NEW YORK – A team of Chinese researchers has developed a novel liquid biopsy-based method to detect urothelial carcinoma that applies low-coverage whole-genome sequencing to detect copy number variations (CNVs) from exfoliated cells in a bladder cancer patient's urine.
To detect cases of bladder cancer, clinicians typically recommend that patients undergo a cystoscopy, which involves inserting a thin tube with a light and a small video camera through the urethra to search for symptoms or collect a tissue biopsy. Clinicians can also perform cytology tests to screen for and identify cancer cells in a patient's urine sample. However, cystoscopies are expensive and invasive, while cytology tests' sensitivities can vary widely for different stages and grades of tumors.
Chuan-Liang Xu, director of the urology department at Changhai Hospital in Shanghai, explained that UroCAD was based on a technology originally developed by Prophet Genomics called Ultrasensitive Chromosome Aberration Detector (UCAD).
Founded in 2017 by CEO Ziliang Qian, Prophet aims to develop its liquid biopsy platform to analyze autosomes for mutations from exfoliated tumor cells in a patient's liquid samples. The company also plans to commercialize the assay developed by Xu and his colleagues, dubbed Urine Exfoliated Cells Copy Number Aberration Detector, or UroCAD, for early detection and monitoring of patients outside China.
"As part of the collaboration [with Prophet Genomics], we applied UCAD technology to our [urothelial carcinoma] patients toward the end of 2018," Xu said in an email. "After promising preliminary results, we decided to initiate the UroCAD clinical trial."
After collecting a patient's urine sample via Prophet Genomics' sample collection kit, Xu's team extracts exfoliated cell DNA and performs LC-WGS on the sample via an Illumina instrument.
The group then analyzes the sequencing data through its custom algorithm to detect significant genomic breakpoints and copy number-altered genomic segments. The algorithm examines each of the patient's 22 autosomes' arms against a pool of non-tumor disease and generates a patient-specific copy number "Z-score." If the Z-score for at least three of the patient's chromosomes is 3.21 or greater, the patient may potentially have bladder cancer.
Xu said that the UroCAD workflow requires six hours for sample preparation, followed by an additional 24 hours for sequencing, downstream analysis, and producing a clinical report.
In a two-part prospective study, published earlier this month in Clinical Cancer Research, Xu and his colleagues collected urine samples from 126 patients with urothelial carcinomas prior to operative treatment, as well as 64 nontumor control samples as part of a discovery phase. The researchers then built the UroCAD model to predict urothelial carcinoma using identified genome-wide CNVs in the patients' urine-exfoliated cell DNA from LC-WGS data.
In the discovery phase, the UroCAD assay had a sensitivity of about 83 percent and a specificity of 97 percent. While the test's sensitivity correlated with tumor grade, the researchers noted that the sensitivity did not correlate with tumor stage or size.
"Usually, high grade tumor cells grow faster and therefore shed from the primary site into the urine," Xu said, and "the sensitivity of UroCAD has shown to be significantly correlated with tumor grade, [for example] 65 percent and 88 percent for low-grade and high-grade tumors, respectively."
The group also validated the diagnostic model by comparing its performance with urine cytology in an external cohort of 56 urothelial cancer patients and 39 controls. The UroCAD assay identified urothelial carcinoma with an improved sensitivity over urine cytology (80 percent vs 34 percent) though slightly lower specificity (95 percent versus 100 percent).
Xu acknowledged that his team dealt with biological and disease-based challenges while adapting the UCAD technology to urothelial carcinoma. Because very few tumor cells exist in urine, the group struggled to reproduce clinical results with the bladder cancer assay in validation cohorts.
However, Xu pointed out that clinical practices require morning void samples from patients, which he said can provide more than enough cells for UroCAD analysis. Initially optimizing the assay in the study by starting with a limited number of cells, Xu believes researchers can reproduce UroCAD's results during different time points and conditions throughout the day.
Xu also noted that patients frequently carry non-tumor diseases — such as stone-related diseases, infectious diseases, and benign prostate hyperplasia — that can affect UroCAD's accuracy. To increase tumor diagnosis specificity, the group expects to recruit a wider variety of non-tumor patient types that represent "most of the benign diseases of interest" in future trials.
"Another interesting finding is the reproducibility of UroCAD when storing urine samples at room temperature for greater than 72 hours," Xu said. "This opened the opportunity for managing urothelial carcinoma at a different level by making UroCAD a home-based point-of-care diagnostic test."
Xu's team believes UroCAD could act as a reliable urothelial carcinoma diagnostic method that offers improved sensitivity and comparable specificity to cytology testing. In addition, Xu believes the assay could be used for recurrence surveillance prior to cystoscopy.
Since completing the study, the researchers launched an additional prospective trial in June to assess the value of the UroCAD assay for urothelial carcinoma surveillance. After collecting urine samples from participants and running them on the UroCAD assay, the researchers will compare the test's sensitivity and specificity to the patient's corresponding pathology.
"We will then collect urine from the same patients every three to six months prior to routine cystoscopy over a two-year period, and [compare] the results to cystoscopy," Xu said. "The goal of the clinical trial is to validate whether UroCAD assay can be used for surveillance of bladder tumors and predict bladder tumor recurrence prior to cystoscopy."
The group has currently recruited about 627 of a planned 750 participants for the longitudinal trial and expects to perform 3,000 urine UroCAD tests over the course of the project.
In addition to the prospective trial, Xu and his colleagues are exploring potential collaborations and implementing additional clinical studies outside of China.
Commercial potential
By acting as a complementary method to cystoscopy, UroCAD can potentially minimize the number of unnecessary cystoscopy procedures per patient, Xu said. UroCAD may also help urologists differentiate between hematuria with unknown reasons and urothelial carcinoma.
"For example, patients with high-grade bladder cancer generally require a cystoscopy every three months for several years," Xu said. "When complemented with the UroCAD assay, the frequency of cystoscopy may be reduced to one every six months."
Xu also believes that UCAD could also eventually be used in the clinical space to analyze CNVs and chromosomal instability markers in other diseases besides urothelial carcinoma. He noted that researchers have previously applied the technology for non-small cell lung cancer, metastatic breast cancer, liver, and cervical cancer.
According to Qian, Prophet has received international IP protection for UCAD and the UroCAD assay and aims to eventually commercialize the assay outside of China. The firm plans to bring the UroCAD test to market in the US as a 510(k)-cleared assay because it "has already generated the clinical trial data," Qian said.
Winston Kuo, president of Prophet Genomics, said that the UCAD technology leverages Qian's industry experience by identifying the unmet needs and "figuring out genomic aberrations that other people have not found yet that can make a difference in the liquid biopsy world."
"We're looking at a specific body fluid, urine, which has its pros and cons from a biology side," Kuo said. "But the technology is very straightforward, since we're looking at all [of a patient's] autosomes."
Prophet has raised a combined $1.5 million from a 2017 seed funding round and a 2018 Series A round, and the firm anticipates launching the UroCAD assay within two to three years depending on discussions with the US Food and Drug Administration. Prophet expects to offer UroCAD as a cost-effective test for end users while ensuring that the assay is priced competitively in different global markets.