Role of circulating tumor cell clusters in patients with metastatic hormone-sensitive prostate cancer receiving a gonadotropin-releasing hormone antagonist: A pilot study

Dear Editor,

Prostate cancer is the most common cancer among men[1].Androgen deprivation therapy (ADT) has remained the primary treatment of metastatic-hormone-sensitive prostate cancer (mHSPC), providing a temporary disease control in the majority of patients.Despite initial ADT response,castration-resistance prostate cancer(CRPC)still develops.Previous study have attempted to determine possible biomarkers for poor prognosis in patients with CRPC [2].For example, circulating tumor cells (CTCs) are thought to detach from primary or secondary tumors of patients with advanced malignant neoplasms [3].Studies have also established that CTCs can be combined with other modalities for monitoring patients with CRPC[4,5].Furthermore,CTC clusters are derived from the multicellular groupings of primary tumor cells and significantly contribute to the metastatic spread of CRPC [6,7].

In contrast, the certainty of the aforementioned mechanisms contributing to treatment resistance in patients with mHSPC receiving ADT remains unclear.One major problem of assessing the role of CTCs and CTC clusters in patients with mHSPC is that the CellSearch system(Menarini Silicon Biosystems Inc., Huntingdon Valley, PA,USA),a major method for identifying CTCs,cannot identify CTCs with low or absent epithelial cell adhesion molecule expression [8].Recently, a study reported that the Celsee system (Celsee Inc., Ann Arbor, MI, USA), a microfluidic device that can be used for CTC enrichment, can capture high CTC values, given its ability to trap CTCs with low or absent epithelial cell adhesion molecule expression [9].This technique has enabled accurate and reproducible liquid biopsies over time.This study assessed the roles of CTCs and CTC clusters detected using the Celsee system for patients with mHSPC treated with a gonadotropin-releasing hormone (GnRH) antagonist.

Patients with mHSPC treated at Tohoku Medical and Pharmaceutical Hospital were prospectively enrolled between April 2019 and April 2020.This study was conducted following the Declaration of Helsinki and had been approved by the clinical research ethics boards of Tohoku Medical and Pharmaceutical Hospital (approval number:2017-2-070).All patients provided written informed consents.At our institution, all patients had a pathological diagnosis of prostate cancer by prostatic biopsy and received a first-line therapy for mHSPC.Evaluation of metastasis was performed through chest, abdominal, and pelvic computed tomography, and technetium-99 mlabelled bone scanning.All patients were diagnosed with mHSPC based on findings of lymph node metastasis outside of the pelvis, bone metastasis, and visceral metastasis.All patients received ADT, comprising subcutaneous injection of the GnRH antagonist, degarelix, as a first-line therapy,and did not receive the combined therapy of androgen receptor signaling pathway inhibitors.Prostate-specific antigen(PSA)progression was defined according to the Prostate Cancer Clinical Trials Working Group 3,where an increase in PSA of over 25% and over 2 ng/mL above nadir at two timepoints at least 3 weeks apart confirmed progression[10].Radiographic progression was defined using the Response Evaluation Criteria in Solid Tumors criteria,which observed the progression of measurable tumors.CRPC was defined as an observed PSA progression or radiographic progression in this study.CTCs were identified through liquid biopsy using the Celsee system [9].Blood samples(10.0 mL)from the patients were drawn into Cell-Free DNA BCT®CE (Streck, La Vista, NE, USA).Circulating cells were collected using the Celsee PREP™400 system (Celsee Inc.,Ann Arbor, MI, USA).With the Celsee Analyzer™imaging station (Celsee Inc., Ann Arbor, MI, USA), these cells were automatically stained with 4′,6-diamidino-2-phenylindole(DAPI) (nucleus), CD45 (a leukocyte marker), and pancytokeratin (an epithelial marker).Cells that tested positive for DAPI and pan-cytokeratin and negative for CD45 were classified as CTCs (Fig.1A—D).CTC clusters can be identified as clustered CTCs that adhered to two or more CTCs (Fig.1C and D).Survival probabilities were estimated using the Kaplan-Meier method.The cutoff for CTCs was defined as the median of each continuous value.All statistical analyses were conducted using JMP®pro 16.0.0(SAS Institute Inc., Cary, NC, USA), withp＜0.05 indicating statistical significance.

Figure 1 Immunofluorescent staining of CTCs and CTC clusters,and the correlations for prognosis of patients with mHSPC.(A—D)Immunofluorescent staining of CTCs (arrows)—DAPI+cells (staining of the nucleus) (A), CD45-cells (a leukocyte marker) (B), pancytokeratin+cells(an epithelial marker)(C),and merged image of DAPI,CD45,and pan-cytokeratin(D);(E—H)Immunofluorescent staining of CTC clusters (arrows)—DAPI+clusters (staining of the nucleus) (E), CD45-clusters (a leukocyte marker) (F), pan-cytokeratin+clusters (an epithelial marker)(G),and merged image of DAPI,CD45, and pan-cytokeratin (H); (I—L)Kaplan-Meier curves of patients with mHSPC receiving ADT—PSA-PFS with ≥median CTCs vs. ＜median CTCs (I), radiographic PFS with ≥median vs.＜median CTCs(J),PSA-PFS with positive vs.negative CTC clusters(K),and radiographic PFS with positive vs.negative CTC clusters(L).DAPI, 4′,6-diamidino-2-phenylindole; PSA, prostate-specific antigen; ADT, androgen deprivation therapy; CTC, circulating tumor cell; PFS, progression-free survival; mHSPC, metastatic-hormone-sensitive prostate cancer.

In this study,17 patients were diagnosed with mHSPC and enrolled.The patients’ clinical characteristics are summarized in the Supplemental Table 1.The mean follow-up duration of patients was 15 months (range 10—17 months).All patients exhibited CTCs (median 7; range 2—33), among whom nine(52.9%)patients showed CTC clusters.Six(35.3%)patients exhibited PSA progression during the follow-up period.Five (29.4%) patients exhibited radiographic progression, all of whom had PSA progression.None of the patients died during the follow-up period.Fig.1I—L details the Kaplan-Meier analysis results for PSA progression-free survival (PFS) and radiographic PFS.Accordingly, no significant difference in PSA-PFS was observed according to the number of CTCs (≥medianvs.＜median;p=0.510).In contrast, patients with CTC clusters had significantly worse PSA-PFS than those without (p=0.047).Furthermore, no significant difference in radiographic-PFS was noted according to the number (≥medianvs.＜median;p=0.760),but patients with CTC clusters had significantly worse radiographic PFS than those without the clusters(p=0.011).

Our pilot study assessed the role of CTCs and CTC clusters detected using the Celsee system for patients with mHSPC treated using a GnRH antagonist.We demonstrated that CTC clusters are an important prognostic factor for patients with mHSPC.CTC clusters in circulation are more infrequent than single cells,but have been correlated with a worse prognosis of malignant neoplasm than single cells.Several studies have suggested that CTC clusters are prognostic factors for CRPC [6,7].Here, the detection of CTC clusters in patients with mHSPC was also associated with their response to GnRH antagonist treatment.In conclusion, this pilot study suggests the potential of CTC clusters as an important prognostic factor for patients with mHSPC.To clarify the results of this study,we will perform a larger cohort study in the future.

Author contributions

Study concept and design: Yuki Kohada, Yasuhiro Nakamura, Makoto Sato.

Data acquisition: Yuki Kohada, Hiroki Kusumito, Takashi Kukimoto, Jotaro Mikami, Jun Ito, Yasuhiro Kaiho.

Data analysis: Katsutoshi Asano, Toru Yaegashi, Kanichi Nakagawara.

Drafting of manuscript:Kanichi Nakagawara,Jun Teishima,Yasuhiro Kaiho, Nobuyuki Hinata, Yasuhiro Nakamura,Makoto Sato.

Critical revision of the manuscript: Yasuhiro Nakamura,Makoto Sato.

Conflicts of interest

The authors declare no conflict of interest.

Acknowledgements

The authors would like to thank Kazue Ise for technical assistance for the data collections with the experiments of this study.We also gratefully acknowledge the technical assistance for CTCs collection of members of Nihon Gene Research Laboratories Inc.

Appendix A.Supplementary data

Supplementary data to this article can be found online at https://doi.org/10.1016/j.ajur.2022.03.009.