Have you ever wondered what exactly happens to a patient sample when it disappears into a laboratory’s ether? Suddenly, a report filled with results magically shows up in your patient’s file, but what happens during that unknown period? The answer is: 

. That sample goes through a complex molecular journey.

This article will walk you through the history of genetic sequencing and polymerase chain reaction (PCR), where they are today, touch on microarrays, explain some standard terminology and the questions those terms are asking, plus describe the clinical laboratory’s workflow.

In today’s molecular testing world, there are 2 very common applications: sequencing and quantitative polymerase chain reaction (qPCR). The first method of sequencing, known as Sanger sequencing, was founded by Frederick Sanger, PhD,
1 of 2 people to win a Nobel Prize twice in the same category. He is considered a pioneer of sequencing DNA for his work with Walter Gilbert, PhD.

 Prior to his work, most research was done on RNA, which is single-stranded and was easily manipulated with RNase enzymes that cut at very specific nucleotide sequences.

With their discovery of DNA, Watson and Crick noted that nucleotides form the building blocks and that adenine binds with thymine and cytosine binds with guanine to form a base pair (bp). These nucleotides are called deoxynucleotides, meaning they are missing a hydroxyl group. In sequencing, in order to interrogate and “read” the genes and DNA of interest, the base pairs are read and identified. Sanger sequencing decided to throw a wrench into a small portion of those nucleotides and make them dideoxynucleotides, removing another hydroxyl group. When those dideoxynucleotides get added, sequencing immediately stops. Imagine you’re building a Lego tower out of regular-sized 2 by 4 pieces but mixed in about 10% of irregular, flat pieces where there aren’t bumps on top to add any more pieces. That’s how dideoxynucleotides work. They stop your Lego tower from growing.

Sanger sequencing was so revolutionary and important at the time, and for the next 20 years, it was used in the Human Genome Project.

 By overlapping the sequences, the human genome was built about 500 to 600 bp at a time, with Sanger sequencing being a critical aspect of the entire project. The method is still used to this day for very fast and inexpensive sequencing results and difficult-to-sequence portions of the genome.

As with any technology, companies are looking to improve the speed, accuracy, and cost of an assay. There have been a few iterations of the next step beyond Sanger, but the one that has become dominant is called next-generation sequencing (NGS), formerly known as massively parallel sequencing. Nowadays, there are 2 versions of sequencing: short reads (Illumina) and long reads (Pacific Biosciences and Oxford Nanopore). Short reads go up to 600 bp together in 1 run, whereas long reads can go beyond 10,000 bp at once, with some reads in the millions of bp. To put this in perspective, the 

 protein is approximately 3000 amino acids in length. By definition, an amino acid is coded for by 3 base pairs, and each base has 2 nucleotides. Thus, there are over 9000 bp (18,000 nucleotides) in the 

Short-read NGS uses a flow cell to hybridize short pieces of DNA to it, replicate that DNA, and then copy it over and over, sometimes hundreds of times. Each nucleotide is fluorescent and will activate upon reading, allowing that nucleotide to be added to the sequence. Remember Lite-Brites when you were a kid? You’d put little pieces on a black board with holes, and the pieces would subsequently glow. Imagine having 1 Lite-Brite as a template, and trying to copy the same image hundreds of times, and each time you add a piece, it glows a specific color assigned to that light, or in this case, nucleotide. Along the way, you consistently make an error in the exact same spot. Because of the consistency of that light being incorrect, that’s not just a mistake. Instead, that becomes an interesting diagnostic possibility because that patient sample has a mutation.

There are 2 common ways to use short-read NGS: whole-genome sequencing (WGS) and targeted sequencing, also known as amplicon sequencing or panel sequencing. WGS refers to just that: sequence the whole genome at a certain level of coverage, which is how often you read a base pair compared with the reference genome. Most of the time, 30 times coverage, meaning each base pair on average was read 30 times, is sufficient for nondisease applications. Gene panel sequencing looks at a specific subset of known disease genes, at a much greater coverage, up to 1000 times but mostly 500 to 600 times. For example, a provider may want to run a gene panel on a patient with a history of colorectal cancer to determine whether there is a hereditary component. Genes included in this panel would include 

, all of which are associated with Lynch syndrome, as well as 

, which are associated with other syndromic patterns where colorectal cancers are common.

 Prostate cancer–specific panels will often include 

Long reads act a little differently from short reads. Instead of creating many short copies on a chip, long reads use a very large circular sequence of DNA and continuously run it through a mechanism, such as a protein pore, to consistently read the same DNA over and over. Comparatively, this is like copying a Lego tower repeatedly using the same colors in the same order vs riding a Ferris wheel and having the operator check each cab every time it passes the bottom. Long reads will catch the same errors as short reads, but also provide some structural variant support and help getting through more difficult areas to read. This allows for some deeper understanding of possible disease states and their proximity to other possible issues.

Kary Mullis, PhD, was a chemist at Cetus Corporation. One night while driving around Mendocino County with his girlfriend, also a chemist at Cetus, he recognized that DNA base pairs were constant in their pairing, and had a random thought to match/hybridize short pieces of DNA that were complementary to long pieces of DNA plus DNA polymerase. This matching added nucleotides to a piece of DNA.

 This allowed a short piece of DNA to be amplified repeatedly using different temperatures, creating billions of copies over many cycles, which would then be studied on an agarose gel 

. Hence, PCR was invented. Mullis was awarded the Nobel Prize in 1993 for this groundbreaking invention, which led to so many discoveries in science. Around the same time, Higuchi et al discovered that increasing amounts of DNA could be directly studied using a fluorescent marker without the need for agarose gel.

In order to use qPCR in diagnostics, the clinic has to know which specific gene is of interest, as the primers have to flank the target sequence to be amplified. The high specificity of this type of assay is both a blessing and a hindrance. It’s a blessing because the clinic can answer a diagnostic question with high confidence but a hindrance because it may miss other possible disease states that are outside the targeted region. Most of the time, a sample will be split up to run multiple different assays at the same time to cover a wider array of diseases. qPCR is also very commonly used to get to the heart of urinary tract infections (UTIs) and their persistent nature. Many companies are offering qPCR diagnostics for UTIs, prostatitis, and more.

 Most of the time, those companies will also offer an NGS panel in addition to cover all diagnostic bases.

Microarrays are small chips with imprinted specific DNA targets of interest (

). The test is run with a reference sample, often labeled with a green fluorescent dye, and the targeted DNA sample, labeled with a red fluorescent dye. Both are then hybridized to the chip, and a comparative analysis is done. If the targeted DNA is expressed at a higher rate, that small area will glow red. If the control DNA is expressed higher (or decreased expression in the target DNA), it will glow green. Finally, if expressed in equal amounts, essentially no mutation, the square will glow yellow.

 The sensitivity is generally low, but the ability to study many targets at once is a highlight. Microarrays are a common technique of many companies that offer testing for determining cultural heritage. Those companies will study up to 700,000 targets at 1 time. However, microarrays are slowly declining because of the greater adoption of NGS assays. These direct-to-consumer companies are really fun and interesting for those who are seeking information about their ancestry but should not be used for cancer risk assessment.

What happens to a sample in the clinical lab?

When a patient has a sample sent for molecular testing, whether it’s tissue, urine, blood, or saliva, that specimen is immediately tagged with a number specific to that patient. The sample is transported to the lab with the appropriate storage. The lab receives the sample and inputs it into their system, also called accessioning. Then, the molecular journey is as follows:

Nucleic acid isolation: convert RNA to DNA if needed

a. Sonication or enzyme digestion to create uniform DNA segments

b. Enrich the target DNA if needed, as target enrichment and amplicon generation workflows used in gene panels

c. Barcode ligation: also known as multiplexing, which is adding unique markers per patient sample so they can be mixed together, then parsed upon software analysis

d. Adapter ligation: allows the DNA to bind to the flow cell

Sequence the DNA: 0.5 days to several days, depending on sequencing type and instrument used

Bioinformatics: Results are parsed and analyzed.

Report generated: Details around the disease state are provided, and sometimes potential treatment scenarios depending on the software’s FDA approvals

What does a clinical laboratory look for in their tests?

Although you’ve learned about generic methods and workflows, labs look for specific issues using molecular methods that cause various disease states. In this section, I’ll lay out a few of the more common terms in the lexicon of genomic testing.

 As the name implies and by definition, this occurs when a single nucleotide change is identified in a particular gene and present in 1% of the population. This SNP results in a gene mutation but may or may not cause an alteration of downstream protein function, depending on whether the change affects the specific amino acid in which it is coding. There is significant interest in looking at a panel of SNPs to determine risk assessment for breast and prostate cancer.

 This is a duplication or deletion of a sequence of nucleotides, not just a single nucleotide like an SNP. Most genes in the human genome have 2 alleles, 1 each inherited from your mother and father. In rare instances, short sequences can be replicated many times. For example, the 

 gene codes for the protein huntingtin. In this case, the trinucleotide CAG can be repeated 36 times or more. The result is abnormal protein production, which can then lead to Huntington disease.

In other cases, entire genes can be repeated or deleted, causing overexpression or underexpression, as in the case of α-amylase 1 and its overexpression because of dietary differences.

 The largest example of this is the trisomy issues that cause Down syndrome.

 Fusions occurs when 2 genes fuse during replication, causing a pseudogene that creates expression issues. One of the earliest discovered examples of this is a reciprocal translocation where the 

 gene (the Philadelphia chromosome), which induces chronic myeloid leukemia.9 This is difficult to detect using molecular testing because there are various fusion loci on each gene, but it can be done with proper techniques, such as digital PCR and NGS.

This partial list of 3 common issues is just a sample of what a molecular lab can discover. Some tests are more involved than others from a workflow and difficulty perspective, whereas others are fairly straightforward. The most challenging part for a lab is to discern the ability of a specific assay type to get the proper answer because some answers are much more difficult to come by.

The world of clinical diagnostics is changing. The development of targeted therapies is increasingly more specific to various molecular changes that are therapeutic resistance drivers. The development of companion diagnostics so patients can receive these new agents is mandatory. In addition, the ability to detect and potentially mitigate disease at a much earlier stage before systemic/metastatic disease has clear upside potential. Therefore, embracing and understanding these new and emerging molecular techniques will improve your patient outcomes and enhance your practice.

has been involved in biotech and clinical sales and marketing for nearly 20 years. He has a Master’s in molecular biology from Washington University in St. Louis and an MBA in strategy and operations from Boston University. He has worked for companies such as Thermo Fisher and Illumina and has started multiple companies outside of the biotech world.

Frederick Sanger - biographical. NobelPrize.org. 2005. Accessed April 7, 2021. 

 The Human Genome Project. NIH National Human Genome Research Institute. Updated December 22, 2020. Accessed April 7, 2021. 

https://www.genome.gov/human-genome-project

 PSM2 gene. MedlinePlus. Updated August 18, 2020. Accessed April 7, 2021. 

https://medlineplus.gov/genetics/gene/pms2/#conditions

 Kary B. Mullis – biographical. NobelPrize.org. 2005. Accessed April 7, 2021. 

 Urology – urinary tract infections, prostatitis, & more. MicroGenDX. Accessed April 7, 2021. 

 Microarray. Scitable. Accessed April 7, 2021. 

https://www.nature.com/scitable/definition/microarray-202/

 HTT huntingtin [ Homo sapiens (human) ]. NCBI. Updated May 18, 2021. Accessed April 7, 2021. 

 AMY1A amylase alpha 1A [ Homo sapiens (human) ]. NCBI. Updated May 18, 2021. Accessed April 7, 2021. 

 Philadelphia chromosome. Wikipedia. Updated April 4, 2021. Accessed April 7, 2021. 

https://en.wikipedia.org/wiki/Philadelphia_chromosome https://www.ncbi.nlm.nih.gov/gene/25

Articles

Embracing and understanding new and emerging molecular techniques will improve patient outcomes.

A primer on DNA sequencing for the practicing urologist

For the most part, for progression to take place, change is inevitable. And change is often very difficult. As someone once told me, if you make a change in your golf swing and it is easy, more than likely you have not changed anything. The urology community has been very progressive over the decades, embracing changes in our clinical world that have led to optimizing patient care while maintaining control, if you will, of the disease states we are accustomed to treating. Although at times this has resulted in fewer operative procedures, which for many physicians is the reason we entered the field, the end result has been better patient outcomes.

Advances in technology generally are the driving force behind these changes, and fortunately, many forward-thinking urologists have been keen in applying them to our field. In the early 1920s, William T. Bovie, working alongside the acknowledged father of neurosurgery, Harvey Cushing, MD, was one of the first pioneers to demonstrate the advantages of electrocautery to stem bleeding in the surgical field.

 A decade later, a urologist in South Carolina, Theodore M. Davis, MD, was able to adapt this technology and incorporate both a cutting and cautery feature into the modern endoscope.

 Thus the birth of transurethral resection of the prostate, which continues to be the gold standard in which all newer therapies, including medical and minimally invasive, must show noninferiority.

For those of us who trained before 1990, prostate biopsy could be considered a form of medieval torture. Blind and either transrectal or transperineal, its complication rates of bleeding and sepsis were extraordinarily high, not to mention the suboptimal sampling due to the lack of imaging and inability to obtain quality samples. Fortunately, a number of our predecessors were prescient enough to adopt prostate ultrasound and template sextant biopsy, which have now led to MRI fusion techniques, again driven by our colleagues. Somewhat forgotten in this discussion is the perseverance of many during that time frame to have practicing urologists trained in prostate ultrasound, wrestling that away from our radiology colleagues. Had those efforts not been successful, it is unclear to me how our current practices might look today.

As alluded to earlier, many of us chose the field of urology for the surgical aspect of the specialty. From an oncologic perspective, mastering the art of nerve-sparing radical retropubic prostatectomy was key to developing a surgical practice. Identification of the neurovascular bundle obviously is a mandate, but controlling bleeding from the Santorini plexus early in the course of the procedure is truly what makes the procedure possible for adequate visualization in a relatively bloodless field. The principles remain the same, but open surgery has now given way to robotically assisted extirpation, which is what most patients opt for when they choose surgical intervention. But as our understanding of the biology of prostate cancer continues to evolve, we must also understand and accept that all prostate cancers do not necessarily need immediate treatment and that active surveillance is truly an option. It is somewhat against our surgical mentality, but it is the correct option for the appropriately identified patient.

In line with this theme, the late 2000s saw the emergence of new therapies for patients with advanced prostate cancer. Many urologists and practices were (and still are) slow to incorporate them into their model for a number of reasons. Probably the most commonly cited is the lack of expertise in prescribing such agents, which was not part of surgical training and belonged more to a “typical” medical oncologist. This paradigm continues to change with the realization that the urologist is perfectly suited to and capable of prescribing and managing these drugs. More importantly, however, our patients have a certain comfort level within the practice.

Part of the shift for many will be incorporating and understanding genetic and genomic testing. Currently, this is heavily weighted to prostate cancer management, but data and literature continue to come forth for both urothelial and renal cancers. That being said, there is no doubt that we will all need to have a working knowledge of molecular testing for risk assessment, family counseling, and therapeutic options. To that end, as a publication, we have opted to include an article in each issue that covers a certain aspect of genomic testing. Our goal is to demystify this area and make it more comprehensible to our readers to achieve better uptake at the provider and practice levels, which currently is very minimal. This, once again, seems to be multifactorial, and we are hoping that educational pieces can assist physicians in their use of such testing and hence their efforts to deliver personalized care.

 Chief Science Officer for U.S. Urology Partners, is editor-in-chief of 

 O’Connor JL, Bloom DA. William T. Bovie and electrosurgery. 

1996;119(4):390-396. doi:10.1016/s0039-6060(96)80137-1

 Goddard JC. That silly operation! The introduction of TURP into Great Britain. The British Association of Urological Surgeons. Accessed May 19, 2021. 

https://www.baus.org.uk/_userfiles/pages/files/museum/Final%20-%20That%20Silly%20Operation.pdf

"As someone once told me, if you make a change in your golf swing and it is easy, more than likely you have not changed anything," writes Raoul S. Concepcion, MD, FACS.

From Dr. Concepcion: Urology's history of embracing change

Patients with muscle-invasive urothelial cancer of the bladder and invasive urothelial cancer of the upper urinary tract are at generally at high risk for metastatic recurrence despite curative intent surgery. A series of clinical trials investigating systemic therapies applied in the adjuvant setting have been performed over the past few decades in an attempt to mitigate this risk.

 Compared with the development of therapeutic approaches in the metastatic setting, the development of therapeutic approaches in the adjuvant setting is potentially more impactful but more complicated. That is, in the metastatic setting, we mainly face the question “Who benefits from treatment?” Whereas in the adjuvant setting, we face both the question “Who needs treatment?” and “Who benefits from treatment?” A large proportion of patients with muscle-invasive urothelial cancer will achieve cure with surgical resection alone. On the other hand, for those patients who harbor micrometastatic disease after surgery, just as in the metastatic setting, not all such cancers will be sensitive to the systemic therapy being applied.

Historically, we have addressed the “Who needs treatment?” question in the adjuvant setting by using pathologic features of the primary tumor and regional lymph nodes to help refine estimates regarding recurrence risk. Such features, in turn, have formed the eligibility criteria for clinical trials. The majority of adjuvant trials in urothelial cancer have enrolled patients with bladder-only primary tumors and have limited enrollment to patients who had not received prior neoadjuvant chemotherapy and had pathologic T3 or higher urothelial cancer or pathologic evidence of regional lymph node involvement. Randomized trials exploring adjuvant cisplatin-based chemotherapy in this context suffered from methodologic flaws and/or closed early due to poor accrual, leaving some doubt regarding the efficacy of this approach despite meta-analyses suggesting a benefit.

Although trials of adjuvant cisplatin-based chemotherapy in high-risk urothelial cancer continued, for a number of reasons, neoadjuvant cisplatin-based chemotherapy became the standard preferred treatment standard supported by level I evidence.

 Therefore, regardless of one’s interpretation of the studies evaluating adjuvant cisplatin-based chemotherapy, additional unmet need populations at high risk for recurrence after surgery arose not addressed by the prior generation of studies: (1) patients with pT3 or higher and/or pN-positive disease who are considered cisplatin ineligible

 and (2) patients with residual ypT3 or higher and/or ypN-positive disease despite having received prior cisplatin-based neoadjuvant chemotherapy. These 2 distinct populations have formed the basis for large phase 3 trials exploring PD-1 and PD-L1 immune checkpoint inhibitors in the adjuvant setting triggered by the safety and efficacy of these therapies in metastatic urothelial cancer.

Three somewhat similarly designed adjuvant trials, with some nuances, have been conducted to test adjuvant PD-1 or PD-L1 blockade. IMvigor010 (NCT02450331) randomized patients with urothelial cancer of the upper tract or bladder at high risk for recurrence (as defined above) after definitive surgery to atezolizumab (Tecentriq) administered for 1 year vs observation.

 This was the first in the series of immune checkpoint blockade adjuvant trials to report and it was presented at the 2020 American Society of Clinical Oncology Annual Meeting, though it has not been published. The trial did not meet the primary end point of improvement in disease-free survival with atezolizumab in the intent-to-treat population, nor was there in an improvement in the subset of patients with tumors harboring increased PD-L1 protein expression.

The Checkmate 274 trial (NCT02632409) employed similar eligibility criteria but randomized patients to 1 year of adjuvant nivolumab (Opdivo) vs placebo (rather than observation).

 This trial, presented at the 2021 Genitourinary Cancers Symposium, met coprimary end points of an improvement in disease-free survival in the intent-to-treat population (HR, 0.70; 95% CI, 0.54-0.89; 

 < .001) and in the subset of patients with tumors harboring increased PD-L1 protein expression (HR, 0.53; 95% CI, 0.34-0.84; 

 < .001). Overall survival, a secondary end point of this study and an event-driven analysis, has not been assessed. A third trial enrolling a similar patient population but randomizing patients to 1 year of adjuvant pembrolizumab (Keytruda) vs observation, dubbed the AMBASSADOR trial (NCT03244384), is still enrolling.

Many questions have arisen regarding the disparate results reported from IMvigor010 and Checkmate 274. Ultimately, these are different drugs explored, employing different PD-L1 biomarkers, in trials that likely enrolled patients with slightly different baseline characteristics and involved some important clinical trial design nuances. For example, the former involving randomization between atezolizumab and observation may have been affected by informed censoring based on the proportion of patients randomized to observation that withdrew early from the study. Checkmate 274 represents the first trial demonstrating an improvement in outcomes with an adjuvant immune checkpoint inhibitor in urothelial cancer, addresses a patient population without previously available systemic therapies to mitigate risk of recurrence, and represents the first big step in a new era of immune checkpoint inhibitor for muscle-invasive urothelial cancer.

is acting chief, Division of Hematology and Medical Oncology, and professor of medicine at Tisch Cancer Institute at the Icahn School of Medicine at Mount Sinai in New York City, New York.

Galsky MD, Herr HW, Bajorin DF. The integration of chemotherapy and surgery for bladder cancer.

. 2005;3(1):45-51. doi:10.6004/jnccn.2005.0003

Sternberg CN, Skoneczna I, Kerst JM, et al. Immediate versus deferred chemotherapy after radical cystectomy in patients with pT3-pT4 or N+ M0 urothelial carcinoma of the bladder (EORTC 30994): an intergroup, open-label, randomised phase 3 trial. 

. 2015;16(1):76-86. doi:10.1016/S1470-2045(14)71160-X

 Grossman HB, Natale RB, Tangen CM, et al. Neoadjuvant chemotherapy plus cystectomy compared with cystectomy alone for locally advanced bladder cancer. 

 2003;349(9):859-866. doi:10.1056/NEJMoa022148

 Griffiths G, Hall R, Sylvester R, Raghavan D, Parmar MK. International phase III trial assessing neoadjuvant cisplatin, methotrexate, and vinblastine chemotherapy for muscle-invasive bladder cancer: long-term results of the BA06 30894 trial.

. 2011;29(16):2171-2177. doi:10.1200/JCO.2010.32.3139

 Galsky MD, Hahn NM, Rosenberg J, et al. A consensus definition of patients with metastatic urothelial carcinoma who are unfit for cisplatin-based chemotherapy. 

. 2011;12(3):211-214. doi:10.1016/S1470-2045(10)70275-8

Hussain MHA, Powles T, Albers P, et al. IMvigor010: primary analysis from a phase III randomized study of adjuvant atezolizumab (atezo) versus observation (obs) in high-risk muscle-invasive urothelial carcinoma (MIUC).

. 2020;38(suppl 15):5000. doi:10.1200/jco.2020.38.15_suppl.5000

Bajorin DF, Witjes JA, Gschwend J. First results from the phase 3 CheckMate 274 trial of adjuvant nivolumab vs placebo in patients who underwent radical surgery for high-risk muscle-invasive urothelial carcinoma (MIUC). 

 2021;339(suppl 6):391. doi:10.1200/JCO.2021.39.6_suppl.391

Recent trial demonstrates an improvement in outcomes with an adjuvant immune checkpoint inhibitor in urothelial cancer.

Adjuvant immune checkpoint blockade in urothelial cancer: A new era?

(provided by Raoul S. Concepcion, MD, FACS)

To contextualize the rapidly evolving landscape of evaluation of patients with prostate cancer, consider a case example: A 62-year-old White man presents for consultation due to an elevated prostate-specific antigen (PSA) of 8.2 ng/dL that was confirmed on repeat testing. He has no prior values available for comparison.

Symptomatically, he has an American Urological Association symptom score of 11 without other complaints. His past medical history is unremarkable with only hypertension, for which he takes amlodipine 5 mg daily and a baby aspirin. He’s undergone a laparoscopic cholecystectomy. He has no known drug allergies and is married with 2 healthy adult male children. Notably, his family history includes prostate cancer in his father (diagnosed at aged 58 years and alive) and breast cancer in his paternal grandmother (diagnosed at aged 50 years and now deceased from the disease).

On physical examination, the prostate is estimated at 50 grams, symmetric, and without nodules or induration. The patient undergoes a prebiopsy multiparametric MRI scan, which demonstrates a prostate volume of 65.3 cc and a PI-RADS (Prostate Imaging Reporting and Data System) 4 lesion at the right peripheral zone (base, 1.2 cm). He then undergoes a fusion biopsy; the pathologic results show:

Target lesion: 2/3 cores (+), grade group (GG) 4, approximately 65% involved

R base: 2/2 cores (+), GG 4, approximately 70% involved

R mid: 1/2 cores (+, GG 4, < 50% involved

R apex: 1/2 cores (+), GG 3, < 50% involved

L base: 2/2 cores (+) GG 3, approximately 60% involved

This patient has newly diagnosed cT1c prostate cancer, which is very high risk—according to the National Comprehensive Cancer Network (NCCN) criteria—because of the presence of more than 4 cores of GG 4 disease.

As we evaluate this patient, our goal is to accurately determine his current and future risk of disease spread to inform treatment, prognosis, and counseling. Current guidelines recommend staging with conventional imaging (CT or MRI, and bone scan) as well as genetic testing. However, advances in staging imaging are allowing more accurate detection of metastatic disease and play an increasingly large role in other parts of the world, with adoption in the United States expected to follow.

Guideline-concordant conventional imaging in patients with high- and very high–risk disease includes abdominopelvic cross-sectional imaging, often a CT scan, and a technetium-99m methylene diphosphonate bone scintigraphy scan. Multiparametric MRI (mpMRI) has emerged in the past 5 to 10 years as a common staging imaging modality, and NCCN guidelines endorse the superiority of mpMRI compared with abdominopelvic CT for staging.1

For this patient, we would follow a current guideline–based approach and complete his staging with a nuclear medicine bone scan to complement his mpMRI. If the bone scan is negative for evidence of metastasis, he falls into a group of men with apparently localized disease who are at high risk for recurrence despite appropriate primary therapy, likely due to the inability of conventional imaging to identify small metastases at low PSA. Closing this gap in diagnostic accuracy is an area of active research.

Newly published data suggest that advanced imaging techniques using PET-CT may complement or supplant conventional imaging in the near future. The use of PET-CT
in prostate cancer in the US has been led, to date, by the approval of 3 radiotracers in the management of recurrent or metastatic prostate cancer: choline C 11, sodium fluoride F 18, and fluciclovine F 18 (Axumin). Newer prostate-specific membrane antigen (PSMA)–based tracers including gallium 68 (68Ga) and DCFPyL F 18 are increasingly being used, first in Australia and Europe and now in the US. These PSMA-based tracers are the subject of several recent clinical trials that support the diagnostic superiority of PSMA PET-CT over conventional and previously approved advanced imaging modalities.

PSMA is a cell surface glycoprotein that is highly overexpressed in most prostate cancer cells. Multiple recent studies have added to the growing body of evidence supporting the use of PSMA PET-CT across the prostate cancer disease spectrum. The proPSMA study was a multicenter clinical trial of patients with high-risk prostate cancer who were randomized to receive conventional imaging vs 68Ga PSMA PET-CT for the purposes of pretreatment staging.

 The investigators found that 68Ga PSMA PET-CT had a 27% greater area under the curve for diagnostic accuracy when compared with conventional imaging, driven by its improved sensitivity (85% vs 38%) and specificity (98% vs 91%). Use of PSMA PET-CT was more likely to effect a significant change in management versus conventional imaging (28% vs 15%, respectively). For this patient, PSMA PET-CT may be more likely to identify small regional or distant metastatic disease that, if found, may influence our treatment decision.

Although proPSMA compared 68Ga PSMA PET-CT to conventional imaging, Calais et al compared fluciclovine F 18 PET-CT with 68Ga PSMA PET-CT in the biochemical recurrence setting among men with PSA lower than 2 ng/mL.

 The indication for imaging in this study was different from proPSMA. Here, imaging was used to detect recurrent disease, whereas proPSMA examined the pretreatment staging setting. The investigators demonstrated that PSMA PET-CT had higher detection rates at low PSA compared with fluciclovine F 18, adding to the evidence of the superior sensitivity of PSMA-based PET-CT over fluciclovine F 18 PET-CT.

A pair of recently published trials, OSPREY and CONDOR, led to the recent FDA approval of F18-DCFPyL (Pylarify), another novel radiotracer used for PSMA-targeted PET-CT, for both initial staging and biochemically recurrent disease.

The multicenter phase 2/3 OSPREY study enrolled 2 cohorts to evaluate the diagnostic performance of DCFPyL F 18 PET-CT: Cohort A—most relevant to our patient—enrolled men with high-risk prostate cancer undergoing radical prostatectomy with pelvic lymphadenectomy to assess its accuracy detecting prostate cancer in the pelvic lymph nodes; cohort B enrolled patients with suspected recurrent or metastatic disease on conventional imaging who then underwent tissue biopsy. In cohort A, DCFPyL F 18 PET-CT had a median specificity of 98%, sensitivity of 40%, positive predictive value (PPV) of 87%, and negative predictive value (NPV) of 83% for detecting pelvic nodal involvement. The performance of DCFPyl F 18 PET-CT was superior compared to conventional imaging with CT/MRI by most measures (3-fold higher PPV: 87% vs 28%, respectively), higher specificity (98% vs 65%), slightly higher NPV (83% vs 78%), and similar sensitivity (40% vs 43%).

The multicenter phase 3 CONDOR study enrolled men with biochemical recurrence after definitive therapy with negative or equivocal conventional imaging and again found that DCFPyL F 18 PET-CT had PPV of 80% to 90% and that 64% of participants experienced a change in intended management after undergoing DCFPyL F 18 PET-CT.

There are some advantages and disadvantages to the different radiotracers from a technical perspective that may guide their real-world adoption. First, as highlighted in the data from Calais and colleagues, 68Ga PSMA PET-CT outperforms fluciclovine F 18 PET-CT.

 Further, 68Ga was the first well-described PSMA-based radiotracer and has, therefore, garnered significant research interest. However, 68Ga requires specialized generators with batch production of 2 to 4 patient doses per generator elution and has a physical half-life of approximately 1 hour, which requires studies to be performed locally because central production with delivery to remote centers would be challenging. On the other hand, F 18–labeled PSMA tracers such as DCFPyL F 18 offer some advantages over 68Ga, namely large-scale radiosynthesis, allowing for a larger number of patient studies; a longer half-life of nearly 2 hours, which could enable centralized production with delivery to more remote centers; and increased maximum spatial resolution due to its lower positron energy than 68Ga.

On December 1, 2020, the FDA approved 68Ga PSMA PET-CT for staging in men with prostate cancer with suspected metastasis before initial definitive therapy and in men with biochemical recurrence, although the approval is, at this time, limited to UCLA and UCSF. DCFPyL F 18 (Pylarify) received FDA approval in May 27, 2021. Although, at this time, NCCN guidelines would not recommend the use of these advanced imaging techniques until after conventional imaging is performed and found to be negative, this may be rapidly changing. The dissemination and availability of DCFPyL F 18, expected to reach routine clinical use towards the end of 2021 dependent on identification of regional radiopharmacies to provide radiotracer, is expected to rapidly bring PSMA imaging from sparse research and clinical trial settings to routine clinical use though this will, as always in US health care, depend on insurer reimbursement decisions.

In addition to staging investigations, another important consideration for this patient is the use of genetic testing. For patients with high- or very high–risk disease, NCCN guidelines recommend germline genetic testing, and contemporary testing may offer actionable information for physicians and patients. 

 mutations, if found, may hold prognostic information; studies have demonstrated that prostate cancer patients with 

 mutations have higher Gleason scores, nodal involvement, metastatic disease, and worse survival.

 In patients with metastatic disease, genetic testing for mutations in homologous recombination repair genes is required to determine suitability for treatment with PARP inhibitors, such as olaparib (Lynparza) or rucaparib (Rubraca). In addition, pembrolizumab (Keytruda) is approved for use in tumors with microsatellite instability or mismatch repair–deficiency, so approximately 5% of metastatic castration-resistant prostate cancer cases could qualify.

Genetic testing is also important for family counseling. Relatives of a patient with a known 

 mutation may be at increased risk for breast, ovarian, pancreatic, colon, and prostate cancer. Data from the IMPACT study

 mutations may benefit from annual PSA screening earlier than noncarriers or using a lower PSA cutoff value.

In this case, the patient’s high-risk disease and positive family history of prostate cancer in his father, diagnosed at a relatively young age, may also point toward a genetic predisposition and support the utility of the recommended germline testing. Although access to genetic counseling remains challenging at many centers and has limited broader adoption, its use is guideline recommended and should be a priority for these patients.

The diagnostic and treatment landscape across the spectrum of disease states in prostate cancer continues to rapidly evolve, as highlighted by this case. In the near future, we will hopefully better understand when and how to use these advanced tests as well as the implications for patient outcomes and survival.

 is an assistant professor of urology at the Vanderbilt University Medical Center in Nashville, Tennessee.

1. NCCN. Clinical Practice Guidelines in Oncology. Prostate cancer, version 2.2021. Accessed March 17, 2021. https://www.nccn.org/professionals/physician_gls/pdf/prostate_blocks.pdf

2. Hofman MS, Lawrentschuk N, Francis RJ, et al; proPSMA Study Group Collaborators. Prostate-specific membrane antigen PET-CT in patients with high-risk prostate cancer before curative-intent surgery or radiotherapy (proPSMA): a prospective, randomised, multicentre study. 

2020;395(10231):1208-1216. doi:10.1016/S0140-6736(20)30314-7

3. Calais J, Ceci F, Eiber M, et al. 18 F-fluciclovine PET-CT and 68 Ga-PSMA-11 PET-CT in patients with early biochemical recurrence after prostatectomy: a prospective, single-centre, single-arm, comparative imaging trial. 

 2019;20(9):1286-1294. doi:10.1016/S1470-2045(19)30415-2

4. Pienta KJ, Gorin MA, Rowe SP, et al. A phase 2/3 prospective multicenter study of the diagnostic accuracy of prostate-specific membrane antigen PET/CT with 18 F-DCFPyL in prostate cancer patients (OSPREY). 

 Published online February 26, 2021. doi:10.1097/JU.0000000000001698

5. Morris MJ, Rowe SP, Gorin MA, et al. Diagnostic performance of 18 F-DCFPYL-PET/CT in men with biochemically recurrent prostate cancer: results from the CONDOR phase 3, multicenter study. 

 Published online February 23, 2021. doi:10.1158/1078-0432.CCR-20-4573

6. Castro E, Goh C, Olmos D, et al. Germline BRCA mutations are associated with higher risk of nodal involvement, distant metastasis, and poor survival outcomes in prostate cancer.

 2013;31(14):1748-1757. doi:10.1200/JCO.2012.43.1882

7. Eeles RA, Bancroft E, Page E, Castro E, Taylor N. Identification of men with a genetic predisposition to prostate cancer: targeted screening in men at higher genetic risk and controls—The IMPACT study.

. 2013;31(suppl 6):12. doi:10.1200/jco.2013.31.6_suppl.12

Advances allow for more accurate detection of metastatic disease.

Update on advanced imaging in prostate cancer

In the 1940s, Huggins and Hodges’ Nobel prize–winning work firmly established the therapeutic effect of testosterone deprivation in the treatment of prostate cancer.

 Since that time, hormonal treatments for prostate cancer have undergone continued advancement. In the modern era, the backbone of therapy is in the form of gonadotropin-releasing hormone (GNRH) agonists and antagonists, which decrease testosterone to the castrate range. These class of agents have distinct mechanisms of action and safety profiles that warrant attention because they optimize treatment for patients with prostate cancer. Herein, we describe the different mechanisms of action and safety profiles of GNRH agonists and antagonists and discuss their use in clinical practice.

GNRH is a peptide hormone synthesized and released by the hypothalamus to stimulate the synthesis and secretion of the gonadotropins luteinizing hormone (LH) and follicle-stimulating hormone (FSH) from the pituitary gland. GNRH is typically produced in a pulsatile fashion, and its synthesis is regulated by circulating levels of testosterone and estrogen. LH acts on Leydig cells in the testes to secrete the majority of the body’s testosterone, whereas FSH targets Sertoli cells in the seminiferous tubules of the testis to regulate spermatogenesis. Androgens then promote prostate cancer growth via activation of cytoplasmic androgen receptors.

GNRH agonists were introduced in 1984 and act via continuous GNRH receptor stimulation that differs from the normal physiologic pulsatile effects of GNRH.

 Continuous signaling initially causes a surge in testosterone and then leads to a drop-off in endogenous hypothalamic GNRH production and downregulation of pituitary GNRH receptors through overstimulation from the GNRH agonist, resulting in decreased LH and FSH levelsand downstream castrate levels of testosterone.

The adverse effects of GNRH analogues are largely due to the impact of these agents on androgen levels. Androgen deprivation therapy (ADT) leads to hypoandrogenism in men, manifested as fatigue, vasomotor symptoms including hot flashes, decreased libido, erectile dysfunction, and gynecomastia. ADT can also impact cardiovascular, bone, and muscle health and cause cognitive/psychiatric changes. Effects on glucose and lipid metabolism have been noted, with data suggesting that low testosterone levels lead to insulin resistance, central obesity, hypercholesterolemia and hypertriglyceridemia, and other precursors of metabolic syndrome.

GNRH antagonists suppress LH and FSH more effectively than LHRH agonists

 and are associated with fewer cardiotoxicities. Several hypotheses have been proposed in attempts to explain how this mechanistic difference affects cardiac outcomes. Stimulation of FSH receptors on the endothelial surface of proliferating tissues could lead to decreased lipid metabolism and increased fat accumulation, leading to increased cardiovascular risk with GNRH agonists. Additionally, GNRH agonists, but not antagonists, bind to and stimulate GNRH receptors on T cells that could result in a hyperinflammatory state causing plaque destabilization.

One disadvantage of GNRH agonists is tumor flare from the initial testosterone surge, which can be seen in up to 63% of patients with advanced prostate cancer.

 Complications from tumor flare can lead to urethral obstruction, worsening pain, and cord compression in more severe cases.

 Antiandrogens are usually given prior to GNRH agonist administration to mitigate this flare phenomenon. GNRH antagonists were evaluated in efforts to find alternative agents not associated with a testosterone surge.

GNRH antagonists act via direct competitive inhibition of the GNRH receptor, thereby blocking its downstream signaling and leading to a rapid decrease in LH, FSH, and testosterone levels. Abarelix (Plenaxis) was the first direct GNRH antagonist approved by the FDA (in 2003) for advanced, symptomatic prostate cancer. The phase 3 clinical trials that led to its approval showed that when compared to leuprolide or leuprolide plus an antiandrogen, abarelix monotherapy had a faster time to medical castration and a greater percentage decrease in prostate-specific antigen.

Unfortunately, abarelix in clinical practice was associated with a higher rate of potentially life-threatening hypersensitivity reactions that eventually led to its withdrawal from the US market in 2005.

It wasn’t until 2008 that the GNRH antagonist degarelix (Firmagon) was approved,

 and in December 2020, relugolix (Orgovyx), the first oral-formulated GNRH analogue, was added to the short list of GNRH antagonists.

ADT with GNRH agonists and antagonists has a role in the treatment of prostate cancer in the local or locally advanced, relapsed/recurrent, advanced/metastatic, and palliative settings.

A pivotal phase 3 clinical study (NCT00295750) compared the efficacy and safety profile of degarelix to leuprolide. The study population was men with prostate cancer of any stage determined to be candidates for ADT, including patients with metastatic disease or biochemical progression with less than 6 months of prior ADT given in the neoadjuvant or adjuvant setting with curative-intent local therapy.

Results showed that degarelix was not only noninferior to leuprolide in the primary end point of suppressing testosterone to 0.5 ng/mL or lower at all monthly measurements for 1 year, but also significantly faster in achieving castrate levels of testosterone in 3 days compared to 28 days for leuprolide. Additionally, degarelix lacked observed testosterone surge and maintained undetectable testosterone levels throughout the 1-year treatment period.

Degarelix was the only approved LHRH antagonist in the US until relugolix was recently added to the treatment armamentarium for prostate cancer. In the phase 3 HERO trial (NCT03085095) that led to the FDA approval of relugolix, patients with advanced prostate cancer received either oral relugolix daily or leuprolide injections every 3 months for 48 weeks. The study population was comprised of prostate cancer patients who were eligible for at least 1 year of continuous ADT in the setting of either biochemical or relapsed disease following local definitive therapy, newly diagnosed hormone-sensitive metastatic disease, or locally advanced disease unlikely to be cured with local definitive therapy.

The addition of an antiandrogen to leuprolide was left to the investigator’s discretion, and 23 of 201 (11%) patients in the leuprolide group received concomitant antiandrogen. Relugolix was found to be superior to leuprolide in its ability to maintain castration-level testosterone (96.7% vs 88.8%, respectively; 

<.001 for superiority), as well as superior in the time to testosterone suppression (4 days vs 29 days) and proportion of patients with testosterone recovery to normal range within 90 days of treatment discontinuation (54% vs 3%).6 Testosterone recovery can be clinically relevant for patients receiving either intermittent or short-course ADT or for those suffering from significant ADT adverse effects or other disease-related complications. However, the HERO trial did not evaluate patients who received ADT with definitive local therapy, and therefore the role in this context is less defined.

The decision to use a GNRH agonist vs antagonist depends on several factors. Given the shorter time to testosterone suppression and lack of testosterone surge, GNRH antagonists may be more clinically beneficial for patients with more advanced disease and at higher risk of developing prostate cancer–related symptoms.

Additionally, the absence of tumor flare with GNRH antagonists may seem especially appealing in patients with impending cord compression or urethral obstruction; however, GNRH agonists in conjunction with antiandrogen agents for flare prophylaxis have been shown to achieve similar effects.

From a practical standpoint, degarelix is given on a monthly basis compared to the less frequent dosing options of GNRH agonists. With oral relugolix, issues of compliance and the likely need for continual testosterone monitoring are possible barriers that are not routinely of concern for patients receiving injection therapy.

Adverse effect profiles also play a large role in the selection of a GNRH agonist or antagonist. Both the phase 3 degarelix trial and the HERO trial showed that adverse events (AEs) were similar across all treatment groups, with flushing/hot flashes being the most common AE. Relugolix had increased reports of diarrhea compared with leuprolide.6 Leuprolide had higher rates of arthralgia and urinary tract infection compared with degarelix, whereas degarelix was associated with more injection-site reactions.

One of the notable findings from the HERO trial was the differential cardiovascular profile between the two agents. Although the trial included patients with a distant history of cardiovascular events, patients with major cardiovascular events 6 months prior to trial enrollment were excluded. Relugolix had a lower incidence of major cardiovascular events in the overall study population (2.9% vs 6.2%, respectively) and in patients with a prior history of a major cardiovascular event (3.6% and 17.8%).

 The latter findings are particularly notable given the association between hormone therapy and cardiovascular events and the aforementioned mechanistic differences between GNRH agonists and antagonists on FSH. Even prior to data from the HERO study, observational studies and meta-analyses demonstrated that patients with preexisting cardiovascular disease have a higher risk of cardiovascular morbidity and mortality with GNRH agonists compared to GNRH antagonists.

The link between cardiotoxicity and ADT prompted a phase 2 trial that prospectively assessed serum cardiac biomarkers in prostate cancer patients with underlying cardiovascular disease who were treated with GNRH agonists or antagonists. Findings from that study showed that patients with baseline elevations in N-terminal pro–brain natriuretic peptide, dimerized plasmin fragment D, C-reactive protein, and high-sensitivity troponin had higher incidences of cardiovascular AEs when treated with LHRH agonists but not antagonists.

 It was suggested by the authors that patients with known cardiovascular disease who are candidates for ADT may benefit more from a GNRH antagonist.

There has yet to be a prospective study evaluating cardiovascular outcomes in ADT-treated patients with cardiovascular disease; however, ongoing trials such as the PRONOUNCE study (NCT02663908) may help further elucidate this. Differences with regard to other ADT-associated AEs are less defined. In a study comparing the GNRH antagonist degarelix to leuprolide, degarelix was associated with more-pronounced reductions in serum alkaline phosphatase; however, whether this translates to an impact on bone strength or fracture risk remains to be determined.

Hormone therapy can significantly impact quality of life, and incorporating strategies to minimize AEs can be crucial. When the more-common AEs of ADT arise—such as muscle loss, weight gain, decreased libido, and fatigue—a supervised exercise program or resistance training can limit or potentially reverse some of them.

Additionally, ADT can accelerate bone resorption, which comprises bone integrity and increases the risk of osteoporotic fractures. Interventions to preserve bone health include calcium and vitamin D supplementation, fracture risk assessments, and use of antiresorptive therapy with denosumab or bisphosphonates. Regarding cardiotoxicity, optimizing preexisting cardiovascular conditions prior to ADT initiation is recommended.

Although not yet investigated, medications such as β-blockers, angiotensin-converting enzyme inhibitors, or low-dose rivaroxaban, as well as modified lifestyle interventions and increased cardiac monitoring, may have beneficial effects in patients receiving ADT.

 Intermittent androgen deprivation (IAD) has also been utilized in clinical practice and involves alternating on- and off-treatment periods. Several phase 2 and phase 3 randomized, controlled trials comparing IAD with continuous androgen deprivation (CAD) have shown that IAD can decrease AEs while maintaining or improving quality of life and preserving the overall survival and progression-free survival benefits of CAD.

 However, these findings must be interpreted with caution because a majority of these studies only included patients with nonmetastatic disease.

In general, many factors play into the decision-making process when choosing between a GNRH agonist or antagonist. Additional studies are needed to better understand the relationship between different ADT agents and their toxicity profiles so that treatment selection and management strategies can be optimized.

is a Hematology/Oncology Fellow at the University of California San Diego, La Jolla, CA.

is an associate professor of medicine and urology at the Moores Cancer Center at the University of California San Diego, La Jolla, CA.

1. Huggins C, Hodges CV. Studies on prostatic cancer: I. the effect of castration, of estrogen and of androgen injection on serum phosphatases in metastatic carcinoma of the prostate. 1941. 

. 2002;168(1):9-12. doi:10.1016/s0022-5347(05)64820-3

2. Massie CE, Lynch A, Ramos-Montoya A, et al. The androgen receptor fuels prostate cancer by regulating central metabolism and biosynthesis. 

 2011;30(13):2719-2733. doi:10.1038/emboj.2011.158

3. Moul JW. Twenty-five year evolution of medical hormonal therapy for prostate cancer. 

 2009;103(2):145-146. doi:10.1111/j.1464-410X.2008.08271.x

4. Freedland SJ, Abrahamsson PA. Androgen deprivation therapy and side effects: are GnRH antagonists safer? 

Many factors play into decision-making process when weighing agents.

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