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Updated: Sep 27, 2026
Cell-Free DNA Integrity Analysis in Urine Samples
Published on: January 5, 2017
Detecting homozygous deletions in the CDKN2A(p16(INK4a))/ARF(p14(ARF)) gene in urinary bladder cancer using real-time
Petra Berggren1, Rajiv Kumar, Shigeru Sakano
1Department of Biosciences at Novum, Karolinska Institute, 141 57 Huddinge, Germany.
Purpose:
9p21 is a major target in the pathogenesis of human urinary bladder cancer. The locus harbors the CDKN2A/ARF tumor suppressor gene, which encodes two cell cycle regulatory proteins cyclin dependent kinase 2A (p16(INK4a)) and alternate reading frame (p14(ARF)). We have designed a real-time quantitative PCR (QPCR) application to study homozygous deletion (HD) of CDKN2A/ARF in 186 urinary bladder cancer patients.
Experimental Design:
Real-time QPCR, based on simultaneous amplification of ARF and a reference gene, GAPDH, was developed and evaluated in three melanoma cell lines with HDs at the CDKN2A/ARF locus (IGR-1, SK-MEL-5, and WM-266-4). In addition, loss of heterozygosity was analyzed at the D9S942, D9S1748, and D12S99 markers. Mutation analysis of the CDKN2A/ARF gene was performed using single-strand conformational polymorphism and sequencing. Results from the present investigation were combined with previous p53 analysis of the same urinary bladder neoplasms.
Results:
Real-time QPCR analysis showed 26 (14%) HDs, 22 (12%) hemizygous deletions, and 3 (2%) multiple duplications. Loss of heterozygosity was determined in 30 (22%) cases at the D9S942 locus, which is located between E1alpha and E1beta of the CDKN2A/ARF gene. No association was established between occurrence of genetic aberrations at 9p21 and tumor stage or grade, supporting previous suggestions that CDKN2A/ARF inactivation is an early event in bladder carcinogenesis.
Conclusions:
We have established a fast and efficient method for detection of HDs. Our data support the notion that inactivation, including HDs, of CDKN2A/ARF is an early event in transitional cell carcinoma. We observed separate and specific targeting of the CDKN2A and ARF genes, respectively, and that simultaneous inactivation of ARF and p53 occurs.
Insights
This study developed a real-time QPCR method to detect homozygous deletions (HDs) of the CDKN2A/ARF tumor suppressor gene in bladder cancer. Results show CDKN2A/ARF inactivation is an early event in transitional cell carcinoma.
Area of Science:
- Oncology
- Molecular Biology
- Genetics
Background:
- The 9p21 locus is crucial in human urinary bladder cancer pathogenesis.
- This locus contains the CDKN2A/ARF tumor suppressor gene, encoding cell cycle regulators p16(INK4a) and p14(ARF).
Purpose of the Study:
- To develop and apply a real-time quantitative PCR (QPCR) method for detecting homozygous deletions (HDs) of the CDKN2A/ARF gene.
- To investigate the role of CDKN2A/ARF inactivation in urinary bladder cancer development.
Main Methods:
- Developed a real-time QPCR assay for simultaneous amplification of ARF and GAPDH.
- Analyzed loss of heterozygosity at D9S942, D9S1748, and D12S99 markers.
- Performed mutation analysis and combined results with prior p53 analysis.
Main Results:
- Identified 14% homozygous deletions (HDs), 12% hemizygous deletions, and 2% duplications in 186 bladder cancer patients.
- Found 22% loss of heterozygosity at the D9S942 locus.
- No correlation between genetic aberrations at 9p21 and tumor stage or grade was observed.
Conclusions:
- Established a rapid and effective method for detecting HDs.
- Data support CDKN2A/ARF inactivation, including HDs, as an early event in transitional cell carcinoma.
- Observed distinct targeting of CDKN2A and ARF, with simultaneous inactivation of ARF and p53.
