Biosens Bioelectron. 2016 Mar 15;77:491-8. doi: 10.1016/j.bios.2015.10.006. Epub 2015 Oct 9.

Measurement of serum prostate cancer markers using a nanopore thin film based optofluidic chip.
 

Alzghoul S1, Hailat M2, Zivanovic S1, Que L3, Shah GV4.
  • 1Louisiana Tech University, United States.
  • 2University of Louisiana-Monroe, United States.
  • 3Iowa State University, United States. Electronic address: lque@iastate.edu.
  • 4University of Louisiana-Monroe, United States. Electronic address: shah@ulm.edu.

 

Abstract

Currently used cancer marker for prostate adenocarcinoma (PC), serum prostate-specific antigen (PSA), greatly overestimates PC population. Patients with high PSA levels have to undergo unnecessary but physically painful and expensive procedure such as prostate biopsies repeatedly. The reliability of PC test can be greatly increased by finding a protein that is secreted selectively by malignant, but not normal, prostate cells. A recently discovered novel protein, referred as neuroendocrine marker (NEM), is secreted only by malignant prostate cells and released in blood circulation. Although NEM seems to be significantly more reliable based on the data obtained from a limited cohort, currently available NEM ELISA is not suitable for undertaking a large study. Therefore, the goal of the present study was to develop an alternative, label-free assay system that can reliably measure NEM and PSA in patient samples. Herein an optofluidic chip that can reliably detect PSA as well as NEM in patient samples has been developed. The optofluidic chip, which consists of arrayed nanopore-based sensors fabricated from anodic aluminum oxide (AAO) thin film, offers improved sensitivity upon the optimization of the concentration of the detector antibodies immobilized on the sensor surface. The results demonstrate that the chip is reliable, extremely sensitive and requires just 1 µl of patient serum (or even less) to measure PSA and NEM even in a non-cancer individual. Compared with the traditional ELISA for PSA, the nanopore-based sensor assay is 50-100 fold more sensitive, and offers many advantages such as elimination of labeled antigen, need for sophisticated equipment and highly trained individuals. These advantages, along with the low cost, should make the technology suitable for point-of-care application to screen elderly male populations for PC and to monitor the progress of patients undergoing PC treatment.

KEYWORDS: Nanopore-based sensor; Optofluidic chip; Prostate cancer; Serum-based biomarkers

PMID: 26457734

 

Supplements:

Prostate Cancer (PC) is the second most common cancer and the sixth leading cause of cancer death among men worldwide [1]. The global incidence of PC has increased dramatically in recent years, largely due to aging of the population and the practice of prostate-specific antigen (PSA) testing [2]. The worldwide PC burden is expected to grow to 1.7 million new cases and 499 000 new deaths by 2030 [1].

PCs display a wide range of clinical characteristics, from slow-growing tumors of no or little clinical significance to aggressively metastatic and lethal diseases. Extensive knowledge of the etiology and progression of PC makes it an ideal disease for cancer detection, prognosis and prevention. Despite the availability of PSA as an established marker for over two decades, routine pathological parameters (such as Gleason score, number or percentage of positive cores and the maximum percentage of tumor involvement in any core) are the only parameters used to assess prognosis at biopsy [2]. This is because PSA is not cancer-specific and is also produced in normal prostate; its levels increase in several benign conditions such as benign prostatic hyperplasia (BPH), prostate inflammation besides PC. Population-based studies suggest that PSA screening overdiagnoses PC, where a large number of detected prostate tumors are either benign or indolent [3]. Because of its unreliability, PSA-based diagnosis requires confirmation by invasive, repetitive and costly procedures such as transrectal ultrasound (TRUS-guided biopsy [4]. Serum PSA levels ≤ 4.0 ng/ml are used as a cut-off point for cancer. However, the levels between 4.1-10 ng/ml are considered “grey zone” because BPH patients often display serum PSA levels of up to 10 ng/ml [5, 6]. All “grey zone” PSA patients are advised to undergo TRUS-guided biopsies. Moreover, TRUS-guided biopsies often miss the tumor and detect cancer in only 40-60% of clinically significant cancers, necessitating several repetitions [7-9]. It is estimated that around 1 million prostate biopsies were performed in the United States alone in 2007 [10-12]. Considering that a total of 186,000 new cases of prostate cancer were reported in 2007, it appears that out of every 5 prostate biopsies, at least 4 were cancer-negative and unnecessary. Dr. Shah’s laboratory has cloned a novel transcript (referred as neuroendocrine marker or NEM) that is selectively expressed in malignant prostate [13]. Comparison of ROC curve analysis of same set of patients suggest that NEM is a more reliable PC marker than PSA (Figure 1). More importantly, PSA-based grey zone patients were clearly stratified by NEM into cancer and non-cancer ones (Figure 2). Based on these results, it appears that combined NEM+PSA test can significantly improve reliability of PC detection and significantly reduce the number of diagnostic biopsies.

 

 

fig1

Figure 1: Comparison of ROC curves of NEM and PSA in same set of patient samples (n=90). Both markers displayed high specificity for PC in these set of samples. However, NEM was more specific than PSA.

 

Early detection still remains essential for good PC prognosis and treatment options. Localized PC can be effectively treated with prostatectomy, radiation therapy, or other local treatments. Advanced PC that has recurred or spread beyond the prostate is difficult to treat. On the other hand, aggressive treatment of indolent PC causes major side effects and reduces a patient’s quality of life for little to no benefit. As the vast majority of men diagnosed with PC elect to undergo definitive therapy, over-diagnosis leads to over-treatment, associated morbidity and adverse effects on the quality of life. NEM has displayed a significant correlation with Gleason score, suggesting that NEM will also help determine prognosis of prostate tumor at the time of detection. This should greatly help in stratifying the patients into those that require aggressive treatment from those who do not.

 

 

fig2

Figure 2: Patient samples with grey zone PSA levels (4.1-10 ng/ml) were analyzed for NEM. The samples could be clearly classified in to cancer and non-cancer, suggesting that NEM can detect cancer in samples that PSA cannot.

 

Hence, the measurements of cancer biomarkers such as NEM and PSA are generally carried out for the following two purposes: (1) to screen the risk populations for the possibility of cancer; and (2) to determine the efficacy of the therapy in patients undergoing cancer treatment. For both purposes, a large number of clinic samples are needed to be tested. Ideally the cancer biomarker screening can be done at a high accurate and a rapid manner, preferably with automation and low cost at the point-of-care. To this end, Dr. Que’s laboratory recently has developed an optofluidic chip-based diagnostic system (Figure 3). This type of chip offers 50-100 fold more sensitivity compared with the traditional ELISA for PSA and NEM. In addition, this type of chip not only can be made disposable thereby avoiding any possible cross-contamination during the test, but also can offer many advantages such as elimination of the labeled antigen, the need of the sophisticated equipment and the highly trained individuals. These advantages make the technology suitable for point-of-care application to screen elderly male populations for PC and to monitor the progress of patients undergoing PC treatment.

 

 

fig3Figure 3: A Photo of 3D printed portable sensing system along with a smart phone. It contains three cartridge-type optofluidic chip, which can be interfaced with a smartphone

 

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