C-MYC COPY NUMBER ANALYSIS IN URINE CELL FREE DNA FROM PRIMARY PROSTATE CANCER PATIENTS: A FEASIBILITY STUDY

Valentina Casadio1, Samanta Salvi1, Filippo Martignano1, Giorgia Gurioli1, Daniele Calistri1, Giacomo Cicchetti2, Massimo Fiori3, Roberta Gunelli3
  • 1 Istituto Scientifico Romagnolo per lo Studio e la Cura dei Tumori (IRST) IRCCS, Laboratorio di Bioscienze (Meldola)
  • 2 Ospedale Bufalini, Unità di Urologia (Cesena)
  • 3 Ospedale Morgagni Pierantoni, Unità di Urologia (Forlì)

Objective

The amplification of 8q, in particular of the region 8q24 containing c-MYC gene, is a frequent event in primary prostate cancer tissues and maybe associated with biochemical recurrence and worse outcome (1; 2). The possibility to have non invasive biomarkers is an important chance for the early diagnosis and monitoring of prostate cancer patients instead of invasive approaches. While the use of circulating cell free DNA from blood has been intensively studied (3) only few data have been published on the role of urinary cell free DNA (UcfDNA) as a noninvasive marker (4; 5).
In the present study we aimed at evaluating copy number variation (CNV) of c-MYC gene in urinary samples collected after radical prostatectomy in a series of patients consecutively enrolled from 2013 to 2014. Our aim was to determine the feasibility of copy number analysis in urine samples using a Real Time PCR approach and to correlate it with clinical pathological characteristics.

Materials and Methods

The study was conducted on a total of 49 individuals, 37 with a first diagnosis of prostate cancer and 12 with benign prostatic disease including prostatitis, inflammation, prostatic benign hyperplasia. Participants were enrolled from the Departments of Urology of Morgagni Pierantoni Hospital (Forlì, Italy) and Bufalini Hospital (Cesena, Italy). First-morning voided urine samples were collected and centrifuged at 850 g for 10min and the supernatants were transferred into cryovials and stored at −80 °C until use.
DNA isolation was performed starting from 4 ml of urine supernatant and using Quick-DNA Urine Kit (Zymo Research), following the manifacturer’s instructions. Urine cell free DNA was quantified using Qubit fluorometer.
c-MYC gene amplification was evaluated by duplex TaqMan quantitative Real Time PCR using two different assays: (ID:Hs01764918 located on exon 3 and ID:HS02602824 located on exon 1) and two different reference genes: AGO1 (ID: Hs02320401) and TCC3 (ID: Hs02765308) both located in chromosomal regions not affected by gain or deletions. For each sample, 1.5 ng of DNA was analyzed in triplicate using TaqMan Universal Master Mix and the primers for target and reference sequences. Three urine DNAs from healthy males over 40 years were singly tested, and then pooled and used as a calibrator, a sample with no CNVs of target and reference genes. In the same run the samples were evaluated for the two loci of the target gene and the two reference genes. When the Ct either for c-MYC or for the reference genes was ≥35.5, samples were considered as “not evaluable”. Copy number variation analysis was performed using CopyCaller Software (Applied Biosystems). Final results were calculated as the average between the copy number values of the two gene loci. CNV values >2.6 were considered as amplification while values <1.4 were considered as deletion.

Results

UcfDNA copy number was feasible on 43 samples. Six samples were considered as “not evaluable” as their Real Time Ct either for c-MYC or for the two reference genes was ≥35.5.
Cancer patients had a pathological stage as follows: 2 pT1, 7 pT2a, 17 pT2b, 6 pT3a and 1 pT3b. Eleven patients had a Gleason score ≤6, 25 had a Gleason score >6. Median PSA was 5.87 for cancer patients and 2.46 for individuals with urological benign pathologies.
Copy number value for c-MYC gene varied from 1.3 to 3.1 in prostate cancer patients with a median value of 2.1 and from 1.1 to 2.4 in patients with benign diseases with a median value of 1.8.
c-MYC gene was gained in 8 of 31 evaluable prostate cancer patients (25.6 %), while it was normal for all individuals with benign pathologies except for two deletions.
Samples with c-MYC copy number gain had all T2 stage tumors. No gain was detected either in T1 or in T3 tumors. Regarding the pathological Gleason score, patients with amplification of c-MYC were: 4 Gleason score 6, 3 Gleason score 7, 1 Gleason score 9. Patients follow-up was available for 24 patients and only two patients experienced a biochemical recurrence until now (one with c-MYC gain).
To date the number of analyzed cases are too low to statistically CNV values with clinical -pathological characteristics or follow-up information.

Discussions

UcfDNA takes its origin either directly from dying cells exfoliated in urine (also prostatic cells) or from the circulation, for this reason it could be a good source of biomarkers especially for urological cancers such as prostate or bladder ones.
In the present study we demonstrated that copy number analysis could be easily performed in cell free DNA isolated from urine supernatant and that no amplification was found in healthy individuals or individuals with benign pathologies.
We found a 26% frequency of copy number gain for c-MYC gene in UcfDNA from prostate cancer patients with different pathological stages and grades. This amplification frequency is in line with those reported in papers previously published regarding 8q24 gain in primary prostate cancer tissues (1; 2). We strangely found no amplification in T3 patients but the number of cases analyzed is still too low to draw any statistical conclusion. The case series will be implemented in the next future.
It will be necessary for prostate cancer patients to analyze CNV of c-MYC in primary tissue and compare the results with those obtained in urine to establish the UcfDNA CNV sensitivity and to eventually adjust the cut off values.

Conclusion

We demonstrated that copy analysis of c-MYC gene is feasible in cell free DNA isolated from urine supernatant. We found that 26% of prostate cancer samples had a gain for c-MYC while all individuals with benign pathologies had normal copy number.
In the next future we will enlarge the case series and compare results with those obtained in the corresponding tissues to test assay sensitivity and correlate with clinical pathological features.

Reference

We demonstrated that copy analysis of c-MYC gene is feasible in cell free DNA isolated from urine supernatant. We found that 26% of prostate cancer samples had a gain for c-MYC while all individuals with benign pathologies had normal copy number.
In the next future we will enlarge the case series and compare results with those obtained in the corresponding tissues to test assay sensitivity and correlate with clinical pathological features.

Argomenti: