Progress Continues for Use of Liquid Biopsies in GU Cancers

Clinical use of liquid biopsies to aid in treatment and management decisions for patients with cancer is a long-held goal of precision medicine.

“Liquid biopsy refers to the use of any type of body fluid to assess an aspect of disease biology or even normal biology,” said Alexander Wyatt, BSc, DPhil, an associate professor in urologic sciences at the University of British Columbia, Canada. “Typically, when we think of liquid biopsy in the context of genitourinary cancer, we are talking about [testing] the blood or the urine.”

There are several components of liquid biopsy in cancer, including circulating tumor cells (CTCs), cell-free circulating nucleic acids in the form of circulating tumor DNA (ctDNA) or RNA, extracellular vesicles, and more.¹ Regardless of the analyte, liquid biopsy provides a minimally invasive method for cancer management with possible diagnostic, prognostic, and predictive applications.

GU Oncology Now recently spoke with several researchers involved in exploring the use and clinical utility of liquid biopsy for genitourinary cancers.

Tissue Versus Liquid

Two major aspects differentiate liquid biopsies from tissue biopsy, the traditional “gold standard,” according to Tyler Stewart, MD, assistant professor of medicine at UC San Diego School of Medicine.

First, is ease of use. Although not completely noninvasive, liquid biopsies are minimally invasive and associated with fewer complications than tissue biopsy, which allows for more frequent real-time monitoring, Dr. Stewart said.

“This can be particularly important in situations where someone is on treatment and having progression of disease and you want to see if there are new emerging mutations,” he noted. “Liquid biopsy is much easier to do, especially in a resistant setting.”

Additionally, tissue is not always available, or if it is, there may not be enough to run the appropriate tests.

The other important differentiating factor is that cancer is a heterogenous disease, even within each patient. One specific area sampled with a tissue biopsy may not pick up a mutation present in other sites.

“Liquid biopsies are not perfect with this either, but they are another way to assess if there are mutations that may have been missed in the tumor site tested,” Dr. Stewart said.

In the early days of liquid biopsy research, the most exciting area was related to CTCs, explained Dr. Wyatt.

“These are cells that break off from the tumor and circulate in the blood stream until they die or seed somewhere else,” Dr. Wyatt said. “The major challenges with CTCs is that there are not many present within a tube of blood. We needed more sensitive approaches.”

Liquid biopsy research continued to evolve over the last 10 years by going after material that would be more commonly found in the blood.

“Cancers are incredibly inefficient and are always turning over. The detritus from those proliferating and dying cell populations can make their way into the blood stream,” Dr. Wyatt said. “Pieces of DNA, RNA, proteins from the cancer cells have leaked out as the cells die. Therefore, liquid biopsy morphed from isolating CTCs into characterizing this free DNA and RNA shed by cancer cells … and the advantage of using that material is there is much more of it present in a typical tube of blood.”

The blood analyte that has “really hit home,” Dr. Wyatt said, is cell-free DNA (cfDNA), which is all the DNA that is shed into the blood and not present in cells, regardless of whether it comes from cancer cells or from healthy normal cells. In a person with cancer, a proportion of that total cfDNA will be tumor-derived, and that is called ctDNA.

“It is that component we are always trying to detect and characterize,” Dr. Wyatt said.

Potential Applications

The potential applications of liquid biopsies in genitourinary cancers fall into 2 buckets, according to Dr. Stewart. One involves the characterization of ctDNA—a qualitative bucket—and one involves the detection of ctDNA—a quantitative bucket.

“The qualitative purpose is probably where liquid biopsies came to the forefront in cancer treatment,” Dr. Stewart said. “This is where you are able to do a liquid biopsy looking at ctDNA and trying to find driver mutations and then, later, resistance mutations.”

In prostate cancer, for example, ctDNA evaluation could be used to find DNA damage repair mutations like BRCA1/2 mutations that could qualify patients for treatment with a poly(adenosine diphosphate–ribose) polymerase inhibitor.

Liquid biopsy could also be employed in patients with metastatic urothelial carcinoma (UC) to evaluate for FGFR3 alterations, for which targeted therapies exist.

Dr. Wyatt and colleagues conducted a study of patients with progressing metastatic UC comparing detection of FGFR alterations in archival tissue with plasma cfDNA samples. The study showed 92% concordance between the 2 methods. Of the 4 discordant results, 1 was a cfDNA test with undetectable FGFR3-TACC3­ fusion that was detected in tissue and 3 were positive ctDNA tests in patients who were FGFR wild-type on the tissue testing.²

In these settings, Dr. Wyatt explained, it is known that the patient has active disease, but the clinician needs more information—or biomarkers—to inform prognosis and therapeutic vulnerability. For these treatment decisions, it is typical to use tumor-naive ctDNA genotyping assays.

“These are broader targeted sequencing panels that are trying to discover mutations in the tumor, without prior knowledge of the tumor,” Dr. Wyatt said.

When used quantitatively, liquid biopsies can detect the presence or absence of disease.

“You can use some of these assays to follow the dynamics of the cancer, its progression or response to treatment,” Dr. Stewart said. “The goal has been to see if we can make these assays so sensitive that we can actually tell if someone has cancer in their body even when the scans are negative and apply that knowledge in the realm of perioperative or neoadjuvant therapy.”

For this application, the liquid biopsy tests are typically tumor-informed—or bespoke patient-specific—assays.

“To apply a tumor-informed ctDNA assay, you must have first resected or sampled the primary tumor and profiled the tumor tissue to find what cancer mutations are present, and then you must search for those exact mutations in a blood sample from the same patient,” Dr. Wyatt said. “The reason to use such a patient-specific approach is because you have to sequence the cfDNA really deeply. You are looking for a needle in a haystack, and if you did not know where in the genome the patient’s cancer mutations fall, it would be a very expensive exercise to deeply sequence all the genome.”

Knowing each patient’s existing mutations allows the assay to be more sensitive while retaining cost-effectiveness.

According to Dr. Stewart, more and more assays under development are striving for the off-the-shelf, tumor-naïve approach with high sensitivity, likely because tumor-informed approaches are limited by the need for tissue and the time it takes to develop a personalized assay. However, he qualified this statement by adding that with the rate of discovery in the field, whatever seems like a front-runner today may not even be in the discussion 5 years from now.

Making Progress

Clinical use of liquid biopsy is close to reality in bladder cancers, where evidence of their utility is mounting as a result of several clinical studies.

The phase 3 IMvigor010 trial was conducted to evaluate atezolizumab as an adjuvant therapy in patients with high-risk muscle invasive bladder cancer (MIBC) but showed no disease-free survival (DFS) benefit compared with observation.³

“Even though this study read out as a negative study, there was an important post hoc analysis of ctDNA at the time of the start of the trial,” said Roger Li, MD, a genitourinary oncologist at Moffitt Cancer Center. “What investigators found was if patients had positive ctDNA in that setting—mind you, this is after the disease has been removed and there are no signs of disease on imaging—they were more likely to derive benefit from adjuvant atezolizumab.”

Specifically, the 37% of patients who were positive for ctDNA had improved DFS (hazard ratio [HR], 0.0.58; 95% CI, 0.43-0.79; P=.0024) and overall survival (OS; HR, 0.59; 95% CI, 0.41-0.86) compared with observation. No differences in DFS or OS were seen in patients who were ctDNA negative.⁴ Updated results showed that ctDNA positivity was associated with a shorter OS (HR, 6.3; 95% CI, 4.3-9.3) compared with ctDNA negativity, and ctDNA positivity identified patients who had an OS benefit with atezolizumab.⁵

“This hints at the fact that ctDNA in the adjuvant setting may act as a prognostic biomarker that can be used to select patients for adjuvant treatment,” Dr. Li said.

The IMvigor011 study will assess the efficacy of atezolizumab compared with placebo in patients with high-risk MIBC who are ctDNA positive after cystectomy.⁶ The ongoing nonrandomized TOMBOLA trial will also evaluate if early initiation of atezolizumab in patients who are ctDNA positive improves OS.⁷  The MODERN trial is a large phase-3 trial for patients with bladder cancer who have undergone resection and are at high risk of recurrence; the trial will evaluate the role of escalating adjuvant therapy for patients who are ctDNA-positive after surgery, and de-escalate therapy for patients who are ctDNA-negative.⁸

Another study published out of Denmark used bespoke ctDNA analysis to track the presence of ctDNA throughout the time of neoadjuvant treatment, Dr. Li said.

“These investigators found that there were some patients who did not have any detectable ctDNA even before implementation of neoadjuvant chemotherapy, pointing to the fact that perhaps these patients don’t have micrometastatic disease and it is safe to forgo the use of neoadjuvant chemotherapy,” Dr. Li said. “Secondly, if they did see that there was no ctDNA at the time of radical cystectomy, these patients did a lot better from a prognostic perspective.”

Presented at the 2023 American Society of Clinical Oncology Annual Meeting, the study included 68 patients with MIBC who received neoadjuvant chemotherapy prior to cystectomy. Researchers showed that patients who were ctDNA negative at both baseline and prior to cystectomy had a significantly higher likelihood of achieving pathologic complete response (pCR) compared with those who were ctDNA positive (P<.0001). ctDNA status at both time points was a better predictor of recurrence-free survival compared with achieving pCR (P<.0001).⁹

“This points to the fact that ctDNA may act as an earlier biomarker for disease recurrence and perhaps can be used for initiation of adjuvant therapy,” Dr. Li said.

Other Applications

In testicular cancer, there is not as much excitement around ctDNA as there is around microRNA (miRNA), according to Dr. Stewart. Embryonic stem cells express various miRNA clusters, among them microRNA-371.

One small study of germ cell tumors showed a 300-fold decrease in microRNA-371 levels postorchiectomy.¹⁰ A subsequent analysis showed that compared with several other miRNAs, microRNA-371 had the best sensitivity (88.7%) and specificity (93.4%) for discrimination of testicular cancer.¹¹

Although there is currently a commercially available kit for measuring microRNA-371 that is certified by the European Union and can be used for quantification of miRNA in patients with testicular germ cell tumors,¹² more studies are needed before its use can be incorporated into clinical practice.¹³

Use of ctDNA is not quite as far along in kidney cancer as it is in bladder cancer—the levels of ctDNA appear to be much lower on average in kidney cancer—but there is emerging evidence of future utility. One small study evaluating plasma cfDNA found that patients with metastatic renal cell carcinoma who were ctDNA positive had shorter OS and progression-free survival (PFS) on first-line therapy compared with ctDNA-negative patients.¹⁴ Several smaller studies have shown associations between ctDNA levels and disease response and disease progression.^15–17

In prostate cancer, where prostate-specific antigen measurement remains a mainstay of disease monitoring, ctDNA measurement could provide additional information. For example, one study found that early changes in ctDNA fraction were associated with the duration of first-line androgen receptor pathway inhibitors and survival in patients with metastatic castration-resistant prostate cancer.¹⁸ Specifically, PFS and OS were shortest in patients with persistent ctDNA present at 4 weeks, independent of other prognostic factors.

Bladder cancers studies of ctDNA are not only looking at the blood, but also liquid urine assays.

“Increasingly there are more and more urine biomarkers that are able to pick up cancer in the urine, and we are studying how to use these to monitor patients for recurrence of localized high-risk NMIBC or MIBC,” Dr. Stewart said. “These biomarkers could be used in the context of bladder preservation for patients with MIBC who received systemic therapy but maybe don’t need their bladder taken out, or perhaps after chemoradiation to monitor for cancer recurrence.”

Translating the Science

While there is a lot of promise in liquid biopsies, these tests are not without challenges.

“This is an exquisitely sensitive test,” Dr. Li said. “It is not only detecting mutations associated with the tumor cells, but in the blood or the urine there could seemingly be mutated or altered genes that are not associated with the tumor, per se.”

Although these assays are very sensitive, there are still questions about if they are sensitive enough.

“There are plenty of patients who are ctDNA negative who still recur,” Dr. Wyatt said. “That suggests a sensitivity problem and that we may have to collect more blood or need a more sensitive assay, because we are clearly not capturing every patient.”

There are also no straightforward answers when it comes to thresholds of ctDNA.

“Right now, these tests say ‘positive’ or ‘negative,’ and many of them give a quantification for that positivity as well,” Dr. Stewart said. “Anecdotally, the higher that quantification is, the more likely I will see something when I obtain imaging.”

However, for patients who are positive but at a low level, it is less clear how to proceed.

“The jury is still out about the clinical impact of using these tests and whether or not we are actually helping people live longer, happier, healthier lives with their use,” Dr. Stewart said. “You can think about it the same way that you would think about more frequent novel imaging, which has not always shown to improve outcomes.”

In the late disease setting, when a clinician may use the test to genotype, some patients will have ctDNA below the level where mutations can be detected. In these cases, the test is not positive or negative, but inconclusive, Dr. Wyatt said.

Lack of education on liquid biopsy is also a challenge. This is especially true regarding how to properly implement these tests and how to interpret them.

“I am a genomic scientist and I still struggle to interpret some of the reports that you get from different genetic or genomic labs,” Dr. Wyatt said. “It would be unfair to expect someone without a genomics background to grasp all the nuances present.”

Dr. Li said that lessons can be learned from the numerous urinary ctDNA tests approved by the US Food and Drug Administration that have not had widespread clinical adoption.

“These tests were rolled out before there was an understanding of the clinical diagnostic performance characteristics,” Dr. Li said. “If left to the community, I think these can be used mistakenly.”

Earlier this year, the Signatera cfDNA test received Centers for Medicare & Medicaid Services coverage for 2 additional indications—ovarian cancer in the adjuvant and surveillance settings and breast cancer in the neoadjuvant setting—adding to existing indications, including recurrence monitoring for colorectal cancer, MIBC, and breast cancer.¹⁹ However, according to Dr. Li, there are a lot of clinicians ordering these tests in clinical practice, but doing so almost at random.

In all, Dr. Li said, “I just don’t think we are there yet.”

References

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3. Bellmunt J, Hussain M, Gschwend JE, et al. Adjuvant atezolizumab versus observation in muscle-invasive urothelial carcinoma (IMvigor010): a multicentre, open-label, randomised, phase 3 trial. Lancet Oncol. 2021;22(4):525-537. doi:10.1016/S1470-2045(21)00004-8
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18. Tolmeijer SH, Boerrigter E, Sumiyoshi T, et al. Early on-treatment changes in circulating tumor DNA fraction and response to enzalutamide or abiraterone in metastatic castration-resistant prostate cancer. Clin Cancer Res. 2023;29(15):2835-2844. doi:10.1158/1078-0432.CCR-22-2998
19. Medicare extends coverage of Natera’s Signatera™ MRD test to ovarian cancer and neoadjuvant breast cancer. Natera. February 26, 2024. Accessed April 2, 2024. https://www.natera.com/company/news/medicare-extends-coverage-of-nateras-signatera-mrd-test-to-ovarian-cancer-and-neoadjuvant-breast-cancer/