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as geNorm [53] or NormFinder [54] are then used to identify small RNAs that represent the
most stable normalizer, or more commonly, the most stable combination of normalizers.
Candidate biomarkers are then assessed relative to the reference gene signal(s) to provide a
reliable difference measurement between sample groups.
In contrast, experimental plans that may include highly multiplexed assays and hundreds
of miRNA targets can benefit from mean centering restricted (MCR) normalization [52] . MCR
normalization calculates the mean value of all detected miRNAs, and assesses expression
differences against this background. An extension of this method, termed concordance cor-
related restricted (CCR) normalization, can be used to migrate candidates identified from
MCR-corrected RT-qPCR array data to a singleplex assay format [52] .
Normalization is an integral component of a larger process that must be thoughtfully con-
structed to achieve the goal of pinpointing miRNAs with potential as biomarkers. Since the
first publication in this area in 2008 [48] , multiple strategies have been devised and tested,
and these methods offer flexibility across a myriad of sample and experimental conditions in
pursuit of robust, disease-specific markers.
5.3 DETECTION OF miRNAs IN CLINICAL SPECIMENS
F OR CANCER DIAGNOSTICS AND PROGNOSTIC S
5.3.1 Pancreatic Cancer as a Model for Development of miRNA-Based Tests
To evaluate the potential of miRNAs as diagnostic tools for cancer, we assessed miRNA
expression in different biopsy types including fresh frozen, FFPE, and fine needle aspiration
(FNA). In this section, we will discuss the use of pancreatic cancer as a model for miRNA-
based assay development. We developed the miRNA-based molecular LDT, miR Inform
Pancreas, that can discriminate between chronic pancreatitis and pancreatic cancer to assist
in obtaining the definitive diagnosis of pancreatic cancer in fixed specimens. In addition, we
are validating additional assays for pancreatic cancer based on the expression of miRNAs
panels for use in patients with benign, inconclusive and non-diagnostic results from endo-
scopic ultrasound-guided fine needle aspiration (EUS-FNA) of solid lesions, as well as an
assay for optimization of surgical treatment in patients with pancreatic cystic lesions.
Despite recent significant advances in diagnostic imaging, staging, surgical techniques,
and perioperative patient care, there is still a pressing need to develop diagnostic tools capa-
ble of identifying patients that are at risk, and to detect tumors in the earliest stages of cancer
development to reduce the high morbidity and mortality associated with this malignancy.
Only serum CA 19-9 has been recommended for use in the routine monitoring of pancreatic
cancer progression in response to therapy [55,56] . However, the clinical utility of CA 19-9 is
compromised by frequent increase of its levels in bile duct, gastric, and colon cancers, as well
as in such non-malignant conditions as pancreatitis and cystic fibrosis, and CA-19 has only a
79-81% sensitivity and 82-90% specificity in symptomatic patients suspected of having pan-
creatic cancer [57] .
Pancreatic cancer is the fourth leading cause of cancer-related deaths in the US, with a five
year overall survival rate of 6% for all stages combined. Over half of patients are diagnosed
at a late stage, when the cancer has already spread, which carries a five year survival rate of
 
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