Liquid Biopsy Approach Research Articles

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 cell-free messenger RNA enables non-invasive pan-tumour monitoring of melanoma therapy independent of the mutational genotype.

Plasma-derived tumour-specific cell-free nucleic acids are increasingly utilized as a minimally invasive, real-time biomarker approach in many solid tumours. Circulating tumour DNA of melanoma-specific mutations is currently the best studied liquid biopsy biomarker for melanoma. However, the combination of hotspot genetic alterations covers only around 80% of all melanoma patients. Therefore, alternative approaches are needed to enable the follow-up of all genotypes, including wild-type. We identified KPNA2, DTL, BACE2 and DTYMK messenger RNA (mRNA) upregulated in melanoma versus nevi tissues by unsupervised data mining (N=175 melanoma, N=20 normal skin, N=6 benign nevi) and experimentally confirmed differential mRNA expression in vitro (N=18 melanoma, N=8 benign nevi). Circulating cell-free RNA (cfRNA) was analysed in 361 plasma samples (collected before and during therapy) from 100 melanoma patients and 18 healthy donors. Absolute cfRNA copies were quantified on droplet digital PCR. KPNA2, DTL, BACE2 and DTYMK cfRNA demonstrated high diagnostic accuracy between melanoma patients' and healthy donors' plasma (AUC > 86%, p<.0001). cfRNA copies increased proportionally with increasing tumour burden independently of demographic variables and even remained elevated in individuals with radiological absence of disease. Re-analysis of single-cell transcriptomes revealed a pan-tumour origin of cfRNA, including endothelial, cancer-associated fibroblasts, macrophages and B cells beyond melanoma cells as cellular sources. Low baseline cfRNA levels were associated with significantly longer progression-free survival (PFS) (KPNA2 HR = .54, p=.0362; DTL HR = .60, p=.0349) and overall survival (KPNA2 HR = .52, p=.0237; BACE2 HR = .55, p=.0419; DTYMK HR = .43, p=.0393). Lastly, we found that cfRNA copies significantly increased during therapy in non-responders compared to responders regardless of therapy and mutational subtypes and that the increase of KPNA2 (HR = 1.73, p=.0441) and DTYMK (HR = 1.82, p=.018) cfRNA during therapy was predictive of shorter PFS. In sum, we identified a new panel of cfRNAs for a pan-tumour liquid biopsy approach and demonstrated its utility as a prognostic, therapy-monitoring tool independent of the melanoma mutational genotype.

Identification of an Inflammatory Biomarker Signature in Plasma-Derived Extracellular Vesicles of Glioblastoma Patients

Abstract AIMS Description of effective biomarkers present in biofluids could prove invaluable in GBM diagnosis. Extracellular vesicles (EVs) are essential to intercellular crosstalk in the tumour bulk and circulating EVs have been described as a potential reservoir of GBM biomarkers. Therefore, EV-based liquid biopsies have been suggested as a promising tool for GBM METHOD Ethical approval was obtained for a prospective study of healthy donors and consenting GBM patients at the University Hospitals Sussex (Brighton). To identify GBM specific proteins, small EVs (sEVs) were isolated from plasma samples using differential ultracentrifugation and validated through Nanoparticles tracking analysis, transmission electron microscopy and detection of known sEVs markers such as CD9, CD63, CD81 and HSP70. sEVs content was characterised through mass spectrometry and bioinformatic tools. RESULTS Our data indicate the presence of a GBM inflammatory biomarker signature comprising members of the complement and regulators of inflammation and coagulation including VWF, FCGBP, C3, PROS1, and SERPINA1. Bioinformatic analysis highlighted that all potential markers exclusively identified in patient samples had already been linked with either GBM diagnosis, prognosis or associated signalling, suggesting that sEVs protein cargo could mirror the landscape of the original tumour and that selective circulating sEV-derived proteins might be used as hallmarks for GBM patients. CONCLUSION this study is a step forward in the development of a non-invasive liquid biopsy approach for the identification of valuable biomarkers that could significantly improve GBM diagnosis and, consequently, patients’ prognosis and quality of life.

Small extracellular vesicles as a multicomponent biomarker platform in urinary tract carcinomas.

Extracellular vesicles are a large group of nano-sized vesicles released by all cells. The variety of possible cargo (mRNAs, miRNAs, lncRNAs, proteins, and lipids) and the presence of surface proteins, signaling molecules, and receptor ligands make them a rich source of biomarkers for malignancy diagnosis. One of the groups gathering the most interest in cancer diagnostic applications is small extracellular vesicles (sEVs), with ≤200 nm diameter, mainly composed of exosomes. Many studies were conducted recently, evaluating the diagnostic potential of sEVs in urinary tract carcinomas (UTCs), discovering and clinically evaluating various classes of biomarkers. The amount of research concerning different types of UTCs understandably reflects their incidence. sEV cargos getting the most interest are non-coding RNAs (miRNA and lncRNA). However, implementation of other approaches such as metabolomic and proteomic analysis is also evaluated. The results of many studies indicate that sEVs have an essential role in the cancer process and possess many possible diagnostic and prognostic applications for UTC. The relative ease of obtaining biofluids rich in sEVs (urine and blood) confirms that sEVs are essential for UTC detection in the liquid biopsy approach. A noticeable rise in research quality is observed as more researchers are aware of the research standardization necessity, which is essential for considering the clinical application of their findings.

Feasibility of Leukemia-Derived Exosome Enrichment and Co-isolated dsDNA Sequencing in Acute Myeloid Leukemia Patients: A Proof of Concept for New Leukemia Biomarkers Detection

Simple SummaryThe present pilot study aimed at investigating the feasibility of a leukemia-derived exosome enrichment approach followed by exosomal dsDNA target re-sequencing for adult Acute Myeloid Leukemias (AML) marker detection. To our knowledge, this is the first time that a proof-of-concept combining a leukemia-derived exosome enrichment strategy based on a commercial CE-IVD kit and next-generation sequencing was applied in a cohort of adult AML patients. The reported approach is easy, quick and user friendly and gives the possibility of obtaining a good quantity of exosomal dsDNA (composed of exosomal cargo and surrounding DNA) suitable for further analysis. The time-effective procedure opens up future effective clinical applications. This pilot study presents the potential of a proof-of-concept based on exosome analysis to be applied in clinical practice, as well as the feasibility of this kind of investigations using a certified kit, avoiding many additional analyses. It may encourage further studies regarding extracellular vesicles in myeloid neoplasia.Exosomes are extracellular vesicles playing a pivotal role in the intercellular communication. They shuttle different cargoes, including nucleic acids from their cell of origin. For this reason, they have been studied as carriers of tumor markers in different liquid biopsy approaches, in particular for solid tumors. Few data are available concerning exosomes as markers of myeloid neoplasia. To better understand their real potential and the best approach to investigate leukemic exosomes, we present the results of a pilot feasibility study evaluating the application of next-generation sequencing analysis of dsDNA derived from exosomes isolated in 14 adult patients affected by acute myeloid leukemias. In particular, leukemia-derived exosome fractions have been analyzed. The concentration of dsDNA co-extracted with exosomes and the number and types of mutations detected were considered and compared with ones identified in the Bone Marrow (BM) and Peripheral Blood (PB) cells. Exosomal DNA concentration, both considering the cargo and the DNA surrounding the lipid membrane resulted in a linear correlation with leukemic burden. Moreover, exosomal DNA mutation status presented 86.5% of homology with BM and 75% with PB. The results of this pilot study confirmed the feasibility of a leukemia-derived exosome enrichment approach followed by exosomal dsDNA NGS analysis for AML biomarker detection. These data point to the use of liquid biopsy in myeloid neoplasia for the detection of active leukemic cells resident in the BM via a painless procedure.

Salivary Exosomal miRNA-1307-5p Predicts Disease Aggressiveness and Poor Prognosis in Oral Squamous Cell Carcinoma Patients.

Background: Salivary exosomal miRNAs as biomarkers facilitate repeated sampling, real-time disease monitoring and assessment of therapeutic response. This study identifies a single salivary exosomal miRNA prognosticator that will aid in improved patient outcome using a liquid biopsy approach. Method: Small RNA and transcriptome sequencing profiles of tumour tissues (n = 12) and salivary exosomes (n = 8) from oral cancer patients were compared to their non-cancerous counterparts. We validated these results using The Cancer Genome Atlas database and performing Real-time PCR on a large patient cohort (n = 19 tissue samples; n = 12 salivary exosomes). Potential target genes and the miRNA–mRNA networks and enriched biological pathways regulated by this microRNA were identified using computational tools. Results: Salivary exosomes (size: 30–50 nm) demonstrated a strong expression of CD47 and detectable expression of tetraspanins CD63, CD81 and CD9 by flow cytometry. miR-1307-5p was exclusively overexpressed in tissues and salivary exosomes of oral cancer patients compared to their non-cancerous counterparts. Enhanced expression of miR-1307-5p clinically correlated with poor patient survival, disease progression, aggressiveness and chemo-resistance. Transcriptome analysis suggested that miRNA-1307-5p could promote oral cancer progression by suppressing THOP1, EHF, RNF4, GET4 and RNF114. Conclusions: Salivary exosomal miRNA-1307-5p is a potential prognosticator for predicting poor survival and poor patient outcome in oral cancers.

Accelerating the Development and Validation of Liquid Biopsy for Early Cancer Screening and Treatment Tailoring.

Liquid biopsy (LB) is a minimally invasive method which aims to detect circulating tumor-derived components in body fluids. It provides an alternative to current cancer screening methods that use tissue biopsies for the confirmation of diagnosis. This paper attempts to determine how far the regulatory, policy, and governance framework provide support to LB implementation into healthcare systems and how the situation can be improved. For that reason, the European Alliance for Personalised Medicine (EAPM) organized series of expert panels including different key stakeholders to identify different steps, challenges, and opportunities that need to be taken to effectively implement LB technology at the country level across Europe. To accomplish a change of patient care with an LB approach, it is required to establish collaboration between multiple stakeholders, including payers, policymakers, the medical and scientific community, and patient organizations, both at the national and international level. Regulators, pharma companies, and payers could have a major impact in their own domain. Linking national efforts to EU efforts and vice versa could help in implementation of LB across Europe, while patients, scientists, physicians, and kit manufacturers can generate a pull by undertaking more research into biomarkers.

Detection of clinically-relevant EGFR variations in de novo small cell lung carcinoma by droplet digital PCR.

Targeted therapy that utilizes tyrosine kinase inhibitors (TKIs), specific to epidermal growth factor receptors (EGFR) has changed the landscape of treatment of non-small cell lung cancer (NSCLC). The success or failure of this approach depends on presence of certain variations in the tyrosine kinase domain of EGFR gene. Generally, patients diagnosed with Small cell lung cancer (SCLC) are considered ineligible for TKI therapy owing to the absence of EGFR variations. . However, there is evidence of these variations being detected in SCLCs, both in de-novo and in transformed SCLCs (TKI-treated adenocarcinomas). Despite the presence of clinically-relevant EGFR variations in SCLCs, the response to TKIs has been inconsistent. Liquid biopsy is a well-established approach in lung cancer management with proven diagnostic, prognostic and predictive applications. It relies on detection of circulating tumor-derived nucleic acids present in plasma of the patient. In this study, a liquid biopsy approach was utilized to screen 118 consecutive lung cancer patients for four clinically-relevant variations in EGFR gene, which included three activating/sensitizing variations (Ex18 G719S, Ex19del E746-A750 and Ex21 L858R) and one acquired/resistance (Ex20 T790M, de novo) variation by droplet digital PCR, the most advanced third generation PCR technique. As expected, clinically-relevant EGFR variations were found in majority of the non-small cell lung cancer cases. However, among the handful of small cell lung cancer samples screened, sensitizing variations (Ex18 G719S and Ex21 L858R) were seen in almost all of them. Interestingly, Ex20 T790M variation was not detected in any of the cases screened. The results of our study indicate that EGFR variations are present in SCLCs and highly sensitive liquid biopsy techniques like ddPCR can be effectively utilized for this purpose of screening EGFR variations in such samples.

Abstract IA09: Role of liquid biopsy in early detection and assessment of response in hepatocellular carcinoma

Abstract Early hepatocellular carcinoma detection is suboptimal with less than 25% of eligible patients enrolled in surveillance. In addition, recommended tools (biannual abdominal ultrasound with or without AFP) have poor sensitivity (63% for early-stage tumors). This has prompted the development of liquid biopsy approaches to improve performance and increase implementation. Methylation analysis of circulating tumor DNA and RNA sequencing in extracellular vesicles have shown promising performance in case control studies. This technology has also been applied to identify predictor biomarkers of response to systemic therapies. With 9 agents approved and without a clear strategy on how to select patients for therapies, the need to develop minimally-invasive biomarkers in this space cannot be overemphasized. Citation Format: Augusto Villanueva-Rodriguez. Role of liquid biopsy in early detection and assessment of response in hepatocellular carcinoma [abstract]. In: Proceedings of the AACR Special Conference: Advances in the Pathogenesis and Molecular Therapies of Liver Cancer; 2022 May 5-8; Boston, MA. Philadelphia (PA): AACR; Clin Cancer Res 2022;28(17_Suppl):Abstract nr IA09.

Clinical relevance of the combined analysis of circulating tumor cells and anti-tumor T-cell immunity in metastatic breast cancer patients.

BackgroundMetastatic breast cancer (mBC) is a heterogeneous disease with varying responses to treatments and clinical outcomes, still requiring the identification of reliable predictive biomarkers. In this context, liquid biopsy has emerged as a powerful tool to assess in real-time the evolving landscape of cancer, which is both orchestrated by the metastatic process and immune-surveillance mechanisms. Thus, we investigated circulating tumor cells (CTCs) coupled with peripheral T-cell immunity to uncover their potential clinical relevance in mBC.MethodsA cohort of 20 mBC patients was evaluated, before and one month after starting therapy, through the following liquid biopsy approaches: CTCs enumerated by a metabolism-based assay, T-cell responses against tumor-associated antigens (TAA) characterized by interferon-γ enzyme-linked immunosorbent spot (ELISpot), and the T-cell receptor (TCR) repertoire investigated by a targeted next-generation sequencing technique. TCR repertoire features were characterized by the Morisita’s overlap and the Productive Simpson Clonality indexes, and the TCR richness. Differences between groups were calculated by Fisher’s, Mann-Whitney or Kruskal-Wallis test, as appropriate. Prognostic data analysis was estimated by Kaplan-Meier method.ResultsStratifying patients for their prognostic level of 6 CTCs before therapy, TAA specific T-cell responses were detected only in patients with a low CTC level. By analyzing the TCR repertoire, the highest TCR clonality was observed in the case of CTCs under the cut-off and a positive ELISpot response (p=0.03). Whereas, at follow-up, patients showing a good clinical response coupled with a low number of CTCs were characterized by the most elevated TCR clonality (p<0.05). The detection of CTCs≥6 in at least one time-point was associated with a lower TCR clonality (p=0.02). Intriguingly, by combining overall survival analysis with TCR repertoire, we highlighted a potential prognostic role of the TCR clonality measured at follow-up (p=0.03).ConclusionThese data, whether validated in a larger cohort of patients, suggest that the combined analysis of CTCs and circulating anti-tumor T-cell immunity could represent a valuable immune-oncological biomarker for the liquid biopsy field. The clinical application of this promising tool could improve the management of mBC patients, especially in the setting of immunotherapy, a rising approach for BC treatment requiring reliable predictive biomarkers.

CfDNA methylome profiling for detection and subtyping of small cell lung cancers

Small cell lung cancer (SCLC) is characterized by morphologic, epigenetic and transcriptomic heterogeneity. Subtypes based upon predominant transcription factor expression have been defined that, in mouse models and cell lines, exhibit potential differential therapeutic vulnerabilities, with epigenetically distinct SCLC subtypes also described. The clinical relevance of these subtypes is unclear, due in part to challenges in obtaining tumor biopsies for reliable profiling. Here we describe a robust workflow for genome-wide DNA methylation profiling applied to both patient-derived models and to patients’ circulating cell-free DNA (cfDNA). Tumor-specific methylation patterns were readily detected in cfDNA samples from patients with SCLC and were correlated with survival outcomes. cfDNA methylation also discriminated between the transcription factor SCLC subtypes, a precedent for a liquid biopsy cfDNA-methylation approach to molecularly subtype SCLC. Our data reveal the potential clinical utility of cfDNA methylation profiling as a universally applicable liquid biopsy approach for the sensitive detection, monitoring and molecular subtyping of patients with SCLC.

Potential utility of miRNAs for liquid biopsy in breast cancer.

Breast cancer (BC) remains the most prevalent malignancy due to its incidence rate, recurrence, and metastasis in women. Conventional strategies of cancer detection– mammography and tissue biopsy lack the capacity to detect the complete cancer genomic landscape. Besides, they often give false- positive or negative results. The presence of this and other disadvantages such as invasiveness, high-cost, and side effects necessitates developing new strategies to overcome the BC burden. Liquid biopsy (LB) has been brought to the fore owing to its early detection, screening, prognosis, simplicity of the technique, and efficient monitoring. Remarkably, microRNAs (miRNAs)– gene expression regulators seem to play a major role as biomarkers detected in the samples of LB. Particularly, miR-21 and miR-155 among other possible candidates seem to serve as favorable biomarkers in the diagnosis and prognosis of BC. Hence, this review will assess the potential utility of miRNAs as biomarkers and will highlight certain promising candidates for the LB approach in the diagnosis and management of BC that may optimize the patient outcome.

Profiling disease and tissue-specific epigenetic signatures in cell-free DNA

Abstract Programmed cell death, accidental cell degradation and active extrusion constantly lead to the release of DNA fragments into human body fluids from virtually all cell and tissue types. It is widely accepted that these cell-free DNA (cfDNA) molecules retain the cell-type specific genetic and epigenetic features. Particularly, cfDNA in plasma or serum has been utilized for molecular diagnostics. The current clinically implemented liquid biopsy approaches are mostly based on detecting genetic differences in cfDNA molecules from healthy and diseased cells. Their diagnostic potential is limited to pathologies involving genetic alterations, by the low proportion of cfDNA molecules carrying the mutation(s) relative to the total cfDNA pool, and by the detection limit of employed techniques. Recently, research efforts turned to epigenetic features of cfDNA molecules and found that the tissue-of-origin of individual cfDNA molecules can be inferred from epigenetic characteristics. Analysis of, e.g., methylation patterns, nucleosome or transcription factor binding site occupancies, fragment size distribution or fragment end motifs, and histone modifications determined the cell or tissue-of-origin of individual cfDNA molecules. With this tissue-of origin-analysis, it is possible to estimate the contributions of different tissues to the total cfDNA pool in body fluids and find tissues with increased cell death (pathologic condition), expanding the portfolio of liquid biopsies beyond genetics and towards a wide range of pathologies, such as autoimmune disorders, cardiovascular diseases, and inflammation, among many others. In this review, we give an overview on the status of tissue-of-origin approaches and focus on what is needed to exploit the full potential of liquid biopsies towards minimally invasive screening methods with broad clinical applications.

Applications of Liquid Biopsies in Non-Small-Cell Lung Cancer.

The concept of liquid biopsy as an analysis tool for non-solid tissue carried out for the purpose of providing information about solid tumors was introduced approximately 20 years ago. Additional to the detection of circulating tumor cells (CTCs), the liquid biopsy approach quickly included the analysis of circulating tumor DNA (ctDNA) and other tumor-derived markers such as circulating cell-free RNA or extracellular vesicles. Liquid biopsy is a non-invasive technique for detecting multiple cancer-associated biomarkers that is easy to obtain and can reflect the characteristics of the entire tumor mass. Currently, ctDNA is the key component of the liquid biopsy approach from the point of view of the prognosis assessment, prediction, and monitoring of the treatment of non-small-cell lung cancer (NSCLC) patients. ctDNA in NSCLC patients carries variants or rearrangements that drive carcinogenesis, such as those in EGFR, KRAS, ALK, or ROS1. Due to advances in pharmacology, these variants are the subject of targeted therapy. Therefore, the detection of these variants has gained attention in clinical medicine. Recently, methods based on qPCR (ddPCR, BEAMing) and next-generation sequencing (NGS) are the most effective approaches for ctDNA analysis. This review addresses various aspects of the use of liquid biopsy with an emphasis on ctDNA as a biomarker in NSCLC patients.

Microsatellite Instable Colorectal Adenocarcinoma Diagnostics: The Advent of Liquid Biopsy Approaches.

The introduction of immunotherapy has revolutionized the oncological targeted therapy paradigm. Microsatellite instability (MSI) identifies a subgroup of colorectal cancers (CRCs) which respond to treatment with immune checkpoint inhibitors. Tissue biopsy is currently the gold standard for the assessment of MSI/Mismatch Repair deficiency (MMRd) by means immunohistochemistry or molecular assays. However, the application of liquid biopsy in the clinic may help to overcome several limitations of tissue analysis and may provide great benefit to the diagnostic scenario and therapeutic decision-making process. In the context of MSI/MMRd CRC, the use of liquid biopsy may allow to establish MSI/MMR status if tissue sampling cannot be performed or in case of discordant tissue biopsies. Liquid biopsy may also become a powerful tool to monitor treatment response and the onset resistance to immunotherapy over time and to stratify of MSI/MMRd patients according to their risk of relapse and metastases. The aim of this review is to summarize the main technical aspects and clinical applications, the benefits, and limitations of the use of liquid biopsy in MSI/MMRd colorectal cancer patients.

Chaperonin containing TCP1 as a marker for identification of circulating tumor cells in blood

Herein we report the use of Chaperonin-Containing TCP-1 (CCT or TRiC) as a marker to detect circulating tumor cells (CTCs) that are shed from tumors during oncogenesis. Most detection methods used in liquid biopsy approaches for enumeration of CTCs from blood, employ epithelial markers like cytokeratin (CK). However, such markers provide little information on the potential of these shed tumor cells, which are normally short-lived, to seed metastatic sites. To identify a marker that could go beyond enumeration and provide actionable data on CTCs, we evaluated CCT. CCT is a protein-folding complex composed of eight subunits. Previously, we found that expression of the second subunit (CCT2 or CCTβ) inversely correlated with cancer patient survival and was essential for tumorigenesis in mice, driving tumor-promoting processes like proliferation and anchorage-independent growth. In this study, we examined CCT2 expression in cancer compared to normal tissues and found statistically significant increases in tumors. Because not all blood samples from cancer patients contain detectable CTCs, we used the approach of spiking a known number of cancer cells into blood from healthy donors to test a liquid biopsy approach using CCT2 to distinguish rare cancer cells from the large number of non-cancer cells in blood. Using a clinically validated method for capturing CTCs, we evaluated detection of intracellular CCT2 staining for visualization of breast cancer and small cell lung (SCLC) cancer cells. We demonstrated that CCT2 staining could be incorporated into a CTC capture and staining protocol, providing biologically relevant information to improve detection of cancer cells shed in blood. These results were confirmed with a pilot study of blood from SCLC patients. Our studies demonstrate that detection of CCT2 could identify rare cancer cells in blood and has application in liquid biopsy approaches to enhance the use of minimally invasive methods for cancer diagnosis.

Nano-omics: nanotechnology-based multidimensional harvesting of the blood-circulating cancerome.

Over the past decade, the development of 'simple' blood tests that enable cancer screening, diagnosis or monitoring and facilitate the design of personalized therapies without the need for invasive tumour biopsy sampling has been a core ambition in cancer research. Data emerging from ongoing biomarker development efforts indicate that multiple markers, used individually or as part of a multimodal panel, are required to enhance the sensitivity and specificity of assays for early stage cancer detection. The discovery of cancer-associated molecular alterations that are reflected in blood at multiple dimensions (genome, epigenome, transcriptome, proteome and metabolome) and integration of the resultant multi-omics data have the potential to uncover novel biomarkers as well as to further elucidate the underlying molecular pathways. Herein, we review key advances in multi-omics liquid biopsy approaches and introduce the 'nano-omics' paradigm: the development and utilization of nanotechnology tools for the enrichment and subsequent omics analysis of the blood-circulating cancerome.

Liquid Biopsy for Pancreatic Cancer Detection Using Infrared Spectroscopy.

Simple SummaryPancreatic tumors are extremely difficult to detect in their early stages due to the lack of early symptomatic evidence and fast, simple, non-invasive, and accurate diagnostic tests. Computed tomography (CT) scans are the usual first step in investigating suspected pancreatic cancer, but may not detect early tumors—especially in asymptomatic patients. Equally, the carbohydrate antigen (CA) 19-9 blood test is not specific to pancreatic cancer, as CA 19-9 levels can be raised in symptomatic patients with other non-malignant diseases, or with other tumors located in the surrounding area. In this proof-of-concept study, infrared spectroscopy was used to discriminate cancers versus healthy controls, as well as cancers versus symptomatic controls. Classification algorithms successfully discriminated the classes with high performances, accounting for detection rates of up to 92%. Simple, minimally invasive, and accurate approaches can represent a powerful aid in the achievement of pancreatic cancer’s earlier detection to improve patients’ prognosis and quality of life.Pancreatic cancer claims over 460,000 victims per year. The carbohydrate antigen (CA) 19-9 test is the blood test used for pancreatic cancer’s detection; however, its levels can be raised in symptomatic patients with other non-malignant diseases, or with other tumors in the surrounding area. Attenuated total reflection Fourier-transform infrared (ATR-FTIR) spectroscopy has demonstrated exceptional potential in cancer diagnostics, and its clinical implementation could represent a significant step towards early detection. This proof-of-concept study, investigating the use of ATR-FTIR spectroscopy on dried blood serum, focused on the discrimination of both cancer versus healthy control samples, and cancer versus symptomatic non-malignant control samples, as a novel liquid biopsy approach for pancreatic cancer diagnosis. Machine learning algorithms were applied, achieving results of up to 92% sensitivity and 88% specificity when discriminating between cancers (n = 100) and healthy controls (n = 100). An area under the curve (AUC) of 0.95 was obtained through receiver operating characteristic (ROC) analysis. Balanced sensitivity and specificity over 75%, with an AUC of 0.83, were achieved with cancers (n = 35) versus symptomatic controls (n = 35). Herein, we present these results as demonstration that our liquid biopsy approach could become a simple, minimally invasive, and reliable diagnostic test for pancreatic cancer detection.

Abstract 2695: Transcriptomic profiling of human patients with acute myeloid leukemia reveals critical effects of growth hormone responsiveness on patient survival and mechanisms of tumor therapy resistance

Abstract Growth hormone (GH) has been reported to drive leukemia cell proliferation as early as 1977, while downstream effectors of the GH signaling pathway (i.e., insulin-like growth factor 1; IGF1) have more recently been widely implicated in the aggressive pathology and robust therapy resistance phenotypes of acute myeloid leukemia (AML). It is also known that patients with acromegaly (high levels of circulating GH and IGF1) develop AML at a significantly higher rate than the general population, while individuals with GH resistance remain completely resistant to all types of cancer. Despite all of the aforementioned evidence pointing to a critical role of GH in AML progression and prognosis, a detailed transcriptomic analysis of human patients is lacking. We aim to address this gap by thorough in silico studies aimed at revealing the nature and extent to which the GH axis can be suitable targeted in AML. Here, we present a summary of our analyses of clinical and transcriptomic data from 200 human patients with AML from The Cancer Genome Atlas database, using multiple bioinformatics software programs in the public domain. Our study revealed that GH receptor (GHR) is expressed more highly in AML than in normal myeloid cells, and a robust correlation exists between GH responsiveness and patient survival. A sex-specific profiling of the patient transcriptomic data identified critical pathways related to therapy resistance, extracellular matrix remodeling, metastasis, and immune evasion to be upregulated differentially in male and female AML patients with relatively elevated GHR expression compared to patients with relatively lower GHR expression. Furthermore, we identified microRNAs which are differentially expressed in AML patients with higher than median GHR expression. We validated these miRNAs to be effective indicators of disease progression and prognosis and can be valuable markers in liquid biopsy approaches. Acknowledgement: This work was supported in part by the State of Ohio’s Eminent Scholar Program that includes a gift from Milton and Lawrence Goll, by the AMVETS, and Ohio University’s Edison Biotechnology Institute. Citation Format: Emily Davis, Reetobrata Basu, John J. Kopchick. Transcriptomic profiling of human patients with acute myeloid leukemia reveals critical effects of growth hormone responsiveness on patient survival and mechanisms of tumor therapy resistance [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 2695.

Abstract 5159: Towards population-scale screening of human cancer using genome-wide fragmentation profiles of cell-free DNA

Abstract Introduction: Earlier detection is a critical clinical intervention to reduce cancer-related mortality. The DELFI liquid biopsy approach (DNA evaluation of fragments for early interception) utilizing low coverage (1-2x) whole genome sequencing (WGS) to analyze cell-free DNA (cfDNA) fragmentation provides a promising avenue for cancer detection. As sequencing costs remain a barrier to adoption of liquid biopsy approaches for early detection, we evaluated WGS for DELFI using 2-channel Illumina NovaSeq sequencing as a more affordable (~7-fold cost savings) alternative to 4-channel HiSeq instruments. Methods: We performed WGS on the prospectively collected LUCAS cohort of 365 individuals at risk for lung cancer using both HiSeq and NovaSeq platforms (Mathios et. al., Nature Communications 2021). Genome-wide fragmentation was summarized in non-overlapping 5 Mb bins by ratio of short (100-150 bp) to long (151-220 bp) fragments.To measure within-sequencer repeatability, we compared fragmentation profiles of non-cancer individuals to the median non-cancer fragmentation profile by Spearman correlation. Principal component analyses were performed to assess the extent to which the sequencer explains variation of fragmentation profiles across samples.For cancer prediction, we used a penalized logistic regression model with fragmentation profiles and other genome-wide characteristics as features. Machine learning performance was assessed by cross-validation and area under the receiver operator characteristic curve (AUC). To evaluate whether we could have developed the classifier from a combination of NovaSeq with HiSeq sequenced samples, we evaluated performance trained on 90:10%, 75:25%, 50:50%, 25:75%, and 10:90% HiSeq:NovaSeq mixtures, respectively. Results: cfDNA fragmentation profiles were highly concordant among non-cancer individuals for both platforms with median correlations of 0.96 (IQR: 0.95 - 0.97) and 0.95 (IQR: 0.94 - 0.96). Visualization of fragmentation principal components did not reveal separation by sequencing platform. The DELFI approach applied to samples sequenced by NovaSeq recapitulated previously published performance measures based on HiSeq (AUC 0.90, 95% CI 0.86 - 0.94). In simulations of mixed-platform datasets, we found the same qualitative performance (AUC range: 0.893-0.902). Conclusions: cfDNA fragmentation profiles were similar between HiSeq and NovaSeq platforms, and classification accuracies from machine learning models trained on these platforms were equivalent. Our results indicate HiSeq and NovaSeq sequenced samples can be combined in models with no discernible loss in classification accuracy provided a balance of non-cancers and cancers are sequenced on both platforms. The lower cost of NovaSeq sequencing may enable wider adoption of genome-wide fragmentation-based approaches for cancer detection. Citation Format: Akshaya V. Annapragada, Dimitrios Mathios, Stephen Cristiano, Jamie E. Medina, Vilmos Adleff, Noushin Niknafs, Jacob Carey, Nic Dracopoli, Peter Bach, Jillian Phallen, Victor E. Velculescu, Robert B. Scharpf. Towards population-scale screening of human cancer using genome-wide fragmentation profiles of cell-free DNA [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 5159.