Mapping cancer biology in space: applications and perspectives on spatial omics for oncology.

Technologies to decipher cellular biology, such as bulk sequencing technologies and single-cell sequencing technologies, have greatly assisted novel findings in tumor biology. Recent findings in tumor biology suggest that tumors construct architectures that influence the underlying cancerous mechanisms. Increasing research has reported novel techniques to map the tissue in a spatial context or targeted sampling-based characterization and has introduced such technologies to solve oncology regarding tumor heterogeneity, tumor microenvironment, and spatially located biomarkers. In this study, we address spatial technologies that can delineate the omics profile in a spatial context, novel findings discovered via spatial technologies in oncology, and suggest perspectives regarding therapeutic approaches and further technological developments.

A Repurposing Programme Evaluating Transdermal Oestradiol Patches for the Treatment of Prostate Cancer Within the PATCH and STAMPEDE Trials: Current Results and Adapting Trial Design.

AIMS: Androgen deprivation therapy (ADT), usually achieved with luteinising hormone releasing hormone analogues (LHRHa), is central to prostate cancer management. LHRHa reduce both testosterone and oestrogen and are associated with significant long-term toxicity. Previous use of oral oestrogens as ADT was curtailed because of cardiovascular toxicity. Transdermal oestrogen (tE2) patches are a potential alternative ADT, supressing testosterone without the associated oestrogen-depletion toxicities (osteoporosis, hot flushes, metabolic abnormalities) and avoiding cardiovascular toxicity, and we here describe their evaluation in men with prostate cancer. MATERIALS AND METHODS: The PATCH (NCT00303784) adaptive trials programme (incorporating recruitment through the STAMPEDE [NCT00268476] platform) is evaluating the safety and efficacy of tE2 patches as ADT for men with prostate cancer. An initial randomised (LHRHa versus tE2) phase II study (n = 251) with cardiovascular toxicity as the primary outcome measure has expanded into a phase III evaluation. Those with locally advanced (M0) or metastatic (M1) prostate cancer are eligible. To reflect changes in both management and prognosis, the PATCH programme is now evaluating these cohorts separately. RESULTS: Recruitment is complete, with 1362 and 1128 in the M0 and M1 cohorts, respectively. Rates of androgen suppression with tE2 were equivalent to LHRHa, with improved metabolic parameters, quality of life and bone health indices (mean absolute change in lumbar spine bone mineral density of -3.0% for LHRHa and +7.9% for tE2 with an estimated difference between arms of 9.3% (95% confidence interval 5.3-13.4). Importantly, rates of cardiovascular events were not significantly different between the two arms and the time to first cardiovascular event did not differ between treatment groups (hazard ratio 1.11, 95% confidence interval 0.80-1.53; P = 0.54). Oncological outcomes are awaited. FUTURE: Efficacy results for the M0 cohort (primary outcome measure metastases-free survival) are expected in the final quarter of 2023. For M1 patients (primary outcome measure - overall survival), analysis using restricted mean survival time is being explored. Allied translational work on longitudinal samples is underway.

Differential impact of cytoplasmic vs. nuclear RAD51 expression on breast cancer progression and patient prognosis.

RAD51 recombinase is one of the DNA damage repair proteins associated with breast cancer risk. Apart from its function to maintain genomic integrity within the cell nucleus, RAD51 localized to the cytoplasm has also been implicated in breast malignancy. However, limited information exists on the roles of cytoplasmic vs. nuclear RAD51 in breast cancer progression and patient prognosis. In the present study, the association of cytoplasmic and nuclear RAD51 with clinical outcomes of patients with breast cancer was analyzed, revealing that elevated cytoplasmic RAD51 expression was associated with breast cancer progression, including increased cancer stage, grade, tumor size, lymph node metastasis and chemoresistance, along with reduced patient survival. By contrast, elevated nuclear RAD51 expression largely had the inverse effect. Results from in vitro investigations supported the cancer­promoting effect of RAD51, showing that overexpression of RAD51 promoted breast cancer cell growth, chemoresistance and metastatic ability, while knockdown of RAD51 repressed these malignant behaviors. The current data suggest that differential expression of subcellular RAD51 had a distinct impact on breast cancer progression and patient survival. Specifically, cytoplasmic RAD51 in contrast to nuclear RAD51 was potentially an adverse marker in breast cancer.

Hema-seq reveals genomic aberrations in a rare simultaneous occurrence of hematological malignancies.

Co-occurrence of multiple myeloma and acute myelogenous leukemia is rare, with both malignancies often tracing back to multipotent hematopoietic stem cells. Cytogenetic techniques are the established baseline for diagnosis and characterization of complex hematological malignancies. In this study, we develop a workflow called Hema-seq to delineate clonal changes across various hematopoietic lineages through the integration of whole-genome sequencing, copy-number variations, cell morphology, and cytogenetic aberrations. In Hema-seq, cells are selected from Wright-stained slides and fluorescent probe-stained slides for sequencing. This technique therefore enables direct linking of whole-genome sequences to cytogenetic profiles. Through this method, we mapped sequential clonal alterations within the hematopoietic lineage, identifying critical shifts leading to myeloma and acute myeloid leukemia (AML) cell formations. By synthesizing data from each cell lineage, we provided insights into the hematopoietic tree's clonal evolution. Overall, this study highlights Hema-seq's capability in deciphering genomic heterogeneity in complex hematological malignancies, which can enable better diagnosis and treatment strategies.

Barcoded multiple displacement amplification for high coverage sequencing in spatial genomics.

Determining mutational landscapes in a spatial context is essential for understanding genetically heterogeneous cell microniches. Current approaches, such as Multiple Displacement Amplification (MDA), offer high genome coverage but limited multiplexing, which hinders large-scale spatial genomic studies. Here, we introduce barcoded MDA (bMDA), a technique that achieves high-coverage genomic analysis of low-input DNA while enhancing the multiplexing capabilities. By incorporating cell barcodes during MDA, bMDA streamlines library preparation in one pot, thereby overcoming a key bottleneck in spatial genomics. We apply bMDA to the integrative spatial analysis of triple-negative breast cancer tissues by examining copy number alterations, single nucleotide variations, structural variations, and kataegis signatures for each spatial microniche. This enables the assessment of subclonal evolutionary relationships within a spatial context. Therefore, bMDA has emerged as a scalable technology with the potential to advance the field of spatial genomics significantly.

IL-1RA promotes oral squamous cell carcinoma malignancy through mitochondrial metabolism-mediated EGFR/JNK/SOX2 pathway.

BACKGROUND: Interleukin-1 receptor antagonist (IL-1RA), a member of the IL-1 family, has diverse roles in cancer development. However, the role of IL-1RA in oral squamous cell carcinoma (OSCC), in particular the underlying mechanisms, remains to be elucidated. METHODS: Tumor tissues from OSCC patients were assessed for protein expression by immunohistochemistry. Patient survival was evaluated by Kaplan-Meier curve analysis. Impact of differential IL-1RA expression on cultured OSCC cell lines was assessed in vitro by clonogenic survival, tumorsphere formation, soft agar colony formation, and transwell cell migration and invasion assays. Oxygen consumption rate was measured by Seahorse analyzer or multi-mode plate reader. PCR array was applied to screen human cancer stem cell-related genes, proteome array for phosphorylation status of kinases, and Western blot for protein expression in cultured cells. In vivo tumor growth was investigated by orthotopic xenograft in mice, and protein expression in xenograft tumors assessed by immunohistochemistry. RESULTS: Clinical analysis revealed that elevated IL-1RA expression in OSCC tumor tissues was associated with increased tumor size and cancer stage, and reduced survival in the patient group receiving adjuvant radiotherapy compared to the patient group without adjuvant radiotherapy. In vitro data supported these observations, showing that overexpression of IL-1RA increased OSCC cell growth, migration/invasion abilities, and resistance to ionizing radiation, whereas knockdown of IL-1RA had largely the opposite effects. Additionally, we identified that EGFR/JNK activation and SOX2 expression were modulated by differential IL-1RA expression downstream of mitochondrial metabolism, with application of mitochondrial complex inhibitors suppressing these pathways. Furthermore, in vivo data revealed that treatment with cisplatin or metformin-a mitochondrial complex inhibitor and conventional therapy for type 2 diabetes-reduced IL-1RA-associated xenograft tumor growth as well as EGFR/JNK activation and SOX2 expression. This inhibitory effect was further augmented by combination treatment with cisplatin and metformin. CONCLUSIONS: The current study suggests that IL-1RA promoted OSCC malignancy through mitochondrial metabolism-mediated EGFR/JNK activation and SOX2 expression. Inhibition of this mitochondrial metabolic pathway may present a potential therapeutic strategy in OSCC.

The STAMPEDE2 Trial: a Site Survey of Current Patterns of Care, Access to Imaging and Treatment of Metastatic Prostate Cancer.

AIMS: The forthcoming STAMPEDE2 trial has three comparisons in metastatic hormone-sensitive prostate cancer. We aim to determine clinical practices among STAMPEDE trial investigators for access to imaging and therapeutic choices and explore their interest in participation in STAMPEDE2. MATERIALS AND METHODS: The survey was developed and distributed online to 120 UK STAMPEDE trial sites. Recipients were invited to complete the survey between 16 and 30 May 2022. The survey consisted of 30 questions in five sections on access to stereotactic ablative body radiotherapy (SABR), 177lutetium-prostate-specific membrane antigen-617 (177Lu-PSMA-617), choice of systemic therapies and use of positron emission tomography/computerised tomography and whole-body magnetic resonance imaging. RESULTS: From 58/120 (48%) sites, 64 respondents completed the survey: 55/64 (86%) respondents were interested to participate in SABR, 44/64 (69%) in 177Lu-PSMA-617 and 56/64 (87.5%) in niraparib with abiraterone comparisons; 45/64 (70%) respondents had access to bone, spine and lymph node metastases SABR delivery and 7/64 (11%) to 177Lu-PSMA-617. In addition to androgen deprivation therapy, 60/64 (94%) respondents used androgen receptor signalling inhibitors and 46/64 (72%) used docetaxel; 29/64 (45%) respondents would consider triplet therapy with androgen deprivation therapy, androgen receptor signalling inhibitors and docetaxel. Positron emission tomography/computerised tomography was available to 62/64 (97%) respondents and requested by 45/64 (70%) respondents for disease uncertainty on conventional imaging and 39/64 (61%) at disease relapse. Whole-body magnetic resonance imaging was available to 24/64 (38%) respondents and requested by 13/64 (20%) respondents in highly selected patients. In low-volume disease, 38/64 (59%) respondents requested scans at baseline and disease relapse. In high-volume disease, 29/64 (45%) respondents requested scans at baseline, best response (at prostate-specific antigen nadir) and disease relapse; 54/64 (84%) respondents requested computerised tomography and bone scan for best response assessment. CONCLUSION: There is noteworthy disparity in clinical practice across current study sites, however most have expressed an interest in participation in the forthcoming STAMPEDE2 trial.

ADSCs stimulated by resistin promote breast cancer cell malignancy via CXCL5 in a breast cancer coculture model.

The tumor microenvironment represents one of the main obstacles in breast cancer treatment owing to the presence of heterogeneous stromal cells, such as adipose-derived stem cells (ADSCs), that may interact with breast cancer cells and promote cancer development. Resistin is an adipocytokine associated with adverse breast cancer progression; however, its underlying mechanisms in the context of the breast tumor microenvironment remain largely unidentified. Here, we utilized a transwell co-culture model containing patient-derived ADSCs and breast cancer cell lines to investigate their potential interaction, and observed that breast cancer cells co-cultured with resistin-treated ADSCs (R-ADSCs) showed enhanced cancer cell growth and metastatic ability. Screening by proteome arrays revealed that C-X-C motif chemokine ligand 5 (CXCL5) was released in the conditioned medium of the co-culture system, and phosphorylated ERK was increased in breast cancer cells after co-culture with R-ADSCs. Breast cancer cells treated with the recombinant proteins of CXCL5 showed similarly enhanced cell migration and invasion ability as occurred in the co-culture model, whereas application of neutralizing antibodies against CXCL5 reversed these phenomena. The orthotopic xenograft in mice by breast cancer cells after co-culture with R-ADSCs had a larger tumor growth and more CXCL5 expression than control. In addition, clinical analysis revealed a positive correlation between the expression of resistin and CXCL5 in both tumor tissues and serum specimens of breast cancer patients. The current study suggests that resistin-stimulated ADSCs may interact with breast cancer cells in the tumor microenvironment via CXCL5 secretion, leading to breast cancer cell malignancy.

CD44 Mediates Oral Squamous Cell Carcinoma-Promoting Activity of MRE11 via AKT Signaling.

Oral cancer is one of the highest-incidence malignancies worldwide, with the occurrence of oral squamous cell carcinoma (OSCC) being the most frequently diagnosed form. A barrier for oral cancer management may arise from tumor cells that possess properties of cancer stemness, which has been recognized as a crucial factor in tumor recurrence and metastasis. As such, understanding the molecular mechanisms underlying these tumor cells may provide insights for improving cancer treatment. MRE11 is the core protein of the RAD50/MRE11/NBS1 complex with a primary role in DNA damage repair, and it has been diversely associated with tumor development including OSCC. In this study, we aimed to investigate the engagement of CD44, a cancer stemness marker functioning in the control of cell growth and motility, in OSCC malignancy under the influence of MRE11. We found that overexpression of MRE11 enhanced CD44 expression and tumorsphere formation in OSCC cells, whereas knockdown of MRE11 reduced these phenomena. In addition, the MRE11-promoted tumorsphere formation or cell migration ability was compromised in OSCC cells carrying siRNA that targets CD44, as was the MRE11-promoted AKT phosphorylation. These were further supported by analyzing clinical samples, where higher CD44 expression was associated with lymph node metastasis. Additionally, a positive correlation between the expression of MRE11 and CD44, or that of CD44 and phosphorylated AKT, was observed in OSCC tumor tissues. Finally, the expression of CD44 was found to be higher in the metastatic lung nodules from mice receiving tail vein-injection with MRE11-overexpressing OSCC cells compared with control mice, and a positive correlation between CD44 and phosphorylated AKT was also observed in these metastatic lung nodules. Altogether, our current study revealed a previously unidentified mechanism linking CD44 and AKT in MRE11-promoted OSCC malignancy, which may shed light to the development of novel therapeutic strategies in consideration of this new pathway in OSCC.

Spatial epitranscriptomics reveals A-to-I editome specific to cancer stem cell microniches.

Epitranscriptomic features, such as single-base RNA editing, are sources of transcript diversity in cancer, but little is understood in terms of their spatial context in the tumour microenvironment. Here, we introduce spatial-histopathological examination-linked epitranscriptomics converged to transcriptomics with sequencing (Select-seq), which isolates regions of interest from immunofluorescence-stained tissue and obtains transcriptomic and epitranscriptomic data. With Select-seq, we analyse the cancer stem cell-like microniches in relation to the tumour microenvironment of triple-negative breast cancer patients. We identify alternative splice variants, perform complementarity-determining region analysis of infiltrating T cells and B cells, and assess adenosine-to-inosine base editing in tumour tissue sections. Especially, in triple-negative breast cancer microniches, adenosine-to-inosine editome specific to different microniche groups is identified.

Reduced tissue and serum resistin expression as a clinical marker for esophageal squamous cell carcinoma.

Esophageal cancer is one of the most common malignancies and leading cause of cancer-associated mortality worldwide. However, the molecular mechanisms underlying esophageal cancer progression and the development of clinical tools for effective diagnosis remain unclear. Resistin, which was originally identified as an adipose tissue-secretory factor, has been associated with obesity-related diseases, including certain types of cancer. Thus, the present study aimed to investigate the expression levels of resistin in tissue and serum specimens from patients with esophageal squamous cell carcinoma (ESCC) to determine the potential biological effects of resistin on ESCC cells. The results demonstrated that both tissue and serum resistin levels were significantly lower in patients with ESCC compared with healthy controls. In addition, resistin expression was positively associated with the body mass index of patients with ESCC. In vitro studies revealed that resistin inhibited the migratory ability of ESCC cells, while having no effect on ESCC cell proliferation. Taken together, these results suggest that resistin may have the potential to be developed into a clinical marker for ESCC. However, further studies are required to investigate resistin receptor expression and determine the potential involvement of resistin-associated biological pathways, which may provide insight for future development of targeted therapies for resistin-mediated ESCC.

Photopatterned microswimmers with programmable motion without external stimuli.

We introduce highly programmable microscale swimmers driven by the Marangoni effect (Marangoni microswimmers) that can self-propel on the surface of water. Previous studies on Marangoni swimmers have shown the advantage of self-propulsion without external energy source or mechanical systems, by taking advantage of direct conversion from power source materials to mechanical energy. However, current developments on Marangoni microswimmers have limitations in their fabrication, thereby hindering their programmability and precise mass production. By introducing a photopatterning method, we generated Marangoni microswimmers with multiple functional parts with distinct material properties in high throughput. Furthermore, various motions such as time-dependent direction change and disassembly of swimmers without external stimuli are programmed into the Marangoni microswimmers.

Nasopharyngeal Type-I Interferon for Immediately Available Prophylaxis Against Emerging Respiratory Viral Infections.

In addition to SARS-CoV-2 and its variants, emerging viruses that cause respiratory viral infections will continue to arise. Increasing evidence suggests a delayed, possibly suppressed, type 1 interferon (IFN-I) response occurs early during COVID-19 and other viral respiratory infections such as SARS and MERS. These observations prompt considering IFN-ß as a prophylactic or early intervention for respiratory viral infections. A rationale for developing and testing intranasal interferon beta (IFN-ß) as an immediately available intervention for new respiratory viral infections that will arise unexpectedly in the future is presented and supported by basic and clinical trial observations. IFN-ß prophylaxis could limit the spread and consequences of an emerging respiratory viral infection in at-risk individuals while specific vaccines are being developed.

MRE11 as a molecular signature and therapeutic target for cancer treatment with radiotherapy.

MRE11, the core of the MRE11/RAD50/NBS1 complex, is one of key DNA damage response proteins. Increasing evidence suggests that its expression in cancer cells is critical to developing radioresistance; as such, MRE11 is an emerging marker for targeted radiosensitization strategies. Elevated MRE11 in tumor tissues has been associated with poor survival in patients undergoing radiotherapy, although in some cancer types, the opposite has been noted. The recent discovery of ionizing radiation-induced truncation of MRE11, which decreases its efficacy, may explain some of these paradoxical findings. The progress of research on the biological modulation of MRE11 expression is also discussed, with the potential application of small molecule or large molecule inhibitors of MRE11 for enhancing radiosensitivity. Current research has further highlighted both nuclease and non-nuclease activities of MRE11 in cancer cells treated with ionizing radiation, and differentiation between these is essential to verify the targeting effects of radiosensitizing agents. These updates clarify our understanding of how MRE11 expression may be utilized in future stratification of cancer patients for radiotherapy, and how it may be leveraged in shaping novel radiosensitization strategies.

Adipocytokines visfatin and resistin in breast cancer: Clinical relevance, biological mechanisms, and therapeutic potential.

Obesity is one of the major modifiable risk factors in breast cancer, with obese adipose tissue showing a pathological role in breast cancer development and malignancy via the release of secretory factors, such as proinflammatory cytokines and adipocytokines. The current article focuses on visfatin and resistin, two such adipocytokines that have emerged over the last two decades as leading breast cancer promoting factors in obesity. The clinical association of circulating visfatin and resistin with breast cancer and their biological mechanisms are reviewed, in addition to their role in the context of tumor-stromal interactions in the breast cancer microenvironment. Recent findings have unraveled several mediators of visfatin and resistin that are involved in the crosstalk between breast cancer cells and adipose tissue in the breast tumor microenvironment, including growth differentiation factor 15 (GDF15), interleukin 6 (IL-6), and toll-like receptor 4 (TLR4). Finally, current therapeutics targeting visfatin and resistin and their respective pathways are discussed, including future therapeutic strategies such as new drug design or neutralizing peptides that target extracellular visfatin or resistin. These hold promise in the development of novel breast cancer therapies and are of increasing relevance as the prevalence of obesity-related breast cancer increases worldwide.

Visfatin Enhances Breast Cancer Progression through CXCL1 Induction in Tumor-Associated Macrophages.

Visfatin, an adipocytokine highly expressed in breast tumor tissues, is associated with breast cancer progression. Recent studies showed that adipocytokines mediate tumor development through adipocytokine tumor-stromal interactions in the tumor microenvironment. This study focused on the interaction between one key stromal constituent-tumor-associated macrophages-and visfatin. Pretreatment of THP-1 and peripheral blood mononuclear cells (PBMCs) with recombinant visfatin resulted in M2-polarization determined by CD163 and CD206 expression. Indirect co-culture with visfatin-treated THP-1 (V-THP-1) promoted the viability, migration, tumorsphere formation, EMT, and stemness of breast cancer cells. Cytokine array identified an increased CXCL1 secretion in V-THP-1 conditioned medium and recombinant CXCL1 enhanced cell migration and invasion, which were abrogated by the CXCL1-neutralizing antibody. Additionally, visfatin induced pERK in THP-1 cells and clinical samples confirmed a positive CXCL1/pERK correlation. In an orthotopic mouse model, the tumor bioluminescent signal of luciferase-expressing MDA-MB-231 (Luc-MDA-MB-231) cells co-cultured with V-THP-1 and the expression of proliferation marker Ki67 were significantly higher than that co-cultured with THP-1. Furthermore, tail vein-injected Luc-MDA-MB-231 pretreated with V-PBMCs conditioned medium metastasized to lungs more frequently compared to control, and this was reversed by CXCL1 blocking antibody. In summary, this study demonstrated that visfatin enhanced breast cancer progression via pERK/CXCL1 induction in macrophages.

DNA Micro-Disks for the Management of DNA-Based Data Storage with Index and Write-Once-Read-Many (WORM) Memory Features.

DNA-based data storage has attracted attention because of its higher physical density of the data and longer retention time than those of conventional digital data storage. However, previous DNA-based data storage lacked index features and the data quality of storage after a single access was not preserved, obstructing its industrial use. Here, DNA micro-disks, QR-coded micro-sized disks that harbor data-encoded DNA molecules for the efficient management of DNA-based data storage, are proposed. The two major features that previous DNA-based data-storage studies could not achieve are demonstrated. One feature is accessing data items efficiently by indexing the data-encoded DNA library. Another is achieving write-once-read-many (WORM) memory through the immobilization of DNA molecules on the disk and their enrichment through in situ DNA production. Through these features, the reliability of DNA-based data storage is increased by allowing selective and multiple accession of data-encoded DNA with lower data loss than previous DNA-based data storage methods.

Trust as a mediator in the relationship between childhood sexual abuse and IL-6 level in adulthood.

Childhood sexual abuse (CSA) has been shown to predict the coupling of depression and inflammation in adulthood. Trust within intimate relationships, a core element in marital relations, has been shown to predict positive physical and mental health outcomes, but the mediating role of trust in partners in the association between CSA and inflammation in adulthood requires further study. The present study aimed to examine the impact of CSA on inflammatory biomarkers (IL-6 and IL-1ß) in adults with depression and the mediating role of trust. A cross-sectional survey data set of adults presenting with mood and sleep disturbance was used in the analysis. CSA demonstrated a significant negative correlation with IL-6 level (r = -0.28, p<0. 01) in adults with clinically significant depression, while trust showed a significant positive correlation with IL-6 level (r = 0.36, p < .01). Sobel test and bootstrapping revealed a significant mediating role for trust between CSA and IL-6 level. CSA and trust in partners were revealed to have significant associations with IL-6 level in adulthood. Counterintuitively, the directions of association were not those expected. Trust played a mediating role between CSA and adulthood levels of IL-6. Plausible explanations for these counterintuitive findings are discussed.

OPENchip: an on-chip in situ molecular profiling platform for gene expression analysis and oncogenic mutation detection in single circulating tumour cells.

Liquid biopsy holds promise towards practical implementation of personalized theranostics of cancer. In particular, circulating tumour cells (CTCs) can provide clinically actionable information that can be directly linked to prognosis or therapy decisions. In this study, gene expression patterns and genetic mutations in single CTCs are simultaneously analysed by strategically combining microfluidic technology and in situ molecular profiling technique. Towards this, the development and demonstration of the OPENchip (On-chip Post-processing ENabling chip) platform for single CTC analysis by epithelial CTC enrichment and subsequent in situ molecular profiling is reported. For in situ molecular profiling, padlock probes that identify specific desired targets to examine biomarkers of clinical relevance in cancer diagnostics were designed and used to create libraries of rolling circle amplification products. We characterize the OPENchip in terms of its capture efficiency and capture purity, and validate the probe design using different cell lines. By integrating the obtained results, molecular analyses of CTCs from metastatic breast cancer (HER2 (ERBB2) gene expression and PIK3CA mutations) and metastatic pancreatic cancer (KRAS gene mutations) patients were demonstrated without any off-chip processes. The results substantiate the potential implementation of early molecular detection of cancer through sequencing-free liquid biopsy.