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Circulating Tumor Cell and Circulating Tumor DNA Assays Reveal

Complementary Information for Patients with Metastatic

Urothelial Cancer

Heather J. Chalfin *, Stephanie A. Glavaris, Michael A. Gorin, Max R. Kates, Megan H. Fong,

Liang Dong, Andres Matoso, Trinity J. Bivalacqua, Michael H. Johnson, Kenneth J. Pienta,

Noah M. Hahn, David J. McConkey

The James Buchanan Brady Urological Institute and Greenberg Institute, Johns Hopkins University School of Medicine, Baltimore, MD, USA

Article info Abstract

Article history: Despite considerable advances in the management of urothelial (UC),

Received 8 April 2019 better risk stratification and enhanced detection of minimal residual are

Received in revised form still urgent priorities to prolong survival while avoiding the morbidity of over-

2 July 2019 treatment. Circulating tumor cells and DNA (CTCs, ctDNA) are two biologically

Accepted August 15, 2019 distinct “liquid ” that may potentially address this need, although they

have been understudied in UC to date and their relative utility is unknown. To this

Associate Editor: Ashish Kamat

end, matched CTC and ctDNA samples were collected for a head-to-head compari-

son in a pilot study of 16 patients with metastatic UC. CTCs were defined as

Keywords:

- and/or EpCAM-positive using the RareCyte direct imaging platform.

Bladder cancer

ctDNA was assayed using the PlasmaSelect64 probe-capture assay. 75% of patients

Liquid

had detectable CTCs, and 73% had detectable somatic mutations, with no correla-

Circulating tumor cells

tion between CTC count and ctDNA. 91% of patients had confirmation of at

Circulating tumor DNA

least one plasma mutation and, importantly, several clinically actionable mutations

were detected in plasma that were not found in the matching tumor. A ctDNA

fraction of >2% was significantly associated with worse overall survival (p = 0.039)

whereas CTC detection was not (p = 0.46). Notably, using a predefined gene panel

for ctDNA detection had a high but not complete detection rate in metastatic UC,

similar to what has been described for a custom tissue-personalized assay ap-

proach. In sum, both liquid biopsies show promise in UC and deserve further

investigation.

Patient summary: New “liquid biopsy” blood tests are emerging for urothelial

cancer aimed at early detection and avoiding overtreatment. Our results suggest

that two such tests provide complementary information: circulating tumor cells

may be best for studying the biological features of a person’s cancer, whereas

circulating tumor DNA may be better for early detection.

Published by Elsevier B.V. on behalf of European Association of Urology.

* Corresponding author. The James Buchanan Brady Urological Institute, Johns Hopkins Univer-

sity School of Medicine, 600 N. Wolfe Street, Baltimore, MD 21287, USA. Tel.: +1 443 7423130,

Fax: +1 410 9550833.

E-mail address: heather.chalfi[email protected] (H.J. Chalfin).

https://doi.org/10.1016/j.euo.2019.08.004

2588-9311/Published by Elsevier B.V. on behalf of European Association of Urology.

Please cite this article in press as: Chalfin HJ, et al. and Circulating Tumor DNA Assays Reveal

Complementary Information for Patients with Metastatic Urothelial Cancer. Eur Urol Oncol (2019), https://doi.org/10.1016/j. euo.2019.08.004

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Urothelial carcinoma (UC) of the bladder is a common single cell level, and thus CTC assays are not simply an

urologic malignancy, with an estimated 79 000 new cases interchangeable alternative for the more widely studied liquid

diagnosed each year in the USA [1]. Despite considerable biopsy of circulating tumor DNA (ctDNA). Furthermore, CTC

advances in the treatment of UC, it remains an aggressive detection reveals that an intact cell has survived in the

disease and the gold standard of cystectomy is a morbid peripheral circulation, which is not biologically equivalent to

procedure. Approximately 40% of patients will enjoy a detection of fragments of shed tumor DNA via a ctDNA assay.

complete response to neoadjuvant chemotherapy with no To date, CTCs and ctDNA have been understudied in UC.

residual tumor found at the time of cystectomy; however, The vast majority of the work investigating CTCs in bladder

these cases cannot be reliably identified before surgery with cancer has used the CellSearch test, with no consensus on

any currently available test [2]. Better detection of minimal their prognostic significance [3]. These discordant observa-

residual disease and better risk stratification are an urgent tions are likely because of the limitations of the CellSearch

priority to minimize overtreatment and prolong survival. technology, which cannot detect epithelial marker–

Many minimally invasive “liquid biopsy” technologies are negative CTCs [4]. Furthermore, the emphasis has been

currently under investigation to address this need, including on counting CTCs as opposed to harnessing the opportunity

circulating tumor cells (CTCs). These are malignant cancer to study cell morphology and biomarkers at the single cell

cells that can be detected in peripheral blood. CTC morpholo- level. More recently, novel selection-free platforms with

gy, expression, and genomics can be assessed at the enhanced detection capabilities and a greater focus on

Fig. 1 – Individual patient CTC and ctDNA results grouped by ctDNA fraction detected. Patients are grouped by high (red box around liquid biopsy time

point) and low (green box) ctDNA fraction. One patient did not have ctDNA results (black box). The ctDNA fraction is shown in red/green, and the CTC

count displayed below in black. Blue boxes represent tumors that were sequenced. Patient 2 had basal cell skin cancer resected. Patient 3 had

seminoma treated with orchiectomy and radiation at age 22 yr. Patient 4 had squamous cell skin cancer resected. Patients 8 and 13 had Gleason

6 (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article).

CHEMO = chemotherapy; EBRT = external beam radiation therapy; LNU = left nephroureterectomy; MET = metastatic; NAC = neoadjuvant chemotherapy;

NephroU = nephroureterectomy; NMIBC = non–muscle-invasive bladder cancer; RC = radical cystectomy; TURBT = transurethral resection of bladder

tumor.

Please cite this article in press as: Chalfin HJ, et al. Circulating Tumor Cell and Circulating Tumor DNA Assays Reveal

Complementary Information for Patients with Metastatic Urothelial Cancer. Eur Urol Oncol (2019), https://doi.org/10.1016/j. euo.2019.08.004

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morphology have started to emerge [5]. For ctDNA, there is correlate with cell-free DNA or the ctDNA fraction (Supple-

also no consensus on the optimal strategy for detection. A mentary Fig. 1). We discovered a similar genomic landscape to

personalized medicine approach of unique droplet digital that previously described, with frequent mutations in TP53,

polymerase chain reaction (ddPCR) assays targeted to each TERT, and ERBB2 (Fig. 2) [7]. With a median follow up of

individual patient’s known mutations should offer greater 5.9 mo, a ctDNA fraction of >2% was associated with worse

sensitivity than a generic next-generation sequencing (NGS) overall survival (OS; p = 0.039) with a median OS of 38 wk

panel. Conversely, an NGS panel may be able to detect versus not reached for ctDNA 2% (Supplementary Fig. 2). By

mutations that have arisen later and are not represented in contrast, CTC detection did not have a significant impact on OS

the primary tumor. Compounding these considerations is (p = 0.46), with a median OS of 59 wk for no CTCs versus

the distinctly lower expression of CTCs and ctDNA in UC 25 wk for 1 CTC (Supplementary Fig. 3). The cutoffs were

than in other malignancies [6]. defined a priori on the basis of previous work [7].

Given the potential of these distinct liquid biopsies for risk Notably, our ctDNA detection rate of 73% is in line with

stratification and the lack of information on their relative that of three other groups that reported 65%, 69%, and 73%

utility in UC, we performed a head-to-head comparison of a for small cohorts [8]. Overall, these numbers are disap-

selection-free -based CTC assay (Rare- pointing, as they represent incomplete detection in

Cyte) and a plasma NGS panel for ctDNA detection in a pilot advanced disease, and the ideal liquid biopsy should be

cohort of 16 consecutive patients with metastatic UC. Fig. 1 sensitive enough for early diagnosis and detection of

shows their clinical course and treatment. We found similar minimal residual disease. Of note, there is one commercially

detection rates for CTCs (75% of patients; median 2.5 CTCs, available NGS ctDNA panel (Guardant360) with a detection

range 0–170) and ctDNA (73% of patients; median of 2 somatic rate of 90% reported for genomic alterations in advanced

mutations, range 0–7) yet interestingly the CTC count did not UC; however, the same group showed that the results for

Fig. 2 – Mutational landscape of metastatic bladder cancer plasma samples. Each column represents one plasma sample for a given patient, with the

corresponding circulating tumor DNA fraction plotted on the bar graph above. Each mutated gene is shown on a row, with the mutation frequency of

that gene in the overall cohort shown on the bar graph to the right. Boxes are color coded with respect to the mutant allele fraction of the mutation.

(For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

Please cite this article in press as: Chalfin HJ, et al. Circulating Tumor Cell and Circulating Tumor DNA Assays Reveal

Complementary Information for Patients with Metastatic Urothelial Cancer. Eur Urol Oncol (2019), https://doi.org/10.1016/j. euo.2019.08.004

EUO-262; No. of Pages 5

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this panel were significantly discordant with tumor described the unique challenge of ctDNA detection in UC and

collected at the same time [9–11]. In the present study noted that the optimal approach must provide sensitivity

we found higher mutant allele frequencies (MAFs) for without losing the ability to detect clonal evolution.

plasma mutations that were concordant with tissue

fi

(11.16 vs 0.40; p  0001) and that were triplicated in Author contributions: Heather J. Chal n had full access to all the data in

the study and takes responsibility for the integrity of the data and the

replicates (43.38 vs 21.09; p = 0.07), suggesting a certain

accuracy of the data analysis.

threshold for detection with the NGS assay. Logically, it

might be expected that a tissue-personalized ddPCR assay

Study concept and design: Chalfin, Glavaris, Gorin, Hahn, McConkey.

would provide greater sensitivity; however, this approach

Acquisition of data: Chalfin, Glavaris, Kates, Fong, Dong, Matoso,

also has limitations due to tumor heterogeneity and clonal

Bivalacqua, Johnson, Pienta, Hahn, McConkey.

evolution. In this context, in the present study several Analysis and interpretation of data: Chalfin, Glavaris, Gorin, Dong, Hahn,

potentially clinically actionable mutations were only McConkey.

fi

detected in plasma and not in matched tumor specimens Drafting of the manuscript: Chal n, Glavaris.

collected at various time points relative to the liquid Critical revision of the manuscript for important intellectual content:

Chalfin, Glavaris, Gorin, Kates, Bivalacqua, Pienta, Hahn, McConkey.

biopsies (91% of patients had tissue confirmation of at least

Statistical analysis: Chalfin, Glavaris, Gorin.

one plasma mutation, Supplementary Figs. 4 and 5; all

Obtaining funding: Chalfin, Hahn, McConkey.

available primary/metastatic tumor was sequenced, Sup-

Administrative, technical, or material support: Glavaris, Fong, Dong,

plementary Fig. 1). The main example of a tissue-personal-

Matoso.

ized ddPCR assay in UC was described by Birkenkamp-

Supervision: Gorin, Kates, Bivalacqua, Johnson, Pienta, Hahn, McConkey.

Demtröder et al [12]. Five of 12 patients (42%) had at least

Other: None.

one undetectable result during metastatic disease, with one

patient having seven negative reads. These patients Financial disclosures: Heather J. Chalfin certifies that all conflicts of

fi fi

received heterogeneous treatments, and the question of interest, including speci c nancial interests and relationships and

fi

sensitivity for the metastatic setting of ddPCR assays af liations relevant to the subject matter or materials discussed in the

manuscript (eg, employment/affiliation, grants or funding, consultan-

designed based on primary tumor alterations deserves

cies, honoraria, stock ownership or options, expert testimony, royalties,

further dedicated study. An additional challenge for NGS

or patents filed, received, or pending), are the following: Kenneth J.

assays is that quantification of mutant genome expression

Pienta has acted as a consultant for Celsee Diagnostics and acts as the

must be extrapolated from the MAFs. By design, MAF is

Chief Medical Officer for Cuebiophrma. The PlasmaSELECT test is owned

affected by the overall coverage of the region of interest,

by Personal Genome Diagnostics (PGDx). The Johns Hopkins University

which is not uniform across the genome. This limitation

has licensed technologies to PGDx and, as a result, has a financial interest

affected the present study, and it must be emphasized that

in the company. This arrangement has been reviewed and approved by

our analysis was certainly impacted by small numbers and the Johns Hopkins University in accordance with its conflict of interest

heterogeneous clinicopathologic factors at baseline. CTC policies. The remaining authors have nothing to disclose.

detection was not significantly associated with OS; howev-

er, this could simply reflect a lack of statistical power Funding/Support and role of the sponsor: H.J.C. is supported by a grant

necessary to detect a relationship that does exist. from the Johns Hopkins Greenberg Bladder Cancer Institute. The sponsor

Despite these challenges, liquid biopsy represents the next played a role in review of the manuscript.

frontier for diagnosis, management, and follow-up for solid

malignancies. Our results suggest that CTCs and ctDNA can

provide complementary information: ctDNA quantification Appendix A. Supplementary data

was relevant for survival and the NGS assay detected

individual patient mutations that were not identified in Supplementary material related to this article can be

tumor, whereas the CTC platform demonstrated feasibility for found, in the online version, at doi:https://doi.org/10.1016/j.

capture of immunoflourescent images revealing the morphol- euo.2019.08.004.

ogy of circulating malignant cells as well as protein expression

at the single cell level. When considering the ideal CTC assay,

the emphasis should shift from simple enumeration to a References

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Please cite this article in press as: Chalfin HJ, et al. Circulating Tumor Cell and Circulating Tumor DNA Assays Reveal

Complementary Information for Patients with Metastatic Urothelial Cancer. Eur Urol Oncol (2019), https://doi.org/10.1016/j. euo.2019.08.004

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Please cite this article in press as: Chalfin HJ, et al. Circulating Tumor Cell and Circulating Tumor DNA Assays Reveal

Complementary Information for Patients with Metastatic Urothelial Cancer. Eur Urol Oncol (2019), https://doi.org/10.1016/j. euo.2019.08.004