Molecular analysis of circulating tumor cells of metastatic castration-resistant Prostate Cancer Patients receiving 177Lu-PSMA-617 Radioligand Therapy

Abstract

Rationale: Lu-177-PSMA-617 radioligand therapy (RLT) is currently under approval for treatment of metastatic castration resistant prostate cancer (mCRPC) patients with late stage disease. However, previous studies demonstrated both heterogeneity of prostate specific membrane antigen (PSMA) expression, as well as response to PSMA treatment among mCRPC patients. Thus, there is an unmet need for identifying predictive parametres prior or under PSMA-RLT treatment. We therefore aimed to correlate several clinical and molecular parameters with response to PSMA treatment in a cohort of mCRPC patients undergoing PSMA RLT followed by a detailed analysis of promising candidates. Methods: Nineteen patients, median age 68.8 years (range: 56.9 - 83.3) with mCRPC were included in this study. We performed baseline analysis of clinical parameters based on PSMA PET/CT, (metabolic tumor volume (MTV), total tumor volume (TTV)), serum PSA, ALP, LDH and gene expression analysis of circulating tumor cells (expression of AR full length (AR-FL), AR splice variant 7 (AR-V7), PSA and PSMA) as well as common markers for neuroendocrine differentiation (NED). Results: Patients presented with bone, lymph node, and visceral metastases (89%, 68%, and 21%, respectively). All patients were pretreated with docetaxel, either abiraterone or enzalutamide, or both. Biochemical response in terms of PSA decline ≥50 or ≥30% was observed in 42% and 63%, respectively. There were significant correlations between PSA and PSMA mRNA expression, as well as tumor volumes (both MTV and TTV), AR-FL and AR-V7 mRNA expression. However, there was no correlation with response to PSMA treatment. Furthermore, none of these parameters was significantly correlated with baseline serum PSA values. Common NED markers were shown to be specifically high expressed and revealed impact on OS independent from AR-V7 gene expression. Conclusion: We demonstrate that AR-FL and its splice variant AR-V7 might serve as prognostic biomarkers displaying high tumor burden in mCRPC patient prior to PSMA-RLT. Contrary, PSMA, which has been discussed as a biomarker for PSMA targeted treatment, does not display strong prognostic ability - at least on the mRNA level. Surprisingly, none of these parameters correlates to response to PSMA treatment. In contrast, commom NED markers such as SYP and ENO2 as well as FOXA1 expression level seem to predict OS, but not PFS, more reliably. We admit that a limitation of our study is the focus on mRNA expression of potential biomarkers only. Further investigations analyzing the potential role of protein expression of these markers are therefore warranted.

Keywords: 177Lu-PSMA-617; CTC; PSMA; prostate cancer.

Figure 1

PSA response on ¹⁷⁷Lu-PSMA therapy and multi-parameter correlation analysis. Best PSA response during therapy with ¹⁷⁷Lu-PSMA is represented by percentual drop of PSA serum levels and shown in bars for each individual patient in a waterfall plot. Levels >0% represent non-responders, <0% equal any response. Dotted lines indicated established response tresholds of ≥30% and ≥50% (A). Selected parameters have been subjected to a multi-parameter correlation analysis using the RStudio corrplot package. Circle size and colour is linked to amount of correlation (blue for positive, red for negative correlation) and significance (non-significant results with p>0.05 are shown as blank fields). Numbers indicate the precise Pearson value. PSA: prostate-specific antigen; BestR: best response; OS: overall survival; cAR: relative copy numbers of androgen receptor mRNA; cAR-V7: relative copy numbers of androgen receptor splice variant V7 mRNA cPSMA: relative copy numbers of prostate-specific membrane antigen mRNA; bPSA: serum prostate-specific antigen at baseline; CTC: circulating tumor cells; MTV: metabolic tumor volume.

Figure 2

AR-V7 expression subgroup analysis. Best PSA Response is presented for AR-V7^pos (A) and AR-V7^neg (B) patients. Dotted lines indicated established response tresholds of >30% and >50%. Representative PSMA PET images are shown for each group. Multiparameter correlation analysis is shown in corrplots created using the RStudio corrplot package. Circle size and colour is proportional to the respective Pearson coefficient (blue for positive, red for negative correlation) and significance (non-significant results with p>0.05 are shown as blank fields). Numbers indicate the precise Pearson value. OS (E) and PFS (F) is shown for AR-V7^pos (blue) and AR-V7^neg (red) patients. PSA: prostate-specific antigen; BestR: best response; OS: overall survival; PFS=progression-free survival; cAR: relative copy numbers of androgen receptor mRNA; cAR-V7: relative copy numbers of androgen receptor splice variant V7 mRNA cPSMA: relative copy numbers of prostate-specific membrane antigen mRNA; bPSA: serum prostate-specific antigen at baseline; CTC: circulating tumor cells; MTV: metabolic tumor volume.

Figure 3

Expression and impact of common NED markers. FOXA1, SYP and ENO2 expression was investigated between healthy controls (HC) and mCRPC patients (A) and between AR-V7^pos and AR-V7^neg patients (B). FOXA1 expression was normalized to RPL37A, SYP and ENO2 expression was normalized to PSA expression. Normalized expression of FOXA1 was plotted as is, in terms of SYP and ENO2 expression a threshold was determined to separate unspecific expression compared to HCs. AR-V7^pos patients were compared to AR-V7^neg individuals regarding serum PSA and PSMA mRNA expression (C) ALP and LDH serum values were plotted in a bar plot (D and E). Correspondingly, OS (F) and PFS (G) was determined for four groups: AR-V7^posNED^hi (black line), AR-V7^posNED^lo (red line), AR-V7^negNED^hi (green line), AR-V7^negNED^lo (blue line). NED: neuroendocrine differentiation; FOXA1: forkhead box A1; ENO2: Enolase 2, SYP: synaptophysin, RPL37A: ribosomal protein 37A; PSA: prostate-specific antigen; OS: overall survival; AR-V7: androgen receptor splice variant V7. P-values (log rank or Mann-Whitney) <0.05 were considered statistically noticeable.