Background

While programmed death 1 (PD-1) and programmed death-ligand 1 (PD-L1) checkpoint inhibitors have activity in a proportion of patients with advanced bladder cancer, strongly predictive and prognostic biomarkers are still lacking. In this study, we evaluated PD-L1 protein expression on circulating tumor cells (CTCs) isolated from patients with muscle invasive (MIBC) and metastatic (mBCa) bladder cancer and explore the prognostic value of CTC PD-L1 expression on clinical outcomes.

Methods

Blood samples from 25 patients with MIBC or mBCa were collected at UCSF and shipped to Epic Sciences. All nucleated cells were subjected to immunofluorescent (IF) staining and CTC identification by fluorescent scanners using algorithmic analysis. Cytokeratin expressing (CK)⁺ and (CK)^-CTCs (CD45^-, intact nuclei, morphologically distinct from WBCs) were enumerated. A subset of patient samples underwent genetic characterization by fluorescence in situ hybridization (FISH) and copy number variation (CNV) analysis.

Results

CTCs were detected in 20/25 (80 %) patients, inclusive of CK⁺ CTCs (13/25, 52 %), CK^-CTCs (14/25, 56 %), CK⁺ CTC Clusters (6/25, 24 %), and apoptotic CTCs (13/25, 52 %). Seven of 25 (28 %) patients had PD-L1⁺ CTCs; 4 of these patients had exclusively CK^-/CD45^-/PD-L1⁺ CTCs. A subset of CTCs were secondarily confirmed as bladder cancer via FISH and CNV analysis, which revealed marked genomic instability. Although this study was not powered to evaluate survival, exploratory analyses demonstrated that patients with high PD-L1⁺/CD45^-CTC burden and low burden of apoptotic CTCs had worse overall survival.

Conclusions

CTCs are detectable in both MIBC and mBCa patients. PD-L1 expression is demonstrated in both CK⁺ and CK^-CTCs in patients with mBCa, and genomic analysis of these cells supports their tumor origin. Here we demonstrate the ability to identify CTCs in patients with advanced bladder cancer through a minimally invasive process. This may have the potential to guide checkpoint inhibitor immune therapies that have been established to have activity, often with durable responses, in a proportion of these patients.

Background

While programmed death 1 (PD-1) and programmed death-ligand 1 (PD-L1) checkpoint inhibitors have activity in a proportion of patients with advanced bladder cancer, strongly predictive and prognostic biomarkers are still lacking. In this study, we evaluated PD-L1 protein expression on circulating tumor cells (CTCs) isolated from patients with muscle invasive (MIBC) and metastatic (mBCa) bladder cancer and explore the prognostic value of CTC PD-L1 expression on clinical outcomes.

Methods

Blood samples from 25 patients with MIBC or mBCa were collected at UCSF and shipped to Epic Sciences. All nucleated cells were subjected to immunofluorescent (IF) staining and CTC identification by fluorescent scanners using algorithmic analysis. Cytokeratin expressing (CK)⁺ and (CK)^-CTCs (CD45^-, intact nuclei, morphologically distinct from WBCs) were enumerated. A subset of patient samples underwent genetic characterization by fluorescence in situ hybridization (FISH) and copy number variation (CNV) analysis.

Results

CTCs were detected in 20/25 (80 %) patients, inclusive of CK⁺ CTCs (13/25, 52 %), CK^-CTCs (14/25, 56 %), CK⁺ CTC Clusters (6/25, 24 %), and apoptotic CTCs (13/25, 52 %). Seven of 25 (28 %) patients had PD-L1⁺ CTCs; 4 of these patients had exclusively CK^-/CD45^-/PD-L1⁺ CTCs. A subset of CTCs were secondarily confirmed as bladder cancer via FISH and CNV analysis, which revealed marked genomic instability. Although this study was not powered to evaluate survival, exploratory analyses demonstrated that patients with high PD-L1⁺/CD45^-CTC burden and low burden of apoptotic CTCs had worse overall survival.

Conclusions

CTCs are detectable in both MIBC and mBCa patients. PD-L1 expression is demonstrated in both CK⁺ and CK^-CTCs in patients with mBCa, and genomic analysis of these cells supports their tumor origin. Here we demonstrate the ability to identify CTCs in patients with advanced bladder cancer through a minimally invasive process. This may have the potential to guide checkpoint inhibitor immune therapies that have been established to have activity, often with durable responses, in a proportion of these patients.

Background: Lung cancer treatment has become increasingly dependent upon invasive biopsies to profile tumors for personalized therapy. Recently, tumor expression of programmed death-ligand 1 (PD-L1) has gained interest as a potential predictor of response to immunotherapy. Circulating biomarkers present an opportunity for tumor profiling without the risks of invasive procedures. We characterized PD-L1 expression within populations of nucleated cells in the peripheral blood of lung cancer patients in hopes of expanding the role of liquid biopsy in this setting.Methods: Peripheral blood samples from a multi-institutional prospective study of patients with clinical diagnosis of lung cancer were subjected to cytomorphometric and immunohistochemical evaluation using single-cell, automated slide-based, digital pathology. PD-L1 expression was determined by immunofluorescence.Results: PD-L1 expression was detected within peripheral circulating cells associated with malignancy (CCAM) in 26 of 112 (23%) non-small cell lung cancer patients. Two distinct populations of nucleated, nonhematolymphoid, PD-L1-expressing cells were identified; cytokeratin positive (CK+, PD-L1+, CD45^-) and cytokeratin negative (CK-, PD-L1+, CD45^-) cells, both with cytomorphometric features (size, nuclear-to-cytoplasm ratio) consistent with tumor cells. Patients with >1.1 PD-L1(+) cell/mL (n = 14/112) experienced worse overall survival than patients with ≤1.1 PD-L1(+) cell/mL (2-year OS: 31.2% vs. 78.8%, P = 0.00159). In a Cox model adjusting for stage, high PD-L1(+) cell burden remained a significant predictor of mortality (HR = 3.85; 95% confidence interval, 1.64-9.09; P = 0.002).Conclusions: PD-L1 expression is detectable in two distinct cell populations in the peripheral blood of lung cancer patients and is associated with worse survival.Impact: These findings could represent a step forward in the development of minimally invasive liquid biopsies for the profiling of tumors. Cancer Epidemiol Biomarkers Prev; 26(7); 1139-45. ©2017 AACR.