PD-L1 Expression In Circulating Tumor Cells Research Articles

Programmed Death Ligand 1 Expression in Circulating Tumor Cells as a Predictor and Monitor of Response to Atezolizumab plus Bevacizumab Treatment in Patients with Hepatocellular Carcinoma.

There remains no reliable biomarker of therapeutic efficacy in hepatocellular carcinoma (HCC) for the PD-L1 inhibitor atezolizumab and bevacizumab (Atezo/Bev). Circulating tumor cells (CTCs) enable the serial collection of living tumor cells. Pre-treatment and serial CTC gene expression changes and tumor histology were evaluated to identify predictors of response to Atezo/Bev. Peripheral blood from 22 patients with HCC treated with Atezo/Bev and 24 patients treated with lenvatinib was serially collected. The RNA expression in CTCs was analyzed using qRT-PCR. Higher PD-L1 expression in pre-treatment CTCs was associated with response and improved prognosis with Atezo/Bev treatment, but not with lenvatinib. There was no correlation between PD-L1 expression in CTCs and that in liver tumor biopsy specimens scored using imaging software. Furthermore, PD-L1 RNA expression in CTCs was dynamically altered by Atezo/Bev, decreasing during effective response and increasing upon progression. CTC-derived RNA collected during Atezo/Bev indicates that patients with higher PD-L1 expression in CTCs at baseline were 3.9 times more responsive to treatment. Therefore, PD-L1 RNA levels in CTCs are an accurate response predictor and may be a monitorable biomarker that changes dynamically to reflect the response during Atezo/Bev treatment.

Abstract 7488: Detection of PD-L1 mRNA expression in circulating tumor cells (CTCs) from patients with metastatic NSCLC, HNSCC and melanoma using a novel highly sensitive commercially available CE-IVD kit

Abstract Background: We have previously reported the development of an RT-qPCR assay for PD-L1 expression in CTCs (Strati et al, Ann Oncol, 2017). This assay is now commercially available as Oncolipsy PD-L1 kit (CE-IVD, Pharmassist, Greece) and we report here it’s application in size-based enriched CTCs from patients with metastatic NSCLC, HNSCC and melanoma, before and during PD-1/PD-L1 blockade therapies. Patients and Methods: The Oncolipsy PD-L1 kit was analytically validated using Mini-Genes as positive controls (IDT, USA). The analytical sensitivity was tested by spiking known numbers of H1975 cells in peripheral blood (PB) samples from healthy donors (HD). In total 95 patients were enrolled in the study: 69 patients with metastatic NSCLC, 19 patients with metastatic HNSCC and 7 patients with metastatic melanoma. 40 PB samples from HD were used as a control group. 10 ml of PB was collected at baseline (V0), two months after immunotherapy (V1) and at the time of disease progression (PD) or 18 months after initiation of immunotherapy (V2). PD-L1 and B2M transcripts were quantified in cDNAs obtained from CTCs isolated with the size-based PARSORTIX (ANGLE, UK) device. A sample was considered as CTC-positive when cDNAs were positive for CK-8, CK-18 or CK-19 expression. Results: Spiking experiments have shown that 10 H1975cells/10ml PB can be detected using the kit. In CTC-positive samples, overexpression of PD-L1 was detected in 15/58(25.8%) patients at V0, in 6/34(17.6%) patients at V1 and in 3/11(27.3%) at V2. Patients with elevated expression of PD-L1 at V1 in respect to V0 and detectable CTCs had longer PFS (18.5mo vs. 13.2mo, p=0.033) and longer OS (24.8mo vs. 12.8mo, p = 0.071) after initiation of immunotherapy. Patients with elevated PD-L1 at V1 compared to V0 benefited from immunotherapy, with a longer DFS (17.8mo vs. 13.4mo) and OS (29.4mo vs. 26.9mo). Conclusions: The commercially available Oncolipsy PD-L1 kit can be used for the quantification of PD-L1 transcripts in CTCs. Expression of PD-L1 in size-based enriched CTCs can predict response to PD-1/PD-L1 blockade therapy. Acknowledgements: This study has been financially supported by the European Union and Greek national funds through the Operational Program Competitiveness, Entrepreneurship and Innovation, under the call RESEARCH - CREATE - INNOVATE (project code: T1RCI-02935). Trial registration: ClinicalTrials.gov Identifier: NCT04490564 Citation Format: Areti Strati, Martha Zavridou, Stavroula Smilkou, Victoria Tserpeli, Eleni Tzanikou, Dimitra Stergiopoulou, Eleni Efthimiadou, Emily Tsaroucha, Amanda Psyrri, Aggeliki Sfika, Evangelos Bournakis, Ioanna Balgkouranidou, Stylianos Kakolyris, Ioannis Boukovinas, Christos Papadimitriou, Loukas Kaklamanis, Ioanna Koukli, Evi Lianidou. Detection of PD-L1 mRNA expression in circulating tumor cells (CTCs) from patients with metastatic NSCLC, HNSCC and melanoma using a novel highly sensitive commercially available CE-IVD kit [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 7488.

P37.10 PD-L1 Expression in Lymphovascular Tumor Emboli in Lung Adenocarcinoma

PD-L1 expression by immunohistochemistry is the most widely used predictive biomarker of response to immune checkpoint inhibitors. Compared with PD-L1 expression in tumor tissue, the characteristics of PD-L1 expression in circulating tumor cells (CTCs) are less well understood. A major obstacle to study PD-L1 expression in CTCs is the lack of standardized method for PD-L1 expression detection in CTCs. All of the USFDA-approved standard PD-L1 assays are intended to use in tissue sections only, and not applicable to CTCs isolated from peripheral blood.

Development and Analytical Validation of a Reverse Transcription Droplet Digital PCR (RT-ddPCR) Assay for PD-L1 Transcripts in Circulating Tumor Cells.

PD-L1, an immune checkpoint protein, is an important biomarker for monitoring cancer patients during the administration of cancer immunotherapy. Droplet digital PCR (ddPCR), is a highly sensitive and accurate tool for the quantification of cancer biomarkers in liquid biopsy. We report the development and analytical validation of a novel duplex RT-ddPCR assay for the simultaneous quantification of PD-L1 and hypoxanthine phosphoribosyltransferase (HPRT) (used as reference gene) transcripts in circulating tumor cells (CTCs). RT-ddPCR experimental conditions were first optimized and the assay was analytically validated using synthetic standards and the BB49 and SCC47 cancer cell lines. The developed assay was further applied in 71 peripheral blood (PB) samples from head and neck squamous cell carcinoma (HNSCC) patients and 20 PB samples from healthy donors. PD-L1 and HPRT transcripts were quantified in cDNAs derived from CTCs isolated by a size-dependent microfluidic device. The developed RT-ddPCR assay was directly compared to RT-qPCR using 71 identical patient cDNA samples. Analytical sensitivity was 0.64 copies/μL, while estimation of intra- and interassay variation revealed a high reproducibility (within-run CV%:4.7-23%; between-run CV%:13%). Using the developed RT-ddPCR assay 33/71(46.5%) HNSCC patients' samples were found positive for PD-L1 expression in CTCs, while by using RT-qPCR fewer samples (23/71, 32.4%) were positive (concordance: 55/71, 77.5%). The developed RT-ddPCR assay for PD-L1 in CTCs is highly sensitive, specific, and reproducible; additionally, it offers improved diagnostic sensitivity over RT-qPCR. The clinical utility of the assay should be prospectively evaluated for the real-time monitoring of CTCs of cancer patients under immunotherapy.

Circulating Tumor Cells as a Screening and Diagnostic Marker for Early-Stage Non-Small Cell Lung Cancer.

BackgroundCirculating tumor cells (CTCs) have become potential diagnostic biomarker for several types of cancer, including lung cancer. In this study, we aim to determine whether CTCs detected by CellCollector can be used for early-stage diagnosis of lung cancer.MethodsIn this study, we recruited 64 volunteers, among whom 44 were suspected lung cancer patients requiring surgical treatment and 20 were healthy volunteers. We simultaneously analyzed PD-L1 expression in CTCs isolated using the GILUPI CellCollector and copy number variation by next-generation sequencing (NGS).ResultsWe enrolled a total of 44 patients with suspected lung cancer who required surgery and 20 healthy volunteers. The patients were classified into 4 groups based on their pathological results: benign disease, in situ cancer, microinvasive, and invasive. The CTCs detection rate for each group was 10.00% (1/10), 45% (5/11), 50% (7/14), and 67% (6/9), respectively. Among the patients with lung cancer, the CTCs detection rate increased with disease progression. The rate of CTCs positivity was 52.94% (18/34) in patients who were diagnosed with lung cancer by pathology and 10% (1/10) in patients with benign disease. CTCs were not detected in the control group. The area under the receiver operating characteristic (ROC) curve, a measure for distinguishing patients with primary lung cancer, was 0.715 (95% CI 0.549–0.880, P=0.041). The sensitivity and specificity of the in vivo CTCs detection strategy for the diagnosis of early-stage lung cancer were 52.94% and 90%, respectively. CTCs were associated with clinical pathology but not with the size and location of the nodules.ConclusionCTCs isolation using the CellCollector in vivo detection method might be effective for distinguishing between benign and malignant nodules and may be used for early-stage diagnosis of lung cancer.

PD-L1 Expression in Circulating Tumor Cells Increases during Radio(chemo)therapy and Indicates Poor Prognosis in Non-small Cell Lung Cancer

Preclinical studies demonstrated that radiation up-regulates PD-L1 expression in tumor cells, providing a rationale for combining PD-1/PD-L1 inhibitors with radiation. However this has not been validated in patients with non-small cell lung cancer due to the difficulty to obtain serial biopsies. Measuring PD-L1 expression in circulating tumor cells (CTCs), may allow real-time monitoring of immune activation in tumor. In this study, whole blood from non-metastatic NSCLC patients was collected before, during, and after radiation or chemoradiation using a microfluidic chip. PD-L1 expression in CTCs was assessed by immunofluorescence and qPCR and monitored through the course of treatment. Overall, PD-L1(+) CTCs were detected in 25 out of 38 samples (69.4%) with an average of 4.5 cells/ml. After initiation of radiation therapy, the proportion of PD-L1(+) CTCs increased significantly (median 0.7% vs. 24.7%, P < 0.01), indicating up-regulation of PD-L1 in tumor cells in response to radiation. In addition, patients positive for PD-L1 (≥5% of CTCs positive for PD-L1) at baseline had shorter PFS. Gene expression analysis revealed that higher levels of PD-L1 were associated with poor prognosis. Therefore, CTCs can be used to monitor dynamic changes of PD-L1 during radiation therapy which is potentially prognostic of response to treatment.

Abstract 5697: Simultaneous assessment of PD-L1 and IFR1 expression on breast cancer circulating tumor cells

Abstract Background: There is a need for noninvasive predictive biomarkers of response to anti-PD1/PDL1 therapies. Assessment of circulating tumor cells (CTCs) is a rational approach to noninvasive sampling of tumors to understand the potential response or nonresponse to anti-PD1/PD-L1 therapies. IFN-gamma signals through the JAK/STAT cascade to induce PD-L1 via the Interferon Regulatory Factor-1 (IRF1) transcription factor, and is a potent inducer of PD-L1 expression in tumor cells. Recent studies have shown that low or absent IRF1 expression can identify melanomas that lack IFN-gamma responsiveness, and that IRF1 can have a higher predictive value of response to anti-PD1/PD-L1 therapy than PD-L1 itself. Using CTC models and the RareCyte platform, we developed a multiparameter assay that allows simultaneous PD-L1 and IRF1 assessment after CTC identification. We used this assay to better understand the relationship between IRF1 and PD-L1 expression in CTC model systems and applied this assay to blood samples from breast cancer patients. Materials and Methods: Peripheral blood from normal donors or cancer patients under an IRB-approved protocol was collected into RareCyte blood collection tubes. PD-L1(+) and PD-L1(-) CTC models were created by culturing A549 overnight with or without 10ng/mL INF-gamma. The A549 cells were spiked into normal donor blood and buffy coats isolated from 7.5mL of blood by AccuCyte® separation and spread onto slides. MDA-MB-231 cells were used as a model to represent breast cancer cells that express PD-L1 in the absence of IFN-gamma signaling. Slides were stained with a 6-marker panel that included antibodies to pan-cytokeratin (CK), EpCAM, CD45, PDL1, IRF1, and a nuclear dye on the Leica Bond Rx auto-stainer. Slides were scanned with CyteFinder® and CTCs identified by CK and/or EPCAM positivity and negative CD45 staining. Confirmed CTCs were then assessed for expression of PD-L1 and IRF1; cellular compartment was recorded for IRF1 staining. Results: Nuclear expression of IRF1 correlated with PD-L1 expression in IFN-gamma stimulated A549 cells. Unstimulated MDA-MB-231 cells expressed high levels of PD-L1, but this did not correlate with nuclear IRF1 expression. Breast cancer patient samples were identified having CTCs that expressed PD-L1. A PDL1 mean fluorescence intensity (MFI) threshold was set at the upper 95% CI of unstimulated A549 cells to define “high” and “low” PD-L1 expression in the patient samples. The population of high-PD-L1 CTCs had higher nuclear IRF1 MFI than low-PD-L1 CTCs, with mean fold-increase of 2.7 (range from 1.4 to 3.3). Conclusions: Nuclear IRF1 expression correlates with PD-L1 expression in breast cancer CTCs. Using CTCs to evaluate expression of IRF1 and PD-L1 together with assessment of CTC mutational load could be a powerful noninvasive assessment of response to anti-PD1/PD-L1 therapies. Citation Format: Lance U'Ren, Nolan Ericson, Ryan Huston, Elisabeth Mahen, VK Gadi, C. Anthony Blau, Eric Kaldjian. Simultaneous assessment of PD-L1 and IFR1 expression on breast cancer circulating tumor cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 5697.

Abstract 1589: Presence of PD-L1+ tumor cells in tumor tissue and blood significantly associated with T/N stage and lymphovascular invasion in colorectal cancer patients

Abstract Background: PD-L1 assessment on tumor tissue is considered as an important predictive marker for anti-PD-1 treatments and has been approved by FDA as companion diagnostics for immunocheckpoint antagonists. Nonetheless, it remains challenging because of the dynamic nature and heterogeneity of PD-L1 expression, and the availability of tumor tissue. Measurement of circulating tumor cells (CTC) serves a form of liquid biopsy and tumor biomarker due to its non-invasiveness and real-time assessment. Here, we analyzed and compared PD-L1 expression in CTCs and tumor cells harvested from surgical specimen from colorectal cancer (CRC) patients. Methods: Mesenteric vein blood (MVB) samples were collected into K2EDTA-containing vacationer tubes and subsequently performed CTC enumeration and PD-L1 analysis by MiSelect R system. Tumor tissues were homogenized via collagenase enzyme digestion to single cell suspension before subjected to MiSelect R analysis. Tumor cells and CTCs are defined as EpCAM+, cytokeratin+ and CD45- with intact nuclei. PD-L1 analysis was performed by anti-PD-L1 primary antibody (clone 28-8). Tumor cells, CTCs and PD-L1+ tumor cells were counted and compared among different clinicopathological parameters. Results: MVB sample were collected intraoperatively from 116 CRC patients across various stages (stage I: 24, II: 38, III:42 and IV:12). CTC presence correlated with T stage and CEA level (P = 0.018 and 0.049, respectively); furthermore, PD-L1+ CTC presence significantly correlated with clinical stage, N stage, microscopic blood vascular and lymphatic vascular invasion (P = 0.0017, 0.013, 0.014 and 0.004, respectively). The percentage of PD-L1+ tumor cells showed positive correlation between tumor tissue and CTC, and both correlated with tumor stage. Conclusion: PD-L1+ cell was detectable in both tumor tissue and CTC in CRC patients and its percentage increased as disease progressed, which might reflect escape of tumor cell from immune surveillance. The percentage of PD-L1 expression on CTCs correlated with that in paired tumor tissues. On the other hand, the expression of PD-L1 on CTC was significantly associated with blood and lymphatic vessel invasion, which might be one of the potential mechanisms for local and distant metastasis of cancer. Further investigation is warranted to better understand the clinical significances of PD-L1-expression on CTCs and its potential utility as prognostic biomarker for cancer immunotherapy. Citation Format: Ju-Yu Tseng, Chwen-Cheng Chen, Yen-Ru Chen, Chia-Ying Lee, Chun-Chi Lin, Shin-Hang Wang, Tzu-Chao Hung, Hong-Ling Wang, Yi Chung, Yen-Lun Tseng, Mu-Yi Chen, Jeng-Kai Jiang. Presence of PD-L1+ tumor cells in tumor tissue and blood significantly associated with T/N stage and lymphovascular invasion in colorectal cancer patients [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 1589.

2371 Validation of a novel PD-L1 assay for bladder cancer circulating tumor cells

OBJECTIVES/SPECIFIC AIMS: Bladder cancer patients being considered for immune checkpoint blockade are often judged on immunohistochemical staining for the checkpoint target protein PD-L1 in the original surgery or biopsy sample. However, sampling error or the clinical evolution of most patients’ cancer can render the original PD-L1 assessment no longer accurate. In contrast, circulating tumor cells (CTCs) allow serial noninvasive sampling of the current tumor status throughout a patient’s clinical course including those with the highest metastatic potential. We therefore sought to develop a method for quantifying PD-L1 expression in CTCs towards addressing inherent limitations of current UC management. METHODS/STUDY POPULATION: This work utilizes both cancer cell lines as well as patient samples. Positive and negative control cancer cell lines were assessed via “industry standard” antibodies for PD-L1 expression via Western blots and immunofluorescence, and a threshold-based method was developed for reliable quantification. PDL-1 expression was additionally verified via interferon-mediated up-regulation. CTCs isolated from bladder cancer patient samples via a density centrifugation method were then assessed for PD-L1 via the same antibodies. RESULTS/ANTICIPATED RESULTS: We will show preliminary preclinical and clinical data that validates the sensitivity and specificity of our assay. A case study will be presented that illustrate the potential useful of the novel approach we describe and which should be complementary to current clinical practices. In a patient with metastatic bladder cancer, this method effectively detected the PD-L1 expression in CTCs taken at a time coincident to when the patient derived an excellent response to the PD-L1 checkpoint inhibitor Pembrolizumab. DISCUSSION/SIGNIFICANCE OF IMPACT: This work highlights the potential utility of CTCs in the management of bladder cancer. It may be the case that this assay in conjunction with current methods of patient selection for immunotherapy may allow for better response prediction than either method alone.

PD-L1 expression in circulating tumor cells of advanced non-small cell lung cancer patients treated with nivolumab

BackgroundInhibitors of the PD-1/PD-L1 immune checkpoint have become a standard of care in non-small cell lung cancer (NSCLC). Patient selection, currently based on PD-L1 expression on tumor tissue, is limited by its temporal and spatial heterogeneity. We hypothesized that liquid biopsy with PD-L1 analysis on circulating tumor cells (CTCs) might overcome this limitation. MethodsBlood samples were prospectively collected from patients with advanced NSCLC before nivolumab treatment and at the time of progression. CTCs were isolated using a cell size-based technology. PD-L1 expression was assessed by immunofluorescence on CTCs and immunohistochemistry on tissue biopsies. Results113 specimens from 96 patients were collected. Baseline PD-L1 expression could be assessed on 72% and 93% of tissue and CTC, respectively. CTCs were more frequently found to be PD-L1 positive than tissue (83% vs. 41%) and no correlation was observed between tissue and CTC PD-L1 expression (r = 0.04, p = 0.77). Pre-treatment high CTC number was associated with increased risk of death and progression (HR1.06, p = 0.03 for OS; HR1.05, p = 0.02 for PFS). The presence of pre-treatment PD-L1+CTC was not significantly correlated with outcomes but a higher baseline PD-L1+ CTC number (≥1%) was observed in the “non-responders” group (PFS <6 months) (p = 0.04) and PD-L1+CTC were seen in all patients at progression. ConclusionAssessment of PD-L1 expression in CTCs is feasible and CTCs are more often positive than in tissue. Pre-treatment PD-L1+CTCs are associated with bad prognosis in patients treated with PD-1 inhibitors.