CD45 Depletion Research Articles

Changes of circulating tumor cells expressing CD90 and EpCAM in early-phase of atezolizumab and bevacizumab for hepatocellular carcinoma

Circulating tumor cells (CTCs) are noninvasive biomarkers that can indicate the therapeutic response and prognosis. The study aimed to investigate the cellular characteristics of CTCs focusing on monitoring during atezolizumab and bevacizumab (Atezo-Bev) therapy in patients with hepatocellular carcinoma (HCC). Peripheral blood samples were collected from 10 healthy controls and 40 patients with HCC. CTCs enriched using RosetteSep™ Human CD45 depletion cocktail were analyzed by multiparametric flow cytometry. CTC isolation was based on PanCK(+)CD45(−) cells, and CTCs exhibiting markers CD90, CD133, EpCAM, or vimentin. The total number of CTCs and the number of CTCs expressing CD90, CD133, EpCAM, and vimentin were correlated with the BCLC stage of HCC. The change in total CTC count accurately reflected the initial response to Atezo-Bev therapy. The numbers and mean fluorescence intensity of the CTC subsets expressing CD90 and EpCAM molecules decreased in patients with partial response/stable disease, and increased in patients with progressive disease and were markedly correlated with overall survival. CD90(+) and EpCAM(+) CTCs may be candidate biomarkers for the early prediction of the treatment response and the overall survival of patients with HCC receiving Atezo-Bev therapy.

Molecular profiling using next generation sequencing with high-purity enrichment of circulating tumor cells.

e15054 Background: In the field of liquid biopsy, coupled with next generation sequencing (NGS), circulating tumor cells (CTCs) and circulating tumor DNA (ctDNA) can serve as an alternative substrate to tumor tissue for mutation detection and companion diagnostic purpose. Emerging evidence suggests that complementary sequential CTCs and ctDNA profiling offers promising benefits for prognosis and treatment. In contrast to well-established ctDNA extraction methods, the process of isolation of intact CTCs for NGS analysis has not yet been fully elucidated. Here we show that isolation and harvesting of viable CTCs using CytoGen's Smart Biopsy™ System achieves high CTC purity for NGS analysis in breast cancer and non-small cell lung cancer (NSCLC). Methods: Targeted panel sequencing for CTC was developed to detect representative mutated genes using the Oncomine™ Comprehensive Assay Plus (501 genes) and the Oncomine™ Pan-Cancer Cell-Free Assay (52 genes). Analytical validation was performed with breast cancer cells MDA-MB-231 (50 to 5 cells) and NSCLC cells H358 (10 and 5 cells) which were spiked into 5 mL of healthy blood to mimic CTCs. CytoGen's Smart Biopsy™ System was applied to isolate the CTCs. Subsequently, CD45 depletion and whole genome amplification were performed. Sequencing was performed on Ion S5™ XL Systems using an Ion 550™ chip kit. The sequencing data were then analyzed using the Ion Torrent software with the default configurations. Results: As a result of targeted panel sequencing with Oncomine™ Comprehensive Assay Plus, representative breast cancer mutation genes (BRAF, KRAS, TP53, NF2 and NOTCH3) were detected in at least 20 MDA-MB-231 cells. In the results using Oncomine™ Pan-Cancer Cell-Free Assay, mutated genes (BRAF, KRAS and TP53) were successfully detected in as few as 5 MDA-MB-231 cells and, in addition, KRAS mutation was detected even with only 5 H358 cells. These results suggest that CytoGen's Smart Biopsy™ System could be applied to detect representative mutated genes in clinical breast cancer and NSCLC patient samples with detection technology that require high sensitivity and specificity. Conclusions: Molecular analysis of CTCs is a challenge since CTCs are very rare in the blood and there are technical limitations in isolating CTCs with sufficient purity for NGS analysis. In this study, we developed the process for NGS analysis on very small number of high-purity CTCs using CytoGen's Smart Biopsy™ System. Taken together with the results from our ongoing clinical trials, this process could provide information for the diagnosis and appropriate treatment of breast cancer and NSCLC patients.

Abstract 3691: Molecular profiling using next generation sequencing with high purity enrichment of circulating tumor cells

Abstract Purpose: In the area of liquid biopsy, coupled with next generation sequencing (NGS), circulating tumor cells (CTCs) and circulating tumor DNA (ctDNA) can serve as an alternative substrate to tumor tissue for mutation detection and companion diagnostic purpose. Emerging evidence shows that complementary sequential CTCs and ctDNA profiling provide promising benefit for prognosis and treatment. In contrast to well-established ctDNA extraction methods, the process of isolating intact CTCs for NGS analysis is yet to be fully elucidated. Here, we show that the isolation and harvest of viable CTCs by CytoGen’s Smart Biopsy™ System enable the attainment of sufficiently high purity for NGS analysis in metastatic breast cancer. Methods: Targeted panel sequencing for CTC was developed to detect representative mutated genes, using the Oncomine™ Comprehensive Assay Plus (501 genes) and the Oncomine™ Pan-Cancer Cell-Free Assay (52 genes). Analytical validation was performed with breast cancer cells MDA-MB-231 (50 to 5 cells) which were spiked into 5 mL healthy blood, to mimic CTC. The CytoGen’s Smart Biopsy™ system was applied to isolate the CTC. Then, CD45 depletion and whole genome amplification were performed. Sequencing was carried out on Ion S5™ XL systems using an Ion 550™ Chip Kit. Subsequently, sequencing data was analyzed using Ion Torrent software with default configurations. Results: As a result of targeted panel sequencing using Oncomine™ Comprehensive Assay Plus, representative mutated genes (BRAF, KRAS, TP53, NF2 and NOTCH3) in breast cancer were detected in at least 20 MDA-MB-231 cells. In the results using Oncomine™ Pan-Cancer Cell-Free Assay, mutated genes (BRAF, KRAS and TP53) were successfully detected even with only 5 MDA-MB-231 cells. These results suggest that clinical sample of breast cancer could be applied with promising sensitivity and specificity for detection of representative mutated genes. Conclusion: Molecular analysis of CTCs is a challenge since CTCs are very rare in the blood and there are technical limitations in isolating CTCs with sufficient purity. In this study, we developed the process for NGS analysis on very low number of CTCs with high purity, isolated using CytoGen's Smart Biopsy™ system. Overall, these process could provide information for diagnosis and appropriate treatment of metastatic breast cancer along with the results of our ongoing clinical trials. Citation Format: Sehwan Park, Jaesung Choi, Aejin Jeong, Hanbyeol Kim, Jiyoun Oh, Ingeon Jang, Hoin Kang, Jihyun Lee, Sehyung Pak, Min-Ji Song, Jung Won Kim, Hai-Chon Lee. Molecular profiling using next generation sequencing with high purity enrichment of circulating tumor cells [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 3691.

Detection of γ-OHPdG in Circulating Tumor Cells of Patients With Hepatocellular Carcinoma as a Potential Prognostic Biomarker of Recurrence

Background and AimsBlood-based biomarkers for hepatocellular carcinoma (HCC) and its recurrence is lacking. We previously showed that hepatic γ-hydroxy-1,N2-propano-2′-deoxyguanosine (γ-OHPdG), an endogenous DNA adduct derived from acrolein by lipid peroxidation, increased during hepatocarcinogenesis. Additionally, higher hepatic γ-OHPdG from HCC patients after surgery were strongly associated with poor survival (P < .0001) and recurrence-free survival (P = .007) (Fu Y et al., Hepatology, 2018). These findings suggest that γ-OHPdG is a potential prognostic biomarker for HCC and its recurrence. To attain the goal of using γ-OHPdG as a biomarker in future preventive and therapeutic trials, we developed a blood-based method to detect γ-OHPdG in circulating liver tumor cells from HCC patient blood. MethodsWe first established the specificity of anti-γ-OHPdG antibody by determining its dose-response in HepG2 cells treated with acrolein. Then, HepG2 cells in spiked blood of healthy volunteers (HV) and circulating tumor cells (CTCs) from 32 HCC patients were isolated using a RosetteSep™ CD45 Depletion Cocktail and Ficoll Paque. The HCC CTCs identified with anti-asialoglycoprotein receptor 1, a surface protein expressed solely in hepatocytes, were stained with an anti-γ-OHPdG antibody. The number of total HCC CTCs and γ-OHPdG-positive CTCs, as well as the staining intensity, were quantified using a MetaMorph software. As an initial effort toward its clinical application, we also evaluated γ-OHPdG in CTCs from these patients along with certain clinical features. ResultsThe γ-OHPdG antibody specificity was demonstrated by an acrolein concentration-dependent increase of γ-OHPdG-positive HepG2 cells and the intensity of γ-OHPdG staining. The recovery of HepG2 cells from spiked blood was ∼50-60%, and the positivity rate of CTCs in blood from 32 patients with advanced HCC was 97%. The MetaMorph analysis showed a wide variation among patients in total number of CTCs, γ-OHPdG positivity, and staining intensity. Statistical analysis revealed γ-OHPdG in CTCs of these patients appear to be associated with multifocality and poor differentiation. ConclusionsA blood-based method was developed and applied to HCC patients to evaluate the potential of γ-OHPdG in CTCs as a prognostic biomarker.

Detection of DNA adduct γ-OHPdG in circulating tumor cells (CTCs) and its use as a prognostic biomarker in patients with hepatocellular carcinoma (HCC).

594 Background: A blood-based biomarker to predict the risk of hepatocellular carcinoma (HCC), and its recurrence is needed. Previously, we showed that γ-OHPdG, a mutagenic DNA adduct formed by lipid peroxidation, in liver biopsies from HCC patients, as detected by IHC, is inversely associated with overall survival (p&lt;0.0001) and recurrence-free survival (p&lt;0.007) after surgical resection. This finding suggests that γ-OHPdG may serve as a prognostic biomarker of HCC and its recurrence. A non-invasive method to detect γ-OHPdG is needed. Liquid biopsy is preferred over tissue biopsy because it is non-invasive, allows repeated sampling, lower risk to patient, and lower medical care cost. We developed a non-invasive method for detecting and quantifying γ-OHPdG using CTCs from HCC patient. Methods: The method involved following steps. First, CTCs from blood samples were isolated using a RosetteSep CD45 Depletion Cocktail and Ficoll Paque-based method. Isolated cells were identified as liver CTCs using asialoglycoprotein receptor 1 (ASGPR1), a cell surface protein expressed solely on the surface of hepatic cells, and γ-OHPdG by immunocytochemistry. The percentage of ASGPR1 and γ-OHPdG-positive stained CTCs and γ-OHPdG staining intensity was quantified using Metamorph software. The staining intensities in livers CTCs will be compared with that found in liver biopsies by IHC. Results: To determine the sensitivity and specificity of testing -γ-OHPdG level in circulating tumor cells using an-anti γ-OHPdG antibody, we showed that the proportion of γ-OHPdG-positively stained cells and staining intensity increased in HepG2 liver cancer cells upon acrolein treatment at increasing concentration. Furthermore, when the HepG2 was used to spike blood of healthy volunteers’, the recovery rate of γ-OHPdG positivity was &gt; 50-60%. CTCs from 32 HCC patients, detected at a positivity rate of ~97%, were tested for γ-OHPdG levels using the method developed in this project. The clinical factors including demographics, risk factors, alpha-fetoprotein (AFP), HCC size, multifocality, vascular invasion, extrahepatic metastasis have been correlated with the levels of γ-OHPdG in CTCs. Conclusions: The CTC method developed for detecting and quantifying γ-OHPdG warrant validations as a prognostic biomarker for predicting HCC risk and recurrence in clinical trials.

New inflammatory indicators for cell-based liquid biopsy: association of the circulating CD44+/CD24− non-hematopoietic rare cell phenotype with breast cancer residual disease

BackgroundBreast cancer residual disease assessment in early-stage patients has been challenging and lacks routine identification of adjuvant therapy benefit and objective measure of therapy success. Liquid biopsy assays targeting tumor-derived entities are investigated for minimal residual disease detection, yet perform low in clinical sensitivity. We propose the detection of CD44−related systemic inflammation for the assessment of residual cancer.MethodsCirculating CD44+/CD45− rare cells from healthy, noncancer- and cancer-afflicted donors were enriched by CD45 depletion and analyzed by immuno-fluorescence microscopy. CD44+ rare cell subtyping was based on cytological feature analysis and referred to as morphological index. AUC analysis was employed for identification of the most cancer-specific CD44+ subtype.ResultsThe EpCam−/CD44+/CD24−/CD71−/CD45−/DNA+ phenotype alludes to a distinct cell type and was found frequently at concentrations below 5 cells per 5 mL in healthy donors. Marker elevation by at least 5 × on average was observed in all afflicted cohorts. The positive predicted value for the prediction of malignancy-associated systemic inflammation of a CD44+ rare cell subtype with a higher morphological index was 87%. An outlook for the frequency of sustained inflammation in residual cancer may be given to measure 78%.ConclusionThe CD44+ rare cell and subtype denotes improvement in detection of residual cancer disease and may provide an objective and alternative measure of disease burden in early-stage breast cancer.

Abstract 1264: CTC-based biomarker analysis and correlation with clinical response to PSMA-TRT in mCRPC

Abstract Prostate specific membrane antigen (PSMA)-targeted radionuclide therapies (PSMA-TRTs) is one of the most promising treatments for metastatic castration-resistant prostate cancer (mCRPC) upon progression on androgen receptor signaling inhibitors (ARPi). However, recent studies show that only about half of patients respond to PSMA-TRTs. Expression of PSMA on cancer cells via PET-imaging is of some use as a predictive biomarker of response to PSMA-TRTs, but there are patients with strong PSMA imaging who do not respond and some with poor PSMA imaging that do respond. PSMA imaging does not fully capture response predictability. Currently, the lack of predictive biomarkers of response limits optimal selection of the patient population most likely to benefit from to PSMA-TRTs. PSMA-TRTs radionuclides emit α or β radiations, which induce DNA damage in targeted cells, followed by immunogenic cell death (ICD), evidenced by calreticulin (CRT) plasma membrane accumulation. To investigate the role of PSMA expression and CRT membrane localization as a biomarker of response to PSMA-TRT, we isolated circulating tumor cells (CTCs) from patients with mCRPC, enrolled in a phase I dose escalation trial of 225Ac-J591 (NCT03276572). Thirty-two (32) patients were enrolled (median age 69. 5, range 52-89; PSA 149.1, range 4.8-7168); 78% and 68% of patients received ≥ 2 prior ARPi treatments or chemotherapy, respectively. 69% and 44% of patients experienced any or &amp;gt;50% PSA decline, respectively, with a median PFS of 5.1 months and median OS of 11.1 months. CTCs were isolated before treatment (baseline) from 30/32 of enrolled patients, and 12 weeks after treatment initiation (on-treatment) from 21/32 patients. Enriched CTCs via CD45 depletion were subsequently immunostained for cytokeratin (CK, standard epithelial marker), CD45 (standard marker to identify contaminating leukocytes), PSMA, calreticulin (CRT) and DAPI. CTCs were subjected to multiplex confocal microscopy and identified as CK+/CD45-/DAPI+ cells. For each patient, at each time point, the following parameters are scored: i) total number of CTCs, ii) number of PSMA+ CTCs, iii) number of CRT+ CTCs, iv) number of CTCs with plasma membrane CRT decoration. Percent of PSMA+ CTC at baseline and changes in percentage of CRT+ CTCs and CTCs with plasma membrane CRT decoration between baseline and on treatment timepoints are correlated with clinical response and time to progression upon 225Ac-J591 treatment. Our preliminary results indicate higher expression of plasma membranous CRT in on-treatment CTCs in responders (PFS&amp;gt;12 months; n= 39 CTCs from 3 patients) than in non-responders (PFS&amp;lt;3 months; n= 20 CTCs from 2 patients). Full analysis of CRT and PSMA expression is currently being completed. Citation Format: Giuseppe Galletti, Prerna Vatsa, Joseph R. Osborne, Neil H. Bander, David M. Nanus, Scott T. Tagawa, Paraskevi Giannakakou. CTC-based biomarker analysis and correlation with clinical response to PSMA-TRT in mCRPC [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 1264.

Abstract 6235: Detection of γ-OHPdG in circulating liver cells and its potential use as a prognostic biomarker in patients with hepatocellular carcinoma recurrence

Abstract Background: Primary liver cancer is the fourth most common cause of cancer-related death. Resection is the most widely used potentially curative treatment for patients with early-stage hepatocellular carcinoma (HCC). Recurrence within 2 years occurs in 30-50% of patients and is a major cause of mortality. A biomarker to predict the risk of HCC and its recurrence is urgently needed. Our studies showed that hepatic levels of γ-OHPdG, an endogenous adduct from lipid peroxidation, closely correlate with hepatocarcinogenesis in various independent animal models, including an obesity model. Importantly, we observed that higher levels of γ-OHPdG in liver specimens from HCC patients who underwent surgical resection were strongly associated with poor survival (p &amp;lt; 0.0001) as well as with poor recurrence-free survival (RFS) (p = 0.007). These finding suggest that γ-OHPdG may serve as a prognostic biomarker of HCC and its recurrence. A non-invasive method to detect γ-OHPdG is highly desirable. There is a surge of interest in using circulating liver cells (CLCs) from patient blood to diagnose cancer stages and predict treatment outcomes, metastatic potential, and recurrence. We developed a non-invasive method for detecting and quantifying γ-OHPdG using CLCs from patient blood. Method: The method was developed by following steps: (i) isolate CLCs from blood samples using a RosetteSep™ Human CD45 Depletion Cocktail and Ficoll Paque, (ii) detect γ-OHPdG by immunocytochemistry, (iii) quantify the images of γ-OHPdG-positive stained CLCs, and (iv) compare with that found in liver biopsies by immunohistochemistry. Results: To determine the specificity of the anti-γ-OHPdG antibody developed previously in our laboratory, we showed that the proportion of γ-OHPdG-positively stained cells and staining intensity increased in HepG2 liver cancer cells upon acrolein treatment. Liver cells were co-stained with asialoglycoprotein receptor 1 (ASGPR1), a cell surface protein expressed solely on the surface of hepatic cells. CLC γ-OHPdG was quantified using Metamorph (BioVision)-based journal software, developed to quantify the percentage of ASGPR1-stained cells that also stain positively, and with what level of intensity, for γ-OHPdG. We then applied this method to 35 HCC patients’ blood samples. This method detects the number of ASGPR1 stained CLC and determines the cellular localization of γ-OHPdG, allowing the quantification of γ-OHPdG in these cells. While the nuclear γ-OHPdG reflects DNA damage, adducts in both the nucleus and the cytosol will be evaluated against IHC staining using biopsies and other clinical endpoints, such as disease stage and recurrence. Conclusion: The CLC method descried above for detecting and quantifying γ-OHPdG can be potentially used to validate this adduct as a prognostic biomarker for predicting HCC risk and recurrence in clinical trials. Citation Format: Monika Aggarwal, Zizhao Zhu, Sophie Gould, Peter Johnson, Samira Beheshtian, Hongbin Fang, Bhaskar Kallakury, Susette C. Mueller, Aiwu Ruth He, Fung-Lung Chung. Detection of γ-OHPdG in circulating liver cells and its potential use as a prognostic biomarker in patients with hepatocellular carcinoma recurrence [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 6235.

Strategies for Isolating and Propagating Circulating Tumor Cells in Men with Metastatic Prostate Cancer.

Selecting a well-suited method for isolating/characterizing circulating tumor cells (CTCs) is challenging. Evaluating sensitive and specific markers for prostate cancer (PCa)-specific CTC identification and analysis is crucial. We used the CellCollector EpCAM-functionalized system (CC-EpCAM) and evaluated and developed a PCa-functionalized version (CC-PCa); we then compared CTC isolation techniques that exploit the physical and biological properties of CTCs. We established two cohorts of metastatic PCa patients (mPCa; 15 in cohort 1 and 10 in cohort 2). CTC cultivation experiments were conducted with two capturing methods (Ficoll and ScreenCell). The most sensitive detection rates and highest CTC counts were reached with the CC-PCa and ScreenCell system. Patients with ≥5 CTCs isolated with CC-EpCAM had an overall survival (OS) of 0.93 years, and patients with ≥5 CTCs isolated with CC-PCa had an OS of 1.5 years in cohort 1. Nevertheless, we observed the highest sensitivity and specificity for 24-month survival by the Ficoll with CD45 depletion and ScreenCell system with May-Grunwald Giemsa (MGG) staining. The EpCAM molecule is an essential factor related to OS for CTC isolation based on biological properties in mPCa patients. The best-suited CTC capture system is not limited to one characteristic of cells but adapted to downstream analysis.

Circulating erythroblast abnormality associated with systemic pathologies may indicate bone marrow damage.

Background: The circulating rare cell population is diverse and rich in diagnostic information. Its characterization and clinical exploitation by cell-based liquid biopsy is an ongoing research task. Bone marrow is one of the major contributors to the peripheral blood rare cell population and, consequently, determines individual rare cell profiles thus depending on bone marrow health status. Bone marrow damage has been associated with aggressive or late-stage systemic diseases and egress of various bone marrow cells into the blood circulation. The association of quantity and heterogeneity of circulating erythroblast with bone marrow damage is of particular interest. Methods: Circulating CD71high/CD45-/Hoechsthigh blast cells from healthy, noncancer- and cancer-afflicted donors were enriched by CD45 depletion and analyzed by immunofluorescence microscopy. Results: A new finding of aberrant and mitotic circulating erythroid-like cells that appear similar across blood donors afflicted with various systemic pathologies is reported. Further presented is a classification of said erythroblast-like cells in nine subcategories according to morphological differences between phenotypically similar cells. Conclusion: Aberrant and mitotic bone marrow-derived rare circulating erythroid-like cells can be detected in the blood of afflicted individuals but not in healthy donors, suggesting the cause of bone marrow damage.

Aberrant activation of the CD45-Wnt signaling axis promotes stemness and therapy resistance in colorectal cancer cells.

Rationale: Chemoradiation (CRT) is commonly used as an adjuvant or neoadjuvant treatment for colorectal cancer (CRC) patients. However, resistant cells manage to survive and propagate after CRT, increasing the risk of recurrence. Thus, better understanding the mechanism of resistant cancer cells is required to achieve better clinical outcomes.Methods: Here, we explored gene expression profiling of CRC patient tumors to identify therapy resistance genes and discovered that protein tyrosine phosphatase receptor type C (PTPRC), which encodes CD45, was increased in remnant tumor tissues after CRT and correlated with metastasis. Through multiple validations using patient tumors and CRC cell lines, we found for the first time the increase of CD45 expression in CRC (EpCAM+) epithelial cells surviving after CRT. Thus, we investigated the biological role and downstream events of CD45 were explored in human CRC cells and CRC mouse models.Results: Increased CD45 expression in cancer cells in pretreated primary tumors accounts for poor regression and recurrence-free survival in CRT-treated patients. High CD45 expression promotes CRC cell survival upon 5-fluorouracil or radiation treatment, while CD45 depletion sensitizes CRC cells to CRT. Intriguingly, CD45 is preferentially expressed in cancer stem-like cells (CSCs), as determined by spheroid culture and the expression of CSC markers, and is required for the distinct functions of CSCs, such as cancer initiation, repopulation, and metastasis. Mechanistically, CD45 phosphatase activity promotes Wnt transcriptional activity by stabilizing the β-catenin protein, which collectively enhances stemness and the therapy-resistant phenotype.Conclusions: Our results highlight a novel function of CD45 as a mediator of CRT resistance and provide a potential therapy strategy for CRC therapy.

Abstract 3384: Characterization of the tumor microenvironment and liquid biopsy in head and neck and non-small cell lung cancer

Abstract Metastasis in cancer patients is reflected by measurable levels of circulating tumor cells (CTCs) in the blood of cancer patients. CTCs represent cancer cells from the primary and metastatic sites, thereby providing a comprehensive representation of the tumor burden of an individual patient. Recent advancements have shown that PD-1/PD-L1 immune checkpoint therapies have durable responses in a number of solid tumor types. Our study was designed to use multiple CTC enrichment platforms for the capture of CTCs and novel culture formulations for the ex vivo expansion of CTCs. Head and Neck cancer (n=350) and lung cancer (n=150) patients were recruited to investigate the prognostic role of CTCs. In parallel, a subset of HNC tumors were profiled using the NanoString GeoMx Digital Spatial Profiling (DSP) technology using a 44-plex antibody cocktail. We evaluated multiple CTC isolation technologies (CellSearch, filtration, CD45 depletion, Spiral, Straight and novel microfluidic chip technology) using matched patient samples which showed that epitope-independent CTC isolation captured a greater proportion of CTCs. Molecular alterations present in the primary tissue were confirmed in the CTCs by 3D-DNA FISH (EGFR-amplification, ALK-translocations). In HNC, the presence of CTC clusters associated with the development of distant metastatic disease (P=0.0313). HNC CTC-positive patients had shorter progression free survival (Hazard ratio [HR]: 4.946; 95% [CI]:1.571-15.57; P=0.0063) and PD-L1-positive CTCs were found to be significantly associated with worse outcome ([HR]:5.159; 95% [CI]:1.011-26.33; P=0.0485). In a proof of principle study, we were able to demonstrate for the first time, short-term patient derived CTC cultures outside the patient's body and exome sequencing of CTCs cultures confirmed the presence of mutational signatures consistent with The Cancer Genome Atlas (TCGA). Spatial characterization of the tumor microenvironment revealed immune subsets predictive of outcome to immunotherapy. Comprehensive characterization of the tumor microenvironment and liquid biopsy allows for the recapitulation of the molecular diversity present within the tumor, understanding of the disease progression and testing of therapies. Citation Format: Arutha Kulasinghe, Joanna Kapeleris, Liz Kenny, Brett Hughes, Majid Warkiani, Ian Vela, Jean-Paul Thiery, Ken O'Byrne, Chamindie Punyadeera. Characterization of the tumor microenvironment and liquid biopsy in head and neck and non-small cell lung cancer [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 3384.

Abstract A03: Isolation, characterization, and expansion of circulating tumor cells in head and neck cancers

Abstract Metastasis in head and neck cancer (HNC) patients is reflected by measurable levels of circulating tumor cells (CTCs) in the blood of patients. CTCs represent cancer cells from the primary and metastatic sites, thereby providing a comprehensive representation of the tumor burden of an individual patient. Recent advancements have shown that PD-1/PD-L1 immune checkpoint therapies have durable responses in HNC. Our study was designed to use multiple CTC enrichment platforms for the capture of CTCs and novel culture formulations for the ex vivo expansion of CTCs. n=300 head and neck cancer patients were recruited to investigate the prognostic role of CTCs. We evaluated multiple CTC isolation technologies (CellSearch, filtration, CD45 depletion, inertial microfluidics) using matched patient samples, which showed that epitope-independent CTC isolation captured a greater proportion of CTCs. Molecular alterations present in the primary tissue were confirmed in the CTCs by 3D-DNA FISH (EGFR-amplification). The presence of CTC clusters was associated with the development of distant metastatic disease (P=0.0313). HNC CTC-positive patients had shorter progression-free survival (PFS) (Hazard ratio [HR]: 4.946; 95% confidence interval [CI]:1.571-15.57; P=0.0063) and PD-L1-positive CTCs were found to be significantly associated with worse outcome ([HR]:5.159; 95% [CI]:1.011-26.33; P=0.0485). In a proof-of-principle study, we were able to demonstrate for the first time short-term patient-derived CTC cultures outside the patient’s body from 7/18 HNC samples (4/7 HPV-positive). Recently, we have preliminary data that suggest that PD-L1 is frequently expressed on CTCs in HNC and an immunoscore may be able to identify patients likely to benefit from immunotherapy—a current unmet clinical need. Expanding CTCs outside the patient’s body allows for the recapitulation of the molecular diversity present within the tumor, understanding of the disease progression, and testing of therapies. Citation Format: Arutha Kulasinghe, Joanna Kapeleris, Liz Kenny, Majid Warkiani, Ian Vela, Jean Paul Thiery, Ken O’Byrne, Chamindie Punyadeera. Isolation, characterization, and expansion of circulating tumor cells in head and neck cancers [abstract]. In: Proceedings of the AACR-AHNS Head and Neck Cancer Conference: Optimizing Survival and Quality of Life through Basic, Clinical, and Translational Research; 2019 Apr 29-30; Austin, TX. Philadelphia (PA): AACR; Clin Cancer Res 2020;26(12_Suppl_2):Abstract nr A03.

Abstract 1333: Isolation, characterization and expansion of circulating tumor cells in solid cancers

Abstract Metastasis in cancer patients is reflected by measurable levels of circulating tumor cells (CTCs) in the blood of cancer patients. CTCs represent cancer cells from the primary and metastatic sites, thereby providing a comprehensive representation of the tumor burden of an individual patient. Recent advancements have shown that PD-1/PD-L1 immune checkpoint therapies have durable responses in a number of solid tumor types. Our study was designed to use multiple CTC enrichment platforms for the capture of CTCs and novel culture formulations for the ex vivo expansion of CTCs. Head and Neck cancer (n=300) and lung cancer (n=80) patients were recruited to investigate the prognostic role of CTCs. We evaluated multiple CTC isolation technologies (CellSearch, filtration, CD45 depletion, inertial microfluidics) using matched patient samples which showed that epitope-independent CTC isolation captured a greater proportion of CTCs. Molecular alterations present in the primary tissue were confirmed in the CTCs by 3D-DNA FISH (EGFR-amplification, ALK-translocations). In HNC, the presence of CTC clusters associated with the development of distant metastatic disease (P=0.0313). HNC CTC-positive patients had shorter progression free survival (PFS) (Hazard ratio [HR]: 4.946; 95% confidence internal [CI]:1.571-15.57; P=0.0063) and PD-L1-positive CTCs were found to be significantly associated with worse outcome ([HR]:5.159; 95% [CI]:1.011-26.33; P=0.0485). In a proof of principle study, we were able to demonstrate for the first time, short-term patient derived CTC cultures outside the patient’s body from 7/18 HNC samples (4/7 HPV-positive). Recently, we have preliminary data that suggests that PD-L1 is frequently expressed on CTCs in HNC and lung cancer and an immunoscore may be able to identify patients likely to benefit from immunotherapy-a current unmet clinical need. Expanding CTCs outside the patient’s body allows for the recapitulation of the molecular diversity present within the tumor, understanding of the disease progression and testing of therapies. Citation Format: Arutha Kulasinghe, Joanna Kapeleris, Liz Kenny, Majid Warkiani, Ian Vela, Jean-Paul Thiery, Ken O'Byrne, Chamindie Punyadeera. Isolation, characterization and expansion of circulating tumor cells in solid cancers [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 1333.

Abstract 454: Transferrin receptor identifies a novel circulating tumor cell population in patients with pancreatic cancer with a unique metastasis-associated molecular signature

Abstract Pancreatic Ductal Adenocarcinomas (PDAC) is the fourth most common cancer, expected to be the second leading cause of cancer mortality by 2030. Most PDAC patients are diagnosed with metastatic disease, with dismal prognosis of less than 1% 5-year survival. A major challenge in PDAC treatment is the absence of actionable molecular targets and the scarcity of available tumor tissue impeding molecular understanding of PDAC progression. Circulating Tumor Cells (CTCs) provide an easily accessible source for tumor tissue. CellSearchTM is the only FDA-cleared platform for CTC isolation based on positive selection of EpCAM+ cells. However, this method performs poorly in PDAC failing to show any prognostic relevance, likely due to EpCAM down-regulation during epithelial-to-mesenchymal transition (EMT), which precedes metastasis. To overcome this limitation, we tested Transferrin Receptor 1 (TfR) in the identification and isolation of CTCs from PDAC patients. TfR is a cell surface protein that mediates iron uptake, is overexpressed in multiple tumors with sustained expression throughout EMT, making it a suitable candidate for CTC isolation. To test this hypothesis, we first assessed TfR expression in a panel of 9 human PDAC cell lines, and we observed that TfR was expressed in 9/9 (100%) in contrast to 6/9 (66.7%) being EpCAM positive. No TfR expression was detected in healthy donor and PDAC patients’ PBMCs. To assess the performance of TfR in CTC identification and isolation from the peripheral blood of PDAC patients, we collected samples from 37 PDAC patients with stage 4 disease, enriched CTCs by negative CD45 depletion (RossetteSepTM) and subsequently stained live CTCs with TfR, EpCAM, and CD45. CTCs were defined as CD45- cells, positive for TfR+ or EpCAM+. We observed that the number of TfR+ CTCs (median: 148, range 2-4182) was significantly higher than EpCAM+ CTCs (median: 68, range 0-1552) within the same patient and across different patients (P-value 0.007). In addition, serial sampling at baseline (chemotherapy-naïve) and at disease progression from 5 PDAC patients revealed that TfR+CTC enumeration correlated with disease progression radiologically and/or CA19-9 (PDAC tumor marker) levels. In contrast, there was no correlation between the number of EpCAM+CTCs and clinical outcomes. RNA-Sequencing of isolated pools of TfR+ or EpCAM+ CTCs, followed by gene set enrichment analysis, revealed significant enrichment in oncogenic pathways, such as EMT, G2M, MYC and KRAS in TfR+ CTCs as compared with EpCAM+ CTCs. Taken together, our results reveal that TfR identifies a clinically relevant CTC subpopulation which correlates with disease progression and is molecularly distinct. Ongoing studies are focused on the function of TfR in disease progression and the potential use of TfR+ CTC molecular profiles in personalized PDAC treatment. Citation Format: Ahmed Halima, Jiaren Zhang, Giuseppe Galletti, Allyson J. Ocean, Paraskevi Giannakakou. Transferrin receptor identifies a novel circulating tumor cell population in patients with pancreatic cancer with a unique metastasis-associated molecular signature [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 454.

Abstract 5584: Circulating tumor cells in the peripheral blood and leukapheresis product of non-small cell lung cancer patients

Abstract Introduction The number of circulating tumor cells (CTC) isolated by the FDA approved CellSearch (CS) system in 7.5 mL of blood of metastatic and non-metastatic non-small cell lung cancer (NSCLC) patients is low or absent. Processing a larger blood volume could yield a greater amount of CTC from these patients. An approach to probe larger volumes is using diagnostic leukapheresis (DLA) where the blood can be split into its components after which selective harvesting of mononuclear cells (MNC) including CTC can take place. Methods Patients with histologically proven NSCLC who either have been recently diagnosed or are eligible for new therapy can participate in this ongoing study. Patients had DLA for MNC collection derived from one total body blood volume. Before and after DLA, whole blood was drawn in a CellSave blood collection tube and analyzed by CS. The obtained DLA product was divided for analysis. 2x108 cells (on average 2mL of DLA product, representing ~100mL of blood) was diluted in 7.5 mL buffer, and analyzed by CS. 9 mL of the DLA product (on average 1x109 cells) was fixed and depleted of leukocytes using the RosetteSep CD45 depletion kit (Stemcell Technologies), allowing processing of a larger volume, followed by CS analysis. Results of molecular characterization of the detected CTC is pending. Results So far, a total of 8 DLA's were performed in 7 stage 4 NSCLC patients. Sample collection took place before (t=0) and/or after (t=1) treatment. Patients underwent DLA in about 110 minutes, at this time an average of 4860 mL of blood was processed. No complications occurred, no post-procedure complaints were recorded. Similar DLA products with an average of 95 mL were obtained. CTC counts for these patient samples are shown in the table below. CTC counts blood and DLAPatientt=#CTC pre blood#CTC post blood#CTC DLA (~2mL, 200x106 cells)#CTC depleted DLA (~9mL, 900x106 cells)101n/a114113020203514602031000040n/a04495000166000029020114 Conclusion DLA is a novel method to collect CTC from the blood. DLA can result in the detection of CTC where the use of 7.5mL of blood is not sufficient. Additionally, DLA shows an increase of CTC yield in patients with CTC detected in 7.5 mL of blood. Citation Format: Kiki C. Andree, Menno Tamminga, Anouk Mentink, T Jeroen Hilterman, Harry J. Groen, Leon W. Terstappen. Circulating tumor cells in the peripheral blood and leukapheresis product of non-small cell lung 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 5584.

Microfluidic enrichment of plasma cells improves treatment of multiple myeloma.

Cytogenetic alterations form the basis for risk stratification for multiple myeloma (MM) and guide the selection of therapy; however, current pathology assays performed on bone marrow samples can produce false‐negatives due to the unpredictable distribution and rarity of MM cells. Here, we report on a microfluidic device used to facilitate CD45 depletion to enhance the detection of cytogenetic alterations in plasma cells (PCs). Bone marrow samples from 48 patients with MM were each divided into two aliquots. One aliquot was subjected to classic flow cytometry and fluorescent in situ hybridization (FISH). The other first went through CD45+ cell depletion, further enriched by microfluidic size selection. The enriched samples were then analyzed using flow cytometry and FISH and compared to those analyzed using the classic method only. Unlike the traditional method, the microfluidic device removed the CD45+ leukocytes and specifically selected PCs from the remaining white blood cells. Therefore, the microfluidic method (MF‐CD45‐TACs) significantly increased the percentage of CD38+/CD138+ cells to 37.7 ± 20.4% (P < 0.001) from 10.3 ± 8.5% in bone marrow. After the MF‐CD45‐TAC enrichment, the detection rate of IgH rearrangement, del(13q14), del(17p), and 1q21 gains, rose to 56.3% (P < 0.001), 37.5% (P < 0.001), 22.9% (P < 0.001), and 41.7% (P = 0.001), respectively; all rates of detection were significantly increased compared to the classically analyzed samples. In this clinical trial, this microfluidic‐assisted assay provided a precise detection of cytogenetic alterations in PCs and improved clinical outcomes.

Novel method for rapid enrichment of high purity circulating tumor cells (CTCs) for prostate cancer (PCa) gene expression profiling.

271 Background: CTC RNA analysis currently involves single cell recovery that is laborious and expensive, or alternatively lysis of preserved whole blood which yields RNA predominantly from leukocytes which vastly outnumber CTCs. To effectively characterize gene expression in large patient cohorts, new enrichment methodologies are needed that yield high purity CTC populations while preserving RNA integrity. Here we describe a simple yet robust method for enrichment of prostate CTCs for gene expression analysis. Methods: Blood was drawn with informed consent under an IRB-approved protocol. For initial optimization, CFSE-stained PCa cells were spiked into healthy blood and recovered using various combinations of 2 methods: microfluidic enrichment (Parsortix™ system) and CD45 depletion. For assay qualification, a prostate-specific multiplexed qRT-PCR gene expression panel was developed. Enrichment and gene expression were tested initially using PCa cell lines spiked into healthy blood, then metastatic castrate resistant prostate cancer (mCRPC) blood samples in parallel with CellSearch enumeration. Results: Optimal enrichment of live cells was achieved with CD45 depletion followed by microfluidic enrichment, resulting in an average spiked cell recovery of 30% and approximately 100 contaminating background leukocytes. Using this enrichment method, prostate specific genes were detectable by multiplexed qRT-PCR down to 25 cells spiked into 7.5 ml whole blood, and transcripts were not measurable in matched healthy blood controls. When applied to mCRPC patient blood containing CTCs by CellSearch, multiplexed qRT-PCR successfully detected prostate specific genes in all samples. Conclusions: We developed a novel enrichment method capable of rapidly and efficiently recovering live CTCs with high purity, free of magnetic beads and with very few background leukocytes. Captured cells yielded high-quality RNA with high sensitivity and specificity for prostate-specific transcripts. This approach is applicable to high throughput gene expression profiling assays and offers an alternative to laborious single cell recovery or non-cancer-specific whole blood fixation.

Biological significance of novel populations of prostate cancer circulating tumor cells (CTC).

315 Background: Decisions regarding selection and sequencing of treatment in advanced prostate cancer (PC) are often made empirically, without information on the PC molecular profile at the time of treatment. CTC provide real time information regarding contemporaneous biologically relevant cancer subtypes. Current assays often rely on EpCAM or other epithelial markers and may underestimate or exclude other CTC populations of clinical relevance. We devised a novel, simple, rapid and cheap method for isolation of CTC subsets including those without epithelial markers. Methods: Five mL of blood was processed as follows: CD45+ depletion (RosetteSep Human CD45 Depletion Cocktail, Stemcell Technologies); Ficoll centrifugation; antibody staining (mix of EpCAM-PE, PSMA-APC, N-cadherin-AF488, E-cadherin-PerCP/Cy5.5, CD45-APC-H7); Sytox Blue live/dead cell stain; followed by sorting (Becton Dickinson FACSAria Fusion cell sorter). Live cells were sorted into 4 categories denoted E, E+P, P, N (Table). N and P are not identifiable using existing conventional CTC techniques. CTC were assayed for prostate markers ( AR, FOLH1 (PSMA), KLK3 (PSA), TMPRSS2, AMACR) and malignant markers ( PTEN copy number) using digital droplet PCR. Results: Preliminary data are derived from 62 patient samples providing 47 sorted populations. Details are in the Table. Spiking experiments reliably identified as few as 10 CTC / 7.5 mL of blood. P cells were detected in all patients, often outnumbering sum of E and E+P; E and N were never co-expressed. P and N samples frequently expressed prostate markers. Only 1/27 showed no expression of any of these five prostate markers. PTEN deletion assays are in progress. Conclusions: “Unconventional” EpCAMneg CTC appear to be derived from PC and can be detected using broader cell selection criteria. These CTC may provide information on treatment resistance or escape mechanisms. [Table: see text]

Abstract 3785: Self-sorting microwells to isolate and expand single circulating tumor cells

Abstract Introduction: Circulating tumor cells (CTCs) can be isolated from blood and serve as a source of tumor material. Expansions of CTCs may permit functional treatment-efficacy tests in combination with genetics, epigenetics and proteomics screening. We present a fast workflow to isolate, capture, grow and image cells inside a self-sorting microwell chip using the VyCAP single cell isolation platform. After seeding single cells in a microwell chip, they can grow inside the chip or can be transferred to a tissue culture plate for clonal expansion or downstream analysis. Materials and methods: The Rosette-Sep™ Human CD45 depletion cocktail (Stem Cell Technologies) was used to enrich spiked cells from 1 ml of fresh EDTA blood. Cells were stained with CellTrace™ violet for tracking spiked cells, calcein for live cells, ethidium bromide for dead cells, α-CD45-PERCP as negative marker and α-EpCAM-AF647 as positive marker. After density gradient separation using Ficoll-Paque™, the cell suspension was measured and quantified using flow cytometry for Rosette-Sep™ efficiency. Cell suspensions were seeded in the self-sorting microwell chips using a negative pressure of 40-70 mbar using the VyCAP Filtration system. After seeding single cells in the microwells, the chips were imaged with the VyCAP Puncher system and either placed immediately into culture medium for cell expansion or selected cells were transferred into a 96 well tissue culture plate. Cell expansion in microwell chips and plates was followed up to 14 days. Results: Cultured MDA-MB-231 and MCF-7 cells were single-cell-trapped in the microwells with an efficiency of 82% ± 9% and 74% ± 15% respectively, after direct filtration without enrichment. Immediately after capturing in microwells 93% of the MCF7 cells were viable. Rosette-Sep™ enriched MDA-MB-231 and MCF-7 cells with an efficiency of 73% ± 6% and 74% ± 7% respectively, when measured with flow cytometry. The Rosette-Sep™ procedure followed by capture of the MDA-MB-231 and MCF-7 cells in the microwells resulted in an efficiency of 50% ± 11% and 52% ± 11% respectively. 92% of the cells were found back in a culture plate after punching of single cells from the chip, and of these cells, 80% were alive 4 hours after punching. 82% of these cells were alive and still growing after 14 days. In total 53% of cells spiked in blood, could be found back in the cups of which after punching into individual wells, 77% were alive and growing. Conclusion: We present a single cell capture and isolation method for clonal expansion of viable tumor cells. The VyCAP single cell isolation platform used for these experiments provides an easy separation of the single cells from a CTC enriched cell suspension and allows on-chip expansion as well as immediate separation of the CTCs into culture plates. About 50% of spiked cells could be retrieved inside the microwell chip and of these 77% were alive after punching into individual wells of a culture plate. Citation Format: Joost F. Swennenhuis, Kiki C. Andree, Fikri Abali, Michiel Stevens, Joska Broekmaat, Fiona F. Passanha, Cees J. van Rijn, Leon W. Terstappen. Self-sorting microwells to isolate and expand single circulating tumor cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 3785. doi:10.1158/1538-7445.AM2017-3785