yield. Because of the larger sample number, they saw more sample variance. However, they also concluded that variance had more to do with collection, processing and donor variability than variance due to tissue type (Bahlo et al., 2010). The collection and processing methods can all eventually be controlled. In most cases there was enough mass from 1 ml of saliva sample to yield at least 4ug of DNA, which is

Readily-accessible and as well-tolerated as punch biopsies (Camidge et al., 2005), skin is comprised of various layers of cells, making it useful for phenotypic and histological studies. Moreover, as a constantly dividing tissue with cells at various stages of development, skin provides insight into important signaling networks such as EGF, Wnt,

Wee1 inhibitors have been examined as a way to bypass the G2 checkpoint, sensitizing p53 negative cells to DNA-damaging agents (Wang et al., 2001). In research conducted by Mizuarai et al., p53 negative rat skin xenograft tumors, p53 positive and negative cultured cancer cells, and p53 positive rat skin tissues were subjected to gemcitabine alone or in combination with the Wee-1 inhibitor MK-1775 (Mizuarai et al., 2009). Gene expression data

Because of its strong potential as a surrogate tissue, it is important to address storage and handling challenges faced when using skin. Due to its protective nature, skin is shielded by nucleases and difficult to homogenize. We have found immediate preservation in RNAlater following the manufacturer's protocol (rather than flash-freezing) and thorough pulverization are paramount to extracting sufficient quantities of high-quality nucleic acid

Synthetic skin is a relatively new surrogate tissue that lends itself to investigation of a wide variety of processes while reducing the need for volunteer recruitment or laboratory animal testing (Poumay & Coquette, 2007). For extracting nucleic acids, we have found that synthetic skin is less susceptible to nucleic acid degradation and more easily homogenized than real human skin (data not shown). Synthetic skin has recently been used to study processes such as wound healing (Koria et al., 2003), epithelial development (Taylor et al., 2009), effects of cosmetics on skin (Faller et al., 2002), and even differential gene expression

identified five genes as potential biomarkers present in both tumor and skin.

enough DNA for most molecular biology assays.

Notch and cell proliferation (Phillips & Sachs, 2005).

**2.4 Skin** 

**2.4.1 Skin tissue** 

(data not shown).

in skin disorders.

**2.4.2 Skin tissue alternatives** 

overcome many of the problems with reduced sensitivity and specificity. For example, the development of whole genome amplification (WGA) protocols allow researchers to perform reliable genome-wide scans using archived residual blood samples from newborn screening programs, which are standard practice in several countries (Hollegaard et al., 2009). Several studies have shown that despite being considered too vulnerable to degradation by ribonucleases, RNA could be recovered from DBS samples that had been stored for 15-20 years, and be successfully amplified by reverse transcription-PCR (Karlsson et al., 2003; Zubakov et al., 2008). Also, dried blood spots recently become the sample type of choice for HIV screening in low-resource settings (Sherman et al., 2005; Uttayamakul et al., 2005).
