ESOU18: Reflex Testing with Serum, Urine and Tissue Biomarkers: Which, When and How Viable?

Amsterdam, The Netherlands ( Dr. Djavan gave an insightful presentation about reflex testing with serum, urine and tissue biomarkers. Prostate cancer (PC) is the most common non-cutaneous cancer diagnosed and the second most common cause of cancer deaths in men worldwide. Unfortunately, over diagnosis of indolent disease, may have a minimal effect on patient survival. Therefore, using suitable genomic and biochemical markers to detect men with high risk PC will enable us to treat the right patients. 
Several commonly mutated genes have been identified in PC and these include BRCA1, BRCA2, HOXB13, and the mismatch repair genes (e.g., MSH2). Other genes like the specific region of the androgen-regulated gene transmembrane protease, serine 2 (TMPRSS2) with v-ets erythroblastosis virus E26 oncogene homolog (ERG) or ets variant 1 (ETV1) were found to be nearly 100% specific. Another important gene is the α-Methylacyl coenzyme A racemase (AMACR) gene, used for immunostaining on prostate needle biopsy specimens to confirm malignancy. There are also single nucleotide polymorphisms (SNPs) of various genes or loci related to PC. 

Additionally, miRNAs are small non-coding RNAs, which negatively regulate the gene expression. Urine miR187 is an independent predictor of PC, which is currently under investigation. Another PC specific urinary biomarker is the prostate cancer antigen 3 (PCA3), which is a non-coding RNA that can be measured by quantitative amplification (reverse transcription polymerase chaining reaction [RT-PCR]. 

Lastly, epigenetic alterations are defined as changes in gene function that do not involve changes in the DNA sequence. Epigenetic alterations refer to changes in a chromosome that affect gene activity and expression. Many hypermethylated genes have been found in PC, including Glutathione-Stransferase, retinoic acid receptors beta 2, adenomatous polyposis coli and RAS association domain family protein isoform A. 

Blood biomarkers:

PSA is a member of the kallikrein gene family. PSA starts as a zymogen, termed prepro PSA. Cleavage of prepro PSA results in proPSA. Later on, cleavage of proPSA results in the active form of PSA. PSA has a two forms in blood: complexed (bound) PSA and free PSA. Free PSA in the blood includes three isoforms: proPSA, BPH-associated PSA (BPSA), and intact fPSA. ProPSA is cleaved by hK2, and produces active PSA.

 Since PSA, there have been many new alterations and modifications of the PSA. These include the 4Kscore test (Opko Health Inc.), which includes four kallikrein forms (tPSA, fPSA, intact PSA, and hK2) used to calculate risk of PC. Another relatively novel test is the Prostate Health Index (PHI). PHI mathematically calculates the risk of PC by means of total PSA, free PSA and [-2] proPSA. Another PC biomarker is the glycoprotein prostate-specific membrane antigen (PSMA) which can be found in serum, urine, or tissue. 

Tumor circulating cells:

During the last decades, circulating tumor cells have long been hyped as promising biomarkers especially by detection of PSA mRNA in PC. Currently, there is only one FDA-approved methodology for identifying circulating tumor cells: CellSearch. Recent studies have shown that the possibility of immune system markers may aid in the diagnosis of PC. 

Splicing the androgen receptor gene may result in a promising biomarker, for example the AR-V7 Mrna, which can be found in circulating tumor cells. To date, in clinical practice, circulating tumor cells tests are limited mostly due to methodological limitations. 

The main PC biomarkers available nowadays include:

  1. PCA3 in urine – analyzed  by RT-PCR is an assay commercially used today for aiding the diagnosis of PC. This has been used especially in patients with a previous negative prostate biopsy. PCA3 has been recommended in the NCCN guidelines for men at a higher risk of PC with previous negative needle biopsy. PCA3 has been combined with TMPRSS2-ERG fusion-gene transcripts assays, resulting in the emergence of these two biomarkers as potentially valuable new biomarkers. 
  2. SelectMDx - A test combining clinical information (PSA levels, PSA density, age and family history) with assessment of the mRNA levels of the DLX1, HOXC6 and KLK3 expression by using RT-PCR assay. This test is performed on post-DRE, first-void urine specimens before biopsy. This estimates the need for performance of a biopsy. Higher levels of these mRNA can predict Gleason score ≥ 7. 
  3. ConfirmMDx -  developed by MDxHealth, is a quantitative assay of methylation specific polymerase chain reaction, that calculates a mixture of epigenetic alterations in the promoters of GSTP1, APC and RASSF1 in needle biopsy tissue. 
  4. DECIPHER – by Genome DX Biosciences and Mayo clinic, is a genomic test composed of a panel of RNA biomarkers of 22 genes. The NCCN guidelines recommend these tests in patients with positive surgical margins, pT3 PC, or biochemical recurrence after radical prostatectomy. 
  5. Oncotype DX - developed by Genomic Health, is a quantitative RT-PCR test performed on paraffin-embedded biopsy specimens analyzing 12 cancer-related gene aberrations, categorized in four pathways. 
  6. Prolaris – developed by Myriad Genetics is another molecular-based 46-gene panel test with a role in helping to decide which low risk patients can be treated with the active surveillance protocol, and which high-risk patients need adjuvant therapy. 
  7. Assessment of DNA repair gene mutations has also emerged as a potential test with a high prognostic role. It has been found that men with metastatic castration resistant PC have a greater frequency of aberrations in DNA repair genes like BRCA2, BRCA1, CDK12, ATM, FANCD2, and RAD51C. 

In summary, despite major improvement and innovation in the field of PC biomarkers, their use in current clinical practice has been limited. Additional research is needed to implement their use in clinical practice.

Speaker: Bob Djavan, MD Medical University of Vienna (Urology) Vienna, Austria

Written By: Hanan Goldberg, MD, Urologic Oncology Fellow (SUO), University of Toronto, Princess Margaret Cancer Centre @GoldbergHanan at The 15th Meeting of the EAU Section of Oncological Urology ESOU18 - January 26-28, 2018 - Amsterdam, The Netherlands