Pancreatic cancer (PaC) has a poor prognosis in most cases due to the lack of early detection owing to its nonspecific, asymptomatic nature. An effective, inexpensive screening tool is needed for early diagnosis (within 15 years of tumor initiation). It is well accepted that genomic instability is a hallmark of cancer. Recent research indicates that less than hundred microRNA (miRNA) sequences specifically generated by tumors are sufficient for early detection of PaC and other cancers for effective intervention with excellent prognosis. MiRNA can be extracted from urine and blood using standard kits. The key challenge is to detect specific biomarkers in the thousand-fold large background of miRNA sequences that body normally produces. While quantitative reverse transcribe-polymerase chain reaction (qRT-PCR) is an effective tool for miRNA profiling, it is prohibitively expensive fo screening. A microarray is an inexpensive alternative. However, due to the small size of miRNA, a conventional microarray is not reliable due to large background from nonspecific binding. A disruptive technology is needed to read microarrays of miRNA at high specificity with minimal background from the normal miRNA sequences and high sensitivity to avoid PCR amplification. Owing to the small size of miRNA, PCR is an added expensive complexity. It is well known that electrochemical detection has excellent specificity with virtually no background from nonspecific binding of targets to microarray of probes. The key limitations of this active detection method are: (a) only one target sequence per electrode can be detected, and (b) the redox current decreases as the sensor electrode size diminishes , making multiplexing difficult. A method developed in Saraf's lab, at the University of Nebraska-Lincoln, can electrochemically read microarray spots on a monolith electrode by simply scanning a laser with a beam size of ~10 m to quantitatively measure the local redox current. Published studies indicate that Scanning Electrometer for Electrical Double-layer (SEED) has (conservative) responsivity of <0.1 atto-moles. Vajra Instruments has licensed the patent for SEED from UNL and built a commercializable microscope size prototype. Using a 12 spot microarray, preliminary results indicate a sensitivity of 0.1 pico-molar (pM) with no signal from non-specific binding at 100% consistency. The goal in Phase I will be to achieve 0.01 pM sensitivity to detect ~105 copies of tumor specific miRNA extracted from 1 ml of serum or plasma (using standard commercial kits). The study will target five miRNA sequence from deidentified human serum/plasma samples of pancreatic patients without using PCR. The success will be based on the 0.01 pM sensitivity with 103 to 104 fold copies of background miRNA; and quantitative comparison with qRT-PCR, the gold standard. The proof-of-concept, Phase I study will be organized into two specific aims: (1) Quantification of SEED performance using synthetic miRNA; and (2) SEED analysis on serum/plasma samples. Phase II will attempt to bring SEED to market by validating the technology for early detection of PaC, by targeting circulating miRNA from blood.
Profiling Circulating miRNA Without PCR for Early Detection of Pancreatic Cancer Narrative MicroRNA circulating in blood have the potential to effectively diagnose pancreatic cancer and other cancers before clinical signs appear. The key is to detect less than 30 miRNA sequences in the midst of thousand fold large background of other miRNA sequences that the body normally produces. The current technologies are either difficult to multiplex or too expensive to be used for screening. The proposed proof-of-concept study is, to directly measure the tumor specific miRNA extracted from about 1 ml of serum or plasma using a disruptive technology called SEED. Measurement by SEED is virtually blind to non-specific binding with 100% consistency. Owing to its electrochemical nature the binding time of targeted miRNA to the probe will reduce from ~18 hours for conventional microarray methods to below one hour.
