Characterization of BCMA Expression in Circulating Rare Single Cells of Patients with Plasma Cell Neoplasms.

B-cell maturation antigen (BCMA), a key regulator of B-cell proliferation and survival, is highly expressed in almost all cases of plasma cell neoplasms and B-lymphoproliferative malignancies. BCMA is a robust biomarker of plasma cells and a therapeutic target with substantial clinical significance. However, the expression of BCMA in circulating tumor cells of patients with hematological malignancies has not been validated for the detection of circulating plasma and B cells. The application of BCMA as a biomarker in single-cell detection and profiling of circulating tumor cells in patients' blood could enable early disease profiling and therapy response monitoring. Here, we report the development and validation of a slide-based immunofluorescence assay (i.e., CD138, BCMA, CD45, DAPI) for enrichment-free detection, quantification, and morphogenomic characterization of BCMA-expressing cells in patients (N = 9) with plasma cell neoplasms. Varying morphological subtypes of circulating BCMA-expressing cells were detected across the CD138(+/-) and CD45(+/-) compartments, representing candidate clonotypic post-germinal center B cells, plasmablasts, and both normal and malignant plasma cells. Genomic analysis by single-cell sequencing and correlation to clinical FISH cytogenetics provides validation, with data showing that patients across the different neoplastic states carry both normal and altered BCMA-expressing cells. Furthermore, altered cells harbor cytogenetic events detected by clinical FISH. The reported enrichment-free liquid biopsy approach has potential applications as a single-cell methodology for the early detection of BCMA+ B-lymphoid malignancies and in monitoring therapy response for patients undergoing anti-BCMA treatments.

Circulating Tumor Cells: High-Throughput Imaging of CTCs and Bioinformatic Analysis.

Circulating tumor cells (CTCs) represent novel biomarkers, since they are obtainable through a simple and noninvasive blood draw or liquid biopsy. Here, we review the high-definition single-cell analysis (HD-SCA) workflow, which brings together modern methods of immunofluorescence with more sophisticated image processing to rapidly and accurately detect rare tumor cells among the milieu of platelets, erythrocytes, and leukocytes in the peripheral blood. In particular, we discuss progress in methods to measure CTC morphology and subcellular protein expression, and we highlight some initial applications that lead to fundamental new insights about the hematogenous phase of cancer, as well as its performance in early-stage diagnosis and treatment monitoring. We end with an outlook on how to further probe CTCs and the unique advantages of the HD-SCA workflow for improving the precision of cancer care.

Circulating tumor cells as a response monitor in stage IV non-small cell lung cancer.

BACKGROUND: Monitoring circulating tumor cells (CTC) has been shown to be prognostic in most solid malignancies. There is no CTC assay in clinical use for lung cancer therapy monitoring due to inconclusive clinical utility data. Limited data has been published outside of the standard CTC enumerations, regarding clinical significance of phenotypic heterogeneity of CTCs in late stage NSCLC and its ability to correlate with treatment outcomes. METHODS: In 81 patients with stage IV NSCLC, multiple timepoints for CTC analysis were collected after initiation of treatment across 139 lines of therapy using single cell high definition diagnostic pathology imaging of all nucleated cells from 362 peripheral blood samples as a liquid biopsy. RESULTS: We analyzed the subset of 25 patients with complete time series data, totaling 117 blood samples, to determine the significance of HD-CTC kinetics during the initiation of treatment. These kinetics follow three distinct patterns: an increase in HD-CTCs with therapy (mean + 118.40 HD-CTCs/mL), unchanged HD-CTCs numbers (stable; mean 0.54 HD-CTCs/mL), and a decrease in HD-CTCs numbers (mean - 81.40 HD-CTCs/mL). Patients with an increasing CTC count during the first 3 months post initiation of new treatment had a better PFS and OS compared to the other groups. There was weak correlation between the absolute number of HD-CTCs at a single time point of therapy and patient outcomes (OS p value = 0.0754). In the whole cohort of 81 patients, HD-CTCs were detected in 51 (63%) patients at initiation of therapy with a median of 2.20 (range 0-509.20) and a mean of 26.21 HD-CTCs/mL (± 15.64). CONCLUSIONS: CTCs are identifiable in most patients with stage IV NSCLC. While absolute HD-CTC counts do not correlate with prognosis, the changes in CTC counts were predictive of survival in patients with metastatic lung cancer receiving chemotherapy. The level and dynamics of CTCs indicate very different biological and pharmacological phenomena at different stages of disease and timepoints of treatment, highlighting the complex role of CTCs in cancer research and clinical management.

Induction of Apoptosis by PQ1, a Gap Junction Enhancer that Upregulates Connexin 43 and Activates the MAPK Signaling Pathway in Mammary Carcinoma Cells.

The mechanism of gap junction enhancer (PQ1) induced cytotoxicity is thought to be attributed to the change in connexin 43 (Cx43) expression; therefore, the effects of Cx43 modulation in cell survival were investigated in mammary carcinoma cells (FMC2u) derived from a malignant neoplasm of a female FVB/N-Tg(MMTV-PyVT)634Mul/J (PyVT) transgenic mouse. PQ1 was determined to have an IC50 of 6.5 µM in FMC2u cells, while inducing an upregulation in Cx43 expression. The effects of Cx43 modulation in FMC2u cell survival was determined through transfection experiments with Cx43 cDNA, which induced an elevated level of protein expression similar to that seen with PQ1 exposure, or siRNA to silence Cx43 protein expression. Overexpression or silencing of Cx43 led to a reduction or an increase in cell viability, respectively. The mitogen-activated protein kinase (MAPK) family has been implicated in the regulation of cell survival and cell death; therefore, the gap junctional intercellular communication (GJIC)-independent function of PQ1 and Cx43 in the Raf/Mitogen-activated protein kinase/ERK kinase/extracellular-signal-regulated kinase (Raf-MEK-ERK) cascade of cellular survival and p38 MAPK-dependent pathway of apoptosis were explored. PQ1 treatment activated p44/42 MAPK, while the overexpression of Cx43 resulted in a reduced expression. This suggests that PQ1 affects the Raf-MEK-ERK cascade independent of Cx43 upregulation. Both overexpression of Cx43 and PQ1 treatment stimulated an increase in the phosphorylated form of p38-MAPK, reduced levels of the anti-apoptotic protein Bcl-2, and increased the cleavage of pro-caspase-3. Silencing of Cx43 protein expression led to a reduction in the phosphorylation of p38-MAPK and an increase in Bcl-2 expression. The mechanism behind PQ1-induced cytotoxicity in FMC2u mammary carcinoma cells is thought to be attributed to the change in Cx43 expression. Furthermore, PQ1-induced apoptosis through the upregulation of Cx43 may depend on p38 MAPK, highlighting that the effect of PQ1 on gap junctions as well as cellular survival via a MAPK-dependent pathway.

The anticancer effect of PQ1 in the MMTV-PyVT mouse model.

Animal models are commonly used to analyze the mechanism of carcinogenesis as well as the development and screening of potent drugs. Here the transgenic strain FVB/N-Tg(MMTV-PyVT)634Mul/J (also known as PyVT) was used as a model system for measuring tumor burden, drug sensitivity, and metastasis of mammary carcinomas. Loss of gap junctional intercellular communication and the down regulation of connexin expression are characteristic of neoplastic cells. The substituted quinoline, 6-methoxy-8-[(3-aminopropyl)amino]-4-methyl-5-(3-trifluoromethyl-phenyloxy)quinolone (PQ1), has been shown to restore GJIC and increase connexin expression in breast cancer cell lines while not affecting normal mammary cells, suggesting that it may provide effective anticancer treatment with less detrimental effects. The PyVT spontaneous mammary tumor mouse model was used to determine the biological and histological effects of PQ1 on tumorigenesis and metastasis at three stages of development: Pretumor, early tumor and late tumor formation. Treatment with PQ1 at all three stages of development significantly reduced tumor growth. PQ1 treatment further increased Cx43 expression during pre- and early-tumor formation, while it prevented an increase in Cx46 expression during late stage tumor formation. This study shows that Cx43 expression and neoplastic cellular growth are inversely related, but that PQ1 can alter tumor growth through targeting gap junction proteins to prove clinical efficacy in the treatment of spontaneous mammary tumors.

Longitudinal tracking of circulating rare events in the liquid biopsy of stage III-IV non-small cell lung cancer patients.

In the United States, lung cancer is the second most common type of cancer with non-small cell lung cancer (NSCLC) encompassing around 85% of total lung cancer cases. Late-stage patients with metastatic disease have worsening prognosis, highlighting the importance of longitudinal disease monitoring. Liquid biopsy (LBx) represents a way for physicians to non-invasively track tumor analytes, such as circulating tumor cells (CTCs), and understand tumor progression in real-time through analyzing longitudinal blood samples. CTCs have been shown to be effective predictive biomarkers in measuring treatment efficacy and survival outcomes. We used the third-generation High-Definition Single Cell Assay (HDSCA3.0) workflow to analyze circulating rare events longitudinally during treatment in a cohort of 10 late-stage NSCLC patients, identifying rare events including circulating cancer cells (i.e., CTCs), and oncosomes. Here, we show (1) that there is a cancer specific LBx profile, (2) there is considerable heterogeneity of rare cells and oncosomes, and (3) that LBx data elements correlated with patient survival outcomes. Additional studies are warranted to understand the biological significance of the rare events detected, and the clinical potential of the LBx to monitor and predict response to treatment in NSCLC patient care.

Liquid biopsy approach to monitor the efficacy and response to CAR-T cell therapy.

BACKGROUND: Chimeric antigen receptor (CAR)-T cells are approved for use in the treatment of hematological malignancies. Axicabtagene ciloleucel (YESCARTA) and brexucabtagene autoleucel (TECARTUS) genetically modified autologous T cells expressing an anti-CD19 scFv based on the FMC63 clone have shown impressive response rates for the treatment of CD19+B cell malignancies, but there remain challenges in monitoring long-term persistence as well as the functional characterization of low-level persisting CAR-T cells in patients. Furthermore, due to CD19-negative driven relapse, having the capability to monitor patients with simultaneous detection of the B cell malignancy and persisting CAR-T cells in patient peripheral blood is important for ensuring timely treatment optionality and understanding relapse. METHODS: This study demonstrates the development and technical validation of a comprehensive liquid biopsy, high-definition single cell assay (HDSCA)-HemeCAR for (1) KTE-X19 CAR-T cell identification and analysis and (2) simultaneously monitoring the CD19-epitope landscape on neoplastic B cells in cryopreserved or fresh peripheral blood. Proprietary anti-CD19 CAR reagents, healthy donor transduced CAR-T cells, and patient samples consisting of malignant B cell fractions from manufacturing were used for assay development. RESULTS: The CAR-T assay showed an approximate limit of detection at 1 cell in 3 million with a sensitivity of 91%. Genomic analysis was additionally used to confirm the presence of the CAR transgene. This study additionally reports the successful completion of two B cell assays with multiple CD19 variants (FMC63 and LE-CD19) and a unique fourth channel biomarker (CD20 or CD22). In patient samples, we observed that CD19 isoforms were highly heterogeneous both intrapatient and interpatient. CONCLUSIONS: With the simultaneous detection of the CAR-T cells and the B cell malignancy in patient peripheral blood, the HDSCA-HemeCAR workflow may be considered for risk monitoring and patient management.

A Systematic Approach toward Enabling Maximal Targeting Efficiency of Cell Surface Proteins with Actively Targeted SERS Nanoparticles.

With their intricate design, nanoparticles (NPs) have become indispensable tools in the quest for precise cellular targeting. Among various NPs, gold NPs stand out with unique features such as chemical stability, biocompatibility, adjustable shape, and size-dependent optical properties, making them particularly promising for molecular detection by leveraging the surface-enhanced Raman scattering (SERS) effect. Their multiplexing abilities for the simultaneous identification of multiple biomarkers are important in the rapidly evolving landscape of diverse cellular phenotypes and biomolecular profiling. However, the challenge is ensuring that SERS NPs can effectively target specific cells and biomarkers among intricate cell types and biomolecules with high specificity. In this study, we improve the functionalization of SERS NPs, optimizing their targeting efficiency in cellular applications for ca. 160 nm NP-based probes. Spherical SERS NPs, conjugated with antibodies targeting epidermal growth factor receptor and human epidermal growth factor receptor 2, were incubated with cells overexpressing these proteins, and their specific binding potential was quantified at each stage by using flow cytometry to achieve optimal targeting efficiency. We determined that maintaining an average of 3.5 × 105 thiols per NP, 300 antibodies per NP, 18,000 NPs per cell, conducting a 15 min staining incubation at 4 °C in a shaker, and using SM(PEG)12 as a cross-linker for the NP conjugation were crucial to achieve the highest targeting efficiency. Fluorescence and Raman imaging were used with these parameters to observe the maximum ability of these NPs to efficiently target suspended cells. These highly sensitive contrast agents demonstrate their pivotal role in effective active targeting, making them invaluable for multiplexing applications across diverse biological environments.

Blood-based liquid biopsy: A promising noninvasive test in diagnosis, surveillance, and prognosis of patients with upper tract urothelial carcinoma.

OBJECTIVES: To assess the efficacy of blood-based liquid biopsy in the diagnosis, surveillance, and prognosis of upper tract urothelial carcinoma (UTUC). METHODS AND MATERIALS: In this prospective study, peripheral blood samples were collected from patients with primary UTUC before surgery with curative intent and follow-up visits at University of Southern California between May 2021 and September 2022. The samples were analyzed using the third-generation comprehensive high-definition single-cell assay (HDSCA3.0) to detect rare events, including circulating tumor cells (CTCs) and oncosomes, based on the immunofluorescence signals of DAPI (D), cytokeratin (CK), CD45/CD31 (CD), and vimentin (V). The findings of pre-surgery liquid biopsies were compared with those of blood samples from normal donors (NDs) and matched follow-up liquid biopsies. The association between liquid biopsy findings and clinical data, including recurrence-free survival (RFS), was also assessed. RESULTS: Twenty-eight patients with UTUC were included, of whom 21 had follow-up samples. Significant differences in specific rare analytes were detected in the preoperative samples compared to the NDs. In the post- vs. presurgery matched analysis, a significant decrease was detected in total-, CK-, and CK|V oncosomes, as well as in D-, D|V-, and D|V|CD cells. With a median follow-up of 11 months, 8 patients had disease recurrence. Survival analysis demonstrated that patients with >1.95 preoperative CK|V oncosomes (p = 0.020) and those with >4.18 D|CK|V cells (p = 0.050) had worse RFS compared to other patients. CONCLUSIONS: This study demonstrated promising initial evidence for the biomarker role of CTCs and oncosomes in the diagnosis and surveillance of patients with UTUC.

Cancer-related cells and oncosomes in the liquid biopsy of pancreatic cancer patients undergoing surgery.

Pancreatic ductal adenocarcinoma (PDAC) has a five-year survival rate of less than 10% due to its late diagnosis, rapid metastasis, and chemotherapeutic resistance. For a small proportion (10-20%) of early-stage patients however, surgical resection of the pancreatic tumor offers the best chance for survival but the effect of surgery on disease dissemination is unknown. The primary objective of this study was to characterize cellular and acellular blood-based analytes in portal and peripheral blood before pancreatic manipulation, during tumor dissection and immediately after surgical resection to determine the effects of the surgery. This study used the non-enriching third generation High-Definition Single Cell Assay (HDSCA3.0) workflow to investigate heterogeneous circulating rare cell population in the blood. Blood from both sites taken before surgical manipulation of the pancreas had significantly greater incidence of total rare cellular and acellular analytes than normal donor samples. Post-surgery portal and peripheral blood had significantly greater incidence of specific cellular and acellular subtypes compared to the matched pre- and during-surgery samples. Our results reveal that in patients with PDAC liquid biopsy analytes are increased in both the portal and peripheral blood; portal blood contains a higher frequency of analytes than in the peripheral blood; total analytes in the portal and peripheral blood samples were significantly associated with the tumor volume and pathological T stage; and the surgical procedure increased the blood levels of circulating cellular and acellular analytes, but not Epi.CTCs or Mes.CTCs. This study demonstrates liquid biopsy's utility in monitoring patients with PDAC with surgically resectable disease.

PSCA-CAR T cell therapy in metastatic castration-resistant prostate cancer: a phase 1 trial.

Despite recent therapeutic advances, metastatic castration-resistant prostate cancer (mCRPC) remains lethal. Chimeric antigen receptor (CAR) T cell therapies have demonstrated durable remissions in hematological malignancies. We report results from a phase 1, first-in-human study of prostate stem cell antigen (PSCA)-directed CAR T cells in men with mCRPC. The starting dose level (DL) was 100 million (M) CAR T cells without lymphodepletion (LD), followed by incorporation of LD. The primary end points were safety and dose-limiting toxicities (DLTs). No DLTs were observed at DL1, with a DLT of grade 3 cystitis encountered at DL2, resulting in addition of a new cohort using a reduced LD regimen + 100 M CAR T cells (DL3). No DLTs were observed in DL3. Cytokine release syndrome of grade 1 or 2 occurred in 5 of 14 treated patients. Prostate-specific antigen declines (>30%) occurred in 4 of 14 patients, as well as radiographic improvements. Dynamic changes indicating activation of peripheral blood endogenous and CAR T cell subsets, TCR repertoire diversity and changes in the tumor immune microenvironment were observed in a subset of patients. Limited persistence of CAR T cells was observed beyond 28 days post-infusion. These results support future clinical studies to optimize dosing and combination strategies to improve durable therapeutic outcomes. ClinicalTrials.gov identifier NCT03873805 .

Plasticity of circulating tumor cells in small cell lung cancer.

Small cell lung cancer (SCLC) is an aggressive neuroendocrine tumor with low five-year survival rates. Recently described molecular phenotypes of SCLC exhibit differential vulnerabilities heralding potential for stratified treatment. Whilst tumor biopsy in SCLC is challenging, circulating tumor cells in the liquid biopsy are prevalent and can be repeatedly sampled accommodating the dynamic plasticity of SCLC phenotypes. The aim of this study was to characterize the heterogeneity of rare circulating cells with confirmed tumor origin and to explore a liquid biopsy approach for future clinical trials of targeted therapies. This study applied the 3rd generation of a previously validated direct imaging platform to 14 chemo-naive SCLC patients and 10 non-cancerous normal donor (ND) samples. Phenotypic heterogeneity of circulating rare cells in SCLC was observed and a patient-level classification model was established to stratify SCLC patients from non-cancerous donors. Eight rare cell groups, with combinations of epithelial, endothelial, and mesenchymal biomarker expression patterns, were phenotypically characterized. The single-cell genomic analysis confirmed the cancer cell plasticity in every rare cell group harboring clonal genomic alterations. This study shows rare cell heterogeneity and confirms cellular plasticity in SCLC providing a valuable resource for better opportunities to discover novel therapeutic targets in SCLC.

Investigation of liquid biopsy analytes in peripheral blood of individuals after SARS-CoV-2 infection.

BACKGROUND: Post-acute COVID-19 syndrome (PACS) is linked to severe organ damage. The identification and stratification of at-risk SARS-CoV-2 infected individuals is vital to providing appropriate care. This exploratory study looks for a potential liquid biopsy signal for PACS using both manual and machine learning approaches. METHODS: Using a high definition single cell assay (HDSCA) workflow for liquid biopsy, we analysed 100 Post-COVID patients and 19 pre-pandemic normal donor (ND) controls. Within our patient cohort, 73 had received at least 1 dose of vaccination prior to SARS-CoV-2 infection. We stratified the COVID patients into 25 asymptomatic, 22 symptomatic COVID-19 but not suspected for PACS and 53 PACS suspected. All COVID-19 patients investigated in this study were diagnosed between April 2020 and January 2022 with a median 243 days (range 16-669) from diagnosis to their blood draw. We did a histopathological examination of rare events in the peripheral blood and used a machine learning model to evaluate predictors of PACS. FINDINGS: The manual classification found rare cellular and acellular events consistent with features of endothelial cells and platelet structures in the PACS-suspected cohort. The three categories encompassing the hypothesised events were observed at a significantly higher incidence in the PACS-suspected cohort compared to the ND (p-value < 0.05). The machine learning classifier performed well when separating the NDs from Post-COVID with an accuracy of 90.1%, but poorly when separating the patients suspected and not suspected of PACS with an accuracy of 58.7%. INTERPRETATION: Both the manual and the machine learning model found differences in the Post-COVID cohort and the NDs, suggesting the existence of a liquid biopsy signal after active SARS-CoV-2 infection. More research is needed to stratify PACS and its subsyndromes. FUNDING: This work was funded in whole or in part by Fulgent Genetics, Kathy and Richard Leventhal and Vassiliadis Research Fund. This work was also supported by the National Cancer InstituteU54CA260591.

Targeted single-cell proteomic analysis identifies new liquid biopsy biomarkers associated with multiple myeloma.

Multiple myeloma (MM) is accompanied by alterations to the normal plasma cell (PC) proteome, leading to changes to the tumor microenvironment and disease progression. There is a great need for understanding the consequences that lead to MM progression and for the discovery of new biomarkers that can aid clinical diagnostics and serve as targets for therapeutics. This study demonstrates the applicability of utilizing the single-cell high-definition liquid biopsy assay (HDSCA) and imaging mass cytometry to characterize the proteomic profile of myeloma. In our study, we analyzed ~87,000 cells from seven patient samples (bone marrow and peripheral blood) across the myeloma disease spectrum and utilized our multiplexed panel to characterize the expression of clinical markers for PC classification, additional potential therapeutic targets, and the tumor microenvironment cells. Our analysis showed BCMA, ICAM3 (CD50), CD221, and CS1 (SLAMF7) as the most abundantly expressed markers on PCs across all myeloma stages, with BCMA, ICAM3, and CD221 having significantly higher expression levels on disease versus precursor PCs. Additionally, we identify significantly elevated levels of expression for CD74, MUM1, CD229, CD44, IGLL5, Cyclin D1, UBA52, and CD317 on PCs from overt disease conditions compared to those from precursor states.

Cell State and Cell Type: Deconvoluting Circulating Tumor Cell Populations in Liquid Biopsies by Multi-Omics.

Bi-directional crosstalk between the tumor and the tumor microenvironment (TME) has been shown to increase the rate of tumor evolution and to play a key role in neoplastic progression, therapeutic resistance, and a patient's overall survival. Here, we set out to use a comprehensive liquid-biopsy analysis to study cancer and specific TME cells in circulation and their association with disease status. Cytokeratin+, CD45- circulating rare cells (CRCs) from nine breast and four prostate cancer patients were characterized through morphometrics, single-cell copy number analysis, and targeted multiplexed proteomics to delineate cancer cell lineage from other rare cells originating in the TME. We show that we can detect epithelial circulating tumor cells (EPI.CTC), CTCs undergoing epithelial-to-mesenchymal transition (EMT.CTC) and circulating endothelial cells (CECs) using a universal rare event detection platform (HDSCA). Longitudinal analysis of an index patient finds that CTCs are present at the time of disease progression, while CECs are predominately present at the time of stable disease. In a small cohort of prostate and breast cancer patients, we find high inter-patient and temporal intra-patient variability in the expression of tissue specific markers such as ER, HER2, AR, PSA and PSMA and EpCAM. Our study stresses the importance of the multi-omic characterization of circulating rare cells in patients with breast and prostate carcinomas, specifically highlighting overlapping and cell type defining proteo-genomic characteristics of CTCs and CECs.

Humoral innate immune response and disease.

The humoral innate immune response consists of multiple components, including the naturally occurring antibodies (NAb), pentraxins and the complement and contact cascades. As soluble, plasma components, these innate proteins provide key elements in the prevention and control of disease. However, pathogens and cells with altered self proteins utilize multiple humoral components to evade destruction and promote pathology. Many studies have examined the relationship between humoral immunity and autoimmune disorders. This review focuses on the interactions between the humoral components and their role in promoting the pathogenesis of bacterial and viral infections and chronic diseases such as atherosclerosis and cancer. Understanding the beneficial and detrimental aspects of the individual components and the interactions between proteins which regulate the innate and adaptive response will provide therapeutic targets for subsequent studies.

Defining A Liquid Biopsy Profile of Circulating Tumor Cells and Oncosomes in Metastatic Colorectal Cancer for Clinical Utility.

Metastatic colorectal cancer (mCRC) is characterized by its extensive disease heterogeneity, suggesting that individualized analysis could be vital to improving patient outcomes. As a minimally invasive approach, the liquid biopsy has the potential to longitudinally monitor heterogeneous analytes. Current platforms primarily utilize enrichment-based approaches for epithelial-derived circulating tumor cells (CTC), but this subtype is infrequent in the peripheral blood (PB) of mCRC patients, leading to the liquid biopsy's relative disuse in this cancer type. In this study, we evaluated 18 PB samples from 10 mCRC patients using the unbiased high-definition single-cell assay (HDSCA). We first employed a rare-event (Landscape) immunofluorescence (IF) protocol, which captured a heterogenous CTC and oncosome population, the likes of which was not observed across 50 normal donor (ND) samples. Subsequent analysis was conducted using a colorectal-targeted IF protocol to assess the frequency of CDX2-expressing CTCs and oncosomes. A multi-assay clustering analysis isolated morphologically distinct subtypes across the two IF stains, demonstrating the value of applying an unbiased single-cell approach to multiple assays in tandem. Rare-event enumerations at a single timepoint and the variation of these events over time correlated with progression-free survival. This study supports the clinical utility of an unbiased approach to interrogating the liquid biopsy in mCRC, representing the heterogeneity within the CTC classification and warranting the further molecular characterization of the rare-event analytes with clinical promise.

Liquid Biopsy Landscape in Patients with Primary Upper Tract Urothelial Carcinoma.

Urothelial carcinomas (UCs) are a broad and heterogeneous group of malignancies, with the prevalence of upper tract urothelial carcinoma (UTUC) being rare, accounting for only 5-10% of total malignancies. There is a need for additional toolsets to assist the current clinical paradigm of care for patients with UTUC. As a non-invasive tool for the discovery of cancer-related biomarkers, the liquid biopsy has the potential to represent the complex process of tumorigenesis and metastasis. Herein, we show the efficacy of the liquid biopsy as a source of biomarkers for detecting UTUC. Using the third-generation high-definition single-cell assay (HDSCA3.0) workflow, we investigate liquid biopsy samples collected from patients with UTUC and normal donors (NDs) to provide critical information regarding the molecular and morphological characteristics of circulating rare events. We document several important findings from the liquid biopsy analysis of patients diagnosed with UTUC prior to surgery: (1) Large extracellular vesicles (LEVs) and circulating tumor cells (CTCs) are detectable in the peripheral blood. (2) The rare-event profile is highly heterogeneous. (3) Clinical data elements correlate with liquid biopsy analytes. Overall, this study provides evidence for the efficacy of the liquid biopsy in understanding the biology of UTUC with the future intent of informing clinical decision making, ultimately improving patient outcomes.

Disease characterization in liquid biopsy from HER2-mutated, non-amplified metastatic breast cancer patients treated with neratinib.

Metastatic breast cancer (mBC) patients have a high risk of progression and face poor prognosis overall, with about one third (34%) surviving five years or more. In rare instances (2-4% of cases) patients with mBC have ERBB2 (HER2) activating mutations but are ERBB2 non-amplified. Neratinib is a potent, irreversible inhibitor that binds HER2 and inhibits downstream signaling. We used the previously validated high-definition single cell assay (HDSCA) workflow to investigate the clinical significance of the liquid biopsy in ERBB2 mutant, non-amplified, post-menopausal mBC patients starting neratinib and fulvestrant combination therapy. Characterization with a comprehensive liquid biopsy methodology (HDSCA) included genomic analysis of both the cell-free DNA (cfDNA) and single circulating tumor cells (CTCs) to monitor tumor evolution and identify potential mutational variants unique to the patient's clinical response. A limited series of five sequentially enrolled patients presented here were from the MutHER ( https://www.clinicaltrials.gov , NCT01670877) or SUMMIT ( https://www.clinicaltrials.gov , NCT01953926) trials. Patients had an average of 5.4 lines of therapy before enrollment, variable hormone receptor status, and ERBB2 mutations at diagnosis and during treatment. CTC enumeration alone was not sufficient to predict clinical response. Treatment pressure was shown to lead to an observable change in CTC morphology and genomic instability (GI), suggesting these parameters may inform prognosis. Single cell copy number alteration (CNA) analysis indicated that the persistence or development of a clonal population of CTCs during treatment was associated with a worse response. Hierarchical clustering analysis of the single cells across all patients and timepoints identified distinct aberrant regions shared among patients, comprised of 26 genes that are similarly affected and may be related to drug resistance. Additionally, the genomic analysis of the cfDNA, identified new mutations in ERBB2, PIK3CA, and TP53 that arose likely due to treatment pressure in a patient with poor response, further providing insights on the dynamics of the cancer genome over the course of therapy. The data presented in this small cohort study demonstrates the feasibility of real-time molecular profiling of the cellular and acellular fractions of the liquid biopsy using the HDSCA methodology. Additional studies are necessary to determine the potential use of morphometric and genomic analysis as a prognostic tool to advance personalized oncology.

Circulating Tumor Cell Kinetics and Morphology from the Liquid Biopsy Predict Disease Progression in Patients with Metastatic Colorectal Cancer Following Resection.

The liquid biopsy has the potential to improve current clinical practice in oncology by providing real-time personalized information about a patient's disease status and response to treatment. In this study, we evaluated 161 peripheral blood (PB) samples that were collected around surgical resection from 47 metastatic colorectal cancer (mCRC) patients using the High-Definition Single Cell Assay (HDSCA) workflow. In conjunction with the standard circulating tumor cell (CTC) enumeration, cellular morphology and kinetics between time-points of collection were considered in the survival analysis. CTCs, CTC-Apoptotic, and CTC clusters were found to indicate poor survival with an increase in cell count from pre-resection to post-resection. This study demonstrates that CTC subcategorization based on morphological differences leads to nuanced results between the subtypes, emphasizing the heterogeneity within the CTC classification. Furthermore, we show that factoring in the time-point of each blood collection is critical, both for its static enumeration and for the change in cell populations between draws. By integrating morphology and time-based analysis alongside standard CTC enumeration, liquid biopsy platforms can provide greater insight into the pathophysiology of mCRC by highlighting the complexity of the disease across a patient's treatment.