Noninvasive HPV16 Urine Test May Help Detect Cervical Cancer
The use of an ultrasensitive assay was able to detect HPV-16-positive SiHa cells at greater than 500 cells/mL for cervical cancer.
The use of an ultrasensitive assay was able to detect HPV-16-positive SiHa cells at greater than 500 cells/mL for cervical cancer.
A noninvasive urine test for quantifying high-risk human papillomavirus (HPV) 16 E7 coproteins might help clinicians screen for cervical cancer, according to a study published in Microorganisms.1
Study results found use of an ultrasensitive enzyme-linked immunosorbent assay (ELISA) was able to detect HPV-16-posiitve SiHa cells at greater than 500 cells/mL. Additionally, use of the assay was associated with the presence of E7 oncoproteins in 80%, 71%, and 38% of urine specimens from patients with HPV16-positive cervical intraepithelial neoplasia (CIN) 1, CIN 2, and CIN 3, respectively.
“Our new urine test can detect HPV16 E7 proteins, which are critical markers of cervical cancer risk, at extremely low levels. This means that women may be able to screen for cervical cancer without the discomfort and inconvenience of a traditional [papsmear] test,” Etsuro Ito, MD, PhD, lead researcher of the study and professor of biology at Waseda University in Japan, said in a news release on the study.2 “We believe that the E7 oncoprotein is critical in the early stages of HPV-related cervical carcinogenesis and E7 may play a more significant role in the progression of CIN1 and CIN2 than in CIN3.”
The single-center study collected 45 urine samples from randomly selected Japanese-based women of 24 to 57 years with HPV-positive CIN lesions who visited Kanazawa Medical University Hospital’s Department of Obstetrics and Gynecology, between January 2019 and December 2021. Each patient had 45 mL of urine centrifuged at 3000 rpm for 5 min, which was resolved with 2 mL of ThinPrep solution and stored at –80°C. Then 2 µg/mL of primary antibody (NM2) against HPV16 E7 oncoprotein in 50 mM sodium carbonate (pH 9.6) was added to each well and placed at room temperature for 1 hour.
Thereafter, each well was blocked by incubation in 1% Tween 20 in tris-buffered saline (TBS) at room temperature for 1 hour. Then 100 µL of the specimen and a TBS diluted antigen (E7, LS-G21591) were added to each well, followed by room temperature incubation for 1 hour. Then a 100 µL solution of a second antibody (NM3) was joined with alkaline phosphatase and adjusted to 6 ng/mL in TBS and was added to each well of the microplates where they sat at room temperature for 1 hour.
Lastly, 100 µL of a thionicotinamide-adenine dinucleotide (thio-NAD) cycling solution was added to each of the microplates. Then a microplate reader at 405 nm normalized to 660nm measured detectable signal amplified in a triangle-number manner.
Limits of detection (LOD) and quantification (LOQ) for the HPV16 E7 oncoprotein were determined through use of linear calibration curves using the data of 60-minute measurements in the ultrasensitive ELISA. The LOD of E7 was 1.18 x 10-18 moles/assay and the LOQ was 3.92 x 10-18 moles/assay at an assay volume of 100 µL and a molecular mass of 11,022 Da. The intra-assay coefficient of variability (CV) was 4.2% for 16 pg/mL E7, and 3.9% for the inter-assay CV.
Out of the 45 urine specimens, 20 showed positive DNA of HPV16 and related types. As opposed to HPV-positive results, when setting the ELISA index indicating E7 oncoprotein presence to 1.35 HeLa cells, 29%, 75%, and 90% of HPV16-negative CIN1, CIN2, and CIN3 specimens contained E7.
“The present study establishes a foundation for the precise quantification of E7 oncoprotein in patient specimens, facilitating an in-depth examination of how E7 oncoprotein levels affect the evolution of cervical cancer precursors. Our findings highlight the potential to evaluate oncogenic activity at the protein level within the context of [CIN],” Ito and the study authors concluded.
References
- Makioka D, Inada M, Awano M, et al. Quantification of HPV16 E7 oncoproteins in urine specimens from women with cervical intraepithelial neoplasia. Microorganisms. 2024;12(6):1205. doi:10.3390/microorganisms12061205
- Noninvasive urine test for cervical cancer shows promise. News release. US News. Published July 9, 2024. Accessed July 15, 2024. https://tinyurl.com/yc4bhj92
