Tracking early lung cancer metastatic dissemination in TRACERx using ctDNA.

Circulating tumour DNA (ctDNA) can be used to detect and profile residual tumour cells persisting after curative intent therapy1. The study of large patient cohorts incorporating longitudinal plasma sampling and extended follow-up is required to determine the role of ctDNA as a phylogenetic biomarker of relapse in early-stage non-small-cell lung cancer (NSCLC). Here we developed ctDNA methods tracking a median of 200 mutations identified in resected NSCLC tissue across 1,069 plasma samples collected from 197 patients enrolled in the TRACERx study2. A lack of preoperative ctDNA detection distinguished biologically indolent lung adenocarcinoma with good clinical outcome. Postoperative plasma analyses were interpreted within the context of standard-of-care radiological surveillance and administration of cytotoxic adjuvant therapy. Landmark analyses of plasma samples collected within 120 days after surgery revealed ctDNA detection in 25% of patients, including 49% of all patients who experienced clinical relapse; 3 to 6 monthly ctDNA surveillance identified impending disease relapse in an additional 20% of landmark-negative patients. We developed a bioinformatic tool (ECLIPSE) for non-invasive tracking of subclonal architecture at low ctDNA levels. ECLIPSE identified patients with polyclonal metastatic dissemination, which was associated with a poor clinical outcome. By measuring subclone cancer cell fractions in preoperative plasma, we found that subclones seeding future metastases were significantly more expanded compared with non-metastatic subclones. Our findings will support (neo)adjuvant trial advances and provide insights into the process of metastatic dissemination using low-ctDNA-level liquid biopsy.

Genetic and phenotypic profiling of single living circulating tumor cells from patients with microfluidics.

Accurate prediction of the efficacy of immunotherapy for cancer patients through the characterization of both genetic and phenotypic heterogeneity in individual patient cells holds great promise in informing targeted treatments, and ultimately in improving care pathways and clinical outcomes. Here, we describe the nanoplatform for interrogating living cell host-gene and (micro-)environment (NICHE) relationships, that integrates micro- and nanofluidics to enable highly efficient capture of circulating tumor cells (CTCs) from blood samples. The platform uses a unique nanopore-enhanced electrodelivery system that efficiently and rapidly integrates stable multichannel fluorescence probes into living CTCs for in situ quantification of target gene expression, while on-chip coculturing of CTCs with immune cells allows for the real-time correlative quantification of their phenotypic heterogeneities in response to immune checkpoint inhibitors (ICI). The NICHE microfluidic device provides a unique ability to perform both gene expression and phenotypic analysis on the same single cells in situ, allowing us to generate a predictive index for screening patients who could benefit from ICI. This index, which simultaneously integrates the heterogeneity of single cellular responses for both gene expression and phenotype, was validated by clinically tracing 80 non-small cell lung cancer patients, demonstrating significantly higher AUC (area under the curve) (0.906) than current clinical reference for immunotherapy prediction.

Proteome Fishing for CRISPR/Cas12a-Based Orthogonal Multiplex Aptamer Sensing.

Detection of serum protein biomarkers is extremely challenging owing to the superior complexity of serum. Here, we report a method of proteome fishing from the serum. It uses a magnetic nanoparticle-protein corona and a multiplexed aptamer panel, which we incubated with the nanoparticle-protein corona for biomarker recognition. To transfer protein biomarker detection to aptamer detection, we established a CRISPR/Cas12a-based orthogonal multiplex aptamer sensing (COMPASS) platform by profiling the aptamers of protein corona with clinical nonsmall cell lung cancer (NSCLC) serum samples. Furthermore, we determined the four out of nine (FOON) panel (including HE4, NSE, AFP, and VEGF165) to be the most cost-effective and accurate panel for COMPASS in NSCLC diagnosis. The diagnostic accuracy of NSCLC by the FOON panel with internal and external cohorts was 95.56% (ROC-AUC = 99.40%) and 89.58% (ROC-AUC = 95.41%), respectively. Our developed COMPASS technology circumvents the otherwise challenging multiplexed serum protein amplification problem and avoids aptamer degradation in serum. Therefore, this novel COMPASS could lead to the development of a facile, cost-effective, intelligent, and high-throughput diagnostic platform for large-cohort cancer screening.

Red blood cells function as reservoirs of tumor DNA.

Novel screening techniques for early detection of lung cancer are urgently needed. Profiling circulating tumor cell-free DNA (ctDNA) has emerged as a promising tool for biopsy-free tumor genotyping. However, both the scarcity and short half-life of ctDNA substantially limit the sensitivity and clinical utility of ctDNA detection methodologies. Our discovery that red blood cells (RBCs) sequester mitochondrial DNA opens a new avenue for detecting circulating nucleic acids, as RBCs represent an unrecognized reservoir of circulating nucleic acid. Here, we show that RBCs acquire tumor DNA following coculture with lung cancer cell lines harboring Kirsten rat sarcoma viral oncogene homolog (KRAS) and epidermal growth factor receptor (EGFR) mutations. RBC-bound tumor DNA is detectable in patients with early-stage non-small cell lung cancer (NSCLC) but not in healthy controls by qPCR. Our results collectively uncover a previously unrecognized yet easily accessible reservoir of tumor DNA, offering a promising foundation for future RBC-based tumor diagnostics.NEW & NOTEWORTHY We present a novel method for lung cancer detection by revealing RBCs as a reservoir for tumor DNA, overcoming the limitations of current circulating tumor ctDNA methodologies. By demonstrating that RBCs can capture tumor DNA, including critical mutations found in lung cancer, we provide a promising, biopsy-free avenue for early cancer diagnostics. This discovery opens up exciting possibilities for developing RBC-based diagnostic tools, significantly enhancing the sensitivity and clinical utility of noninvasive cancer detection.

Detection of circulating tumor DNA with ultradeep sequencing of plasma cell-free DNA for monitoring minimal residual disease and early detection of recurrence in early-stage lung cancer.

BACKGROUND: In early-stage non-small cell lung cancer (NSCLC), recurrence is frequently observed. Circulating tumor DNA (ctDNA) has emerged as a noninvasive tool to risk stratify patients for recurrence after curative intent therapy. This study aimed to risk stratify patients with early-stage NSCLC via a personalized, tumor-informed multiplex polymerase chain reaction (mPCR) next-generation sequencing assay. METHODS: This retrospective cohort study included patients with stage I-III NSCLC. Recruited patients received standard-of-care management (surgical resection with or without adjuvant chemotherapy, followed by surveillance). Whole-exome sequencing of NSCLC resected tissue and matched germline DNA was used to design patient-specific mPCR assays (Signatera, Natera, Inc) to track up to 16 single-nucleotide variants in plasma samples. RESULTS: The overall cohort with analyzed plasma samples consisted of 57 patients. Stage distribution was 68% for stage I and 16% each for stages II and III. Presurgery (i.e., at baseline), ctDNA was detected in 15 of 57 patients (26%). ctDNA detection presurgery was significantly associated with shorter recurrence-free survival (RFS; hazard ratio [HR], 3.54; 95% confidence interval [CI], 1.00-12.62; p = .009). In the postsurgery setting, ctDNA was detected in seven patients, of whom 100% experienced radiological recurrence. ctDNA positivity preceded radiological findings by a median lead time of 2.8 months (range, 0-12.9 months). Longitudinally, ctDNA detection at any time point was associated with shorter RFS (HR, 16.1; 95% CI, 1.63-158.9; p < .0001). CONCLUSIONS: ctDNA detection before surgical resection was strongly associated with a high risk of relapse in early-stage NSCLC in a large unique Asian cohort. Prospective studies are needed to assess the clinical utility of ctDNA status in this setting.

Digital Circulating Tumor Cells Quantification.

Circulating tumor cells (CTCs) are an emerging but vital biomarker for cancer management. An efficient methodology for accurately quantifying CTCs remains challenging due to their rareness. Here, we develop a digital CTC detection strategy using partitioning instead of enrichment to quantify CTCs. By utilizing the characteristics of droplet microfluidics that can rapidly generate a large number of parallel independent reactors, combined with Poisson distribution, we realize the quantification of CTCs in the blood directly. The limit of detection of our digital CTCs quantification assay is five cells per 5 mL of whole blood. By simultaneously detecting multiple genetic mutations, our approach achieves highly sensitive and specific detection of CTCs in peripheral blood from NSCLC patients (AUC = 1). Our digital platform offers a potential approach and strategy for the quantification of CTCs, which could contribute to the advancement of cancer medical management.

Ultrasensitive Method Enables Liquid Biopsy for the Precise Detection of Circulating MicroRNAs.

Due to invasive and serial examinations of bioactive molecules, liquid biopsy (LB) has emerged as a rapid and reliable solution for early disease detection and monitoring. Developing portable devices with high specificity and sensitivity for LB is highly valuable. To realize a generalized approach to increase the sensitivity of LB, we developed an ultrasensitive diagnostic biochip based on the amplification of miRNA by recombinase polymerase amplification and the significant enhancement of fluorescence signals by photonic crystal (PC) materials. The PCs-RPA biochip has a detection limit as low as 0.24 aM, a wide linear range of 8 orders of magnitude, and excellent specificity. Such advantages realize the accurate detection of circulating miRNAs with very low content in clinical serum samples for the precise diagnosis of nonsmall cell lung cancer.

Exosome-transported circ_0061407 and circ_0008103 play a tumour-repressive role and show diagnostic value in non-small-cell lung cancer.

BACKGROUND: Circular RNAs (circRNAs), one of the major contents of exosomes, have been shown to participate in the occurrence and progression of cancers. The role and the diagnostic potential of exosome-transported circRNAs in non-small-cell lung cancer (NSCLC) remain largely unknown. METHODS: The NSCLC-associated exosomal circ_0061407 and circ_0008103 were screened by circRNA microarray. The role of circ_0061407 and circ_0008103 in NSCLC was examined in vitro and in vivo. The encapsulation of the two circRNAs into exosomes and the transport to recipient cells were observed by confocal microscopy. The effects of exosome-transported circ_0061407 and circ_0008103 on recipient cells were investigated using a co-culture device. Bioinformatics analyses were performed to predict the mechanisms by which circ_0061407 and circ_0008103 affected NSCLC. The quantitative polymerase chain reaction was used to quantify the exosome-containing circ_0061407 and circ_0008103 in the serum samples of healthy, pneumonia, benign lung tumours, and NSCLC. The diagnostic efficacy was evaluated using receiver operating characteristic curves. RESULTS: The levels of circ_0061407 and circ_0008103 within exosomes were down-regulated in the serum of patients with NSCLC. The up-regulation of circ_0061407 and circ_0008103 inhibited the proliferation, migration/invasion, cloning formation of NSCLC cells in vitro and inhibited lung tumour growth in vivo. Circ_0061407 and circ_0008103 were observed to be packaged in exosomes and transported to recipient cells, where they inhibited the proliferation, migration/invasion, and cloning formation abilities of the recipient cells. Moreover, circ_0061407 and circ_0008103 might be involved in the progression of NSCLC by interacting with microRNAs and proteins. Additionally, lower serum exosomal circ_0061407 and circ_0008103 levels were associated with advanced pathological staging and distant metastasis. CONCLUSIONS: This study identified two novel exosome-transported circRNAs (circ_0061407 and circ_0008103) associated with NSCLC. These findings may provide additional insights into the development of NSCLC and potential diagnostic biomarkers for NSCLC.

Integrated omics characterization reveals reduced cancer indicators and elevated inflammatory factors after thermal ablation in non-small cell lung cancer patients.

BACKGROUND: Thermal ablation is a minimally invasive treatment for non-small cell lung cancer (NSCLC). Aside from causing an immediate direct tumour cell injury, the effects of thermal ablation on the internal microenvironment are unknown. This study aimed to investigate the effects of thermal ablation on the plasma internal environment in patients with NSCLC. METHODS: 128 plasma samples were collected from 48 NSCLC (pre [LC] and after thermal ablation [LC-T]) patients and 32 healthy controls (HCs). Olink proteomics and metabolomics were utilized to construct an integrated landscape of the cancer-related immune and inflammatory responses after ablation. RESULTS: Compared with HCs, LC patients exhibited 58 differentially expressed proteins (DEPs) and 479 differentially expressed metabolites (DEMs), which might participate in tumour progression and metastasis. Moreover, 75 DEPs were identified among the HC, LC, and LC-T groups. Forty-eight highly expressed DEPs (eg, programmed death-ligand 1 [PD-L1]) in the LC group were found to be downregulated after thermal ablation. These DEPs had significant impacts on pathways such as angiogenesis, immune checkpoint blockade, and pro-tumour chemotaxis. Metabolites involved in tumour cell survival were associated with these proteins at the expression and functional levels. In contrast, 19 elevated proteins (eg, interleukin [IL]-6) were identified after thermal ablation. These proteins were mainly associated with inflammatory response pathways (NF-κB signalling and tumour necrosis factor signalling) and immune cell activation. CONCLUSIONS: Thermal ablation-induced changes in the host plasma microenvironment contribute to anti-tumour immunity in NSCLC, offering new insights into tumour ablation combined with immunotherapy. Trial registration This study was registered on the Chinese Clinical Trial Registry ( https://www.chictr.org.cn/index.html ). ID: ChiCTR2300076517. Registration Date: 2023-10-11.

Therapeutic drug monitoring of osimertinib in EGFR mutant non-small cell lung cancer by dried blood spot and plasma collection: A pilot study.

AIMS: Therapeutic drug monitoring (TDM) has led to significant improvements in individualized medical care, although its implementation in oncology has been limited to date. Tyrosine kinase inhibitors (TKIs) are a group of therapies for which TDM has been suggested. Osimertinib is one such therapy used in the treatment of epidermal growth factor receptor (EGFR) mutation-driven lung cancer. Herein, we describe a prospective pilot study involving 21 patients on osimertinib primarily as a preliminary evaluation of drug levels in a real-world setting. METHODS: Concentrations of the drug and its primary metabolites were measured with a validated liquid chromatography-mass spectrometry (LC-MS) assay across serial timepoints. As part of this study, inter-individual variability by dose and ethnicity as well as intra-individual variability across timepoints are explored. Furthermore, we attempted to validate dried blood spot (DBS)-based quantitation as an accurate alternative to plasma quantitation. RESULTS: Successful quantitation of osimertinib and primary metabolites was achieved for our subjects. Compound plasma levels were highly correlated to DBS levels. There was no significant difference in concentrations with ethnicity or dosing or intra-individual variability across timepoints. CONCLUSIONS: As such, we demonstrate that TDM for osimertinib is practical for future trials. We also validated the use of DBS as an alternative to conventional quantitation for exploration of TDM for osimertinib in larger trials and for other targeted therapies.

Serum IL-24 combined with CA125 as screening and prognostic biomarkers for NSCLC.

Noninvasive and effective methods for early screening of non-small cell lung cancer (NSCLC) still need to be developed. At present, a reasonable conclusion is that a combination of tumor markers is a superior predictor of screening. Cytokines, as important regulators of cancer development, have great potential for the screening and prognosis of NSCLC. This study screened novel biomarkers related to the early screening and prognosis of NSCLC. In the present study, the biological significance and immunoregulation of interleukin-24 (IL-24) were analyzed based on The Cancer Genome Atlas data. Next, 150 serum samples from initially treated patients with NSCLC and 70 controls were collected, and we obtained pathological sections from 60 patients with NSCLC. The ELISA and immunohistochemistry results showed the differential expression of IL-24 and carbohydrate antigen 125 (CA125). The results show that IL-24 is an important tumor suppressor in NSCLC that helps to improve the poor prognosis of these patients. A significantly negative correlation between IL-24 and CA125 levels was also found. Notably, serum IL-24 levels were significantly negatively correlated with the TNM stage of patients with NSCLC, consistent with an important role for tumor suppressors in NSCLC. The receiver operating characteristic curve analysis showed that a combination of IL-24 and CA125 was an effective panel for discriminating patients with NSCLC from HD, and individuals with other lung diseases. Serum IL-24 and CA125 levels were identified as independent prognostic markers for NSCLC. The IL-24 and CA125 panel exhibited good performance in the screening of NSCLC.

Diagnostic potential of protein serum biomarkers for distinguishing small and non-small cell lung cancer in patients with suspicious lung lesions.

BACKGROUND: Biomarkers play a role in identifying, managing, and predicting cancer outcomes. In lung cancer, they are used at various time points. Doubts remain regarding their accuracy for differential diagnosis and histological subtyping. A diagnostic test study was conducted. It included malignant lesions and controls with benign lesions. Before lung biopsy, all patients had the following biomarkers measured in serum (Pro-GRP,NSE,CYFRA21-1,SCC-Ag,CEA). METHODS: The predictive capacity of serum biomarkers was evaluated to discriminate between lung cancer and benign pathology. The accuracy was also assessed for distinguishing between SCLC and NSCLC and explored their ability to perform histological subtyping. RESULTS: 93 patients were included, 60 with lung cancer, 33 with benign pathology. Pro-GRP and NSE were elevated in SCLC compared with NSCLC or nonmalignant disease. The most accurate for differentiating between malignant and benign pathology were CEA and CYFRA21-1. Pro-GRP had a poor predictive capacity for distinguishing NSCLC from SCLC. However, combined with CEA and CYFRA21-1, performance improved. For SCLC, the diagnostic capacity of Pro-GRP increased by combining with biomarkers, such as NSE/CYFRA21-1. CONCLUSIONS: Biomarkers lacked the sensitivity and specificity for independent differential diagnosis or histological subtyping. However, the observed patterns in biomarker levels associated with specific histological subtypes suggest potential utility in a multi-biomarker approach or in conjunction with other diagnostic tools. This insight could guide future research to improve diagnostic accuracy and personalized treatment strategies in lung cancer.

Biomarkers are crucial for identifying, managing, and predicting outcomes in lung cancer, though they lack accuracy in differentiating histological subtypes.CEA and CYFRA21-1 were the most accurate biomarkers for distinguishing between malignant and benign pathology.Pro-GRP and NSE levels were elevated in SCLC compared to NSCLC. Pro-GRP alone had poor predictive capacity for differentiating NSCLC from SCLC, but combining it with CEA and CYFRA21-1 improved diagnostic performance.Patterns in biomarker levels suggest that a multi-biomarker approach, especially when combined with other diagnostic tools, could improve diagnostic accuracy.

A Validated Assay to Quantify Osimertinib and Its Metabolites, AZ5104 and AZ7550, from Microsampled Dried Blood Spots and Plasma.

BACKGROUND: Osimertinib is an oral small-molecule tyrosine kinase receptor inhibitor used to treat non-small cell lung cancer (NSCLC) with a sensitizing epidermal growth factor receptor mutation. Patients may experience drug toxicity and require dose deescalation. The study aimed to quantitate osimertinib and its 2 active metabolites, AZ5104 and AZ7550, in microsampled dried blood spots (DBS) collected from patients with NSCLC using a hemaPEN device and compare them with plasma drug levels. METHODS: A 6-min ultrahigh-performance liquid chromatography-tandem mass spectrometry method was developed and validated using plasma and DBS. The accuracy, selectivity, matrix effect, recovery, and stability were assessed using bioanalytical validation criteria. The hematocrit effect was investigated in DBS. Drug levels were measured in 15 patients with NSCLC, and the Bland-Altman method was used to compare measurements between plasma and DBS. RESULTS: The validated assay determined accurate and precise quantities, respectively, for osimertinib in both plasma (93.2%-99.3%; 0.2%-2.3%) and DBS (96.7%-99.6%; 0.5%-10.3%) over a concentration of 1-729 ng/mL. The osimertinib metabolites, AZ5104 and AZ7550, were similarly validated in accordance with bioanalytical guidelines. For 30%-60% patient hematocrit, no hematocrit bias was observed with DBS for all analytes. The Bland-Altman method showed high concordance between plasma and DBS analyte levels. Stability experiments revealed that osimertinib and its metabolites were poorly stable in plasma at room temperature, whereas all analytes were stable in DBS for 10 days at room temperature. CONCLUSIONS: The measurement of osimertinib, AZ5104, and AZ7550 from hemaPEN microsampled DBS is a convenient and reliable approach for therapeutic drug monitoring that produces measurements consistent with plasma drug levels.

Utility of neutrophil-to-lymphocyte ratio as an indicator of tumor immune status in non-small cell lung cancer.

BACKGROUND: Neutrophil-to-lymphocyte ratio (NLR) has been reported as a prognostic biomarker in non-small cell lung cancer (NSCLC); however, the underlying biological rationale remains unclear. The present study aimed to explore the potential utility of NLR as a surrogate biomarker for immune response to cancer and to elucidate the underlying mechanism. METHODS: This retrospective study included the medical records of 120 patients with NSCLC who underwent surgery at the study institution in 2012. NLR in peripheral blood was determined from blood test within 30 days before surgery. Tumor immune status was evaluated using immunohistochemical staining to identify CD3+, CD8+ and FOXP3+ tumor-infiltrating lymphocytes (TILs), and the relationship of NLR, with clinicopathologic characteristics including 5-year overall survival (OS), and the tumor immune status was investigated. The median values of NLR and TIL count were used as cutoff points. RESULTS: The 5-year OS was significantly better in patients with low NLR (<2.2) than in those with high NLR (≥2.2) (70.1% vs. 56.8%, P = 0.042) and in patients with high CD3+ TIL count (≥242) than in those with low CD3+ TIL count (<242) (70% vs. 56.8%, P = 0.019). Additionally, the CD3+ TIL count was negatively correlated with preoperative NLR (P = 0.005). CONCLUSION: NLR might potentially reflect the immune status of tumor microenvironment, explaining its impact on prognosis of patients with NSCLC.

Changes in Red Cell Morphology and Haematological Laboratory Parameters Associated With Alectinib.

BACKGROUND: Alectinib is a second-generation anaplastic lymphoma kinase (ALK) inhibitor indicated for ALK-mutated non-small-cell lung cancer. Recently, the association between alectinib and red cell morphological abnormalities has been reported in a few case series. This retrospective observational study aims to determine the frequency of occurrence of acanthocytosis in patients taking alectinib and to evaluate the red cell indices, biochemical markers of haemolysis and eosin-5-maleimide (EMA) binding assay results in patients receiving alectinib. METHODS: Patients who were on alectinib and had a complete blood count test performed in Queen Elizabeth Hospital Haematology Laboratory between 1 May 2021 and 31 August 2021 were included in the study. Haematological investigations that had been performed before and after the commencement of alectinib were reviewed. RESULTS: Fifty patients receiving alectinib were evaluated in this analysis. One hundred per cent of patients showed 3+ acanthocytes on the peripheral blood smears. Compared with the test results before starting alectinib, the post-alectinib blood tests showed a significantly lower haemoglobin concentration, red blood cell count and haematocrit; and a significantly higher mean corpuscular haemoglobin, mean corpuscular haemoglobin concentration and red cell distribution width. All the tested patients showed a marked reduction in EMA mean channel fluorescence compared with normal control. CONCLUSION: Our cohort revealed that alectinib caused significant acanthocytosis in all patients. Alectinib was also associated with changes in red cell indices and biochemical markers of haemolysis, compatible with a spherocytic and anisopoikilocytic morphology with haemolysis. Patients on alectinib had reduced EMA binding.

circIARS: a potential plasma biomarker for diagnosing non-small cell lung cancer.

Non-small cell lung cancer (NSCLC) is one of the most prevalent cancers in the world, and early diagnosis can effectively improve patient survival. Here, differentially expressed circIARS genes are screened from the sequencing results, and their molecular characteristics are examined by Sanger sequencing, RNase R assay, agarose gel electrophoresis (AGE), and fluorescence in situ hybridization (FISH). Real-time fluorescence quantitative polymerase chain reaction (qRT-PCR) is performed to detect the expression level of circIARS. The diagnostic value of the signature is analyzed using a subject operating characteristic (ROC) curve. Moreover, plasma is collected from postsurgical, chemotherapy, and relapse patients to investigate the prognostic value of circIARS in NSCLC. The expression of circIARS is greater in both the plasma and tissues of NSCLC patients than in those of healthy individuals, and could be used to distinguish NSCLC patients from patients with benign pulmonary disease (BPD), small cell lung cancer (SCLC) patients, and healthy individuals. The expression level of circIARS relatively decreases after antitumor therapy, such as chemotherapy, and relatively increases after recurrence. ROC analysis reveals that circIARS has better detection efficiency than traditional markers. In addition, circIARS expression level is strongly correlated with several clinicopathological parameters. Finally, we tentatively predict the downstream miRNAs or RBP that might bind to circIARS. Plasma circIARS is significantly greater in NSCLC patients and has good stability and specificity as a diagnostic marker, which could aid in the adjuvant diagnosis and dynamic monitoring of NSCLC.

Hexokinase 2 discerns a novel circulating tumor cell population associated with poor prognosis in lung cancer patients.

Unlike other epithelial cancer types, circulating tumor cells (CTCs) are less frequently detected in the peripheral blood of non-small cell lung cancer (NSCLC) patients using epithelial marker-based detection approaches despite the aggressive nature of NSCLC. Here, we demonstrate hexokinase-2 (HK2) as a metabolic function-associated marker for the detection of CTCs. In 59 NSCLC patients bearing cytokeratin-positive (CKpos) primary tumors, HK2 enables resolving cytokeratin-negative (HK2high/CKneg) CTCs as a prevalent population in about half of the peripheral blood samples with positive CTC counts. However, HK2high/CKneg tumor cells are a minority population in pleural effusions and cerebrospinal fluids. Single-cell analysis shows that HK2high/CKneg CTCs exhibit smaller sizes but consistent copy number variation profiles compared with CKpos counterparts. Single-cell transcriptome profiling reveals that CK expression levels of CTCs are independent of their epithelial-to-mesenchymal transition (EMT) status, challenging the long-standing association between CK expression and EMT. HK2high/CKneg CTCs display metastasis and EGFR inhibitor resistance-related molecular signatures and are selectively enriched in patients with EGFRL858R driver oncogene mutation as opposed to EGFR19Del , which is more frequently found in patients with prevalent CKpos CTCs in the blood. Consistently, treatment-naïve patients with a larger number or proportion of HK2high/CKneg CTCs in the blood exhibit poor therapy response and shorter progression-free survival. Collectively, our approach resolves a more complete spectrum of CTCs in NSCLC that can potentially be exploited to identify patient prognosis before therapy.

Novel Blood Biomarkers for Response Prediction and Monitoring of Stereotactic Ablative Radiotherapy and Immunotherapy in Metastatic Oligoprogressive Lung Cancer.

Up to 80% of patients under immune checkpoint inhibitors (ICI) face resistance. In this context, stereotactic ablative radiotherapy (SABR) can induce an immune or abscopal response. However, its molecular determinants remain unknown. We present early results of a translational study assessing biomarkers of response to combined ICI and SABR (I-SABR) in liquid biopsy from oligoprogressive patients in a prospective observational multicenter study. Cohort A includes metastatic patients in oligoprogression to ICI maintaining the same ICI due to clinical benefit and who receive concomitant SABR. B is a comparative group of oligometastatic patients receiving only SABR. Blood samples are extracted at baseline (T1), after the first (T2) and last (T3) fraction, two months post-SABR (T4) and at further progression (TP). Response is evaluated by iRECIST and defined by the objective response rate (ORR)-complete and partial responses. We assess peripheral blood mononuclear cells (PBMCs), circulating cell-free DNA (cfDNA) and small RNA from extracellular vesicles. Twenty-seven patients could be analyzed (cohort A: n = 19; B: n = 8). Most were males with non-small cell lung cancer and one progressing lesion. With a median follow-up of 6 months, the last ORR was 63% (26% complete and 37% partial response). A decrease in cfDNA from T2 to T3 correlated with a good response. At T2, CD8+PD1+ and CD8+PDL1+ cells were increased in non-responders and responders, respectively. At T2, 27 microRNAs were differentially expressed. These are potential biomarkers of response to I-SABR in oligoprogressive disease.

Cytokine profiling identifies circulating IL-6 and IL-15 as prognostic stratifiers in patients with non-small cell lung cancer receiving anti-PD-1/PD-L1 blockade therapy.

Whether circulating levels of specific cytokines at baseline link with treatment efficacy of immune checkpoint blockade (ICB) therapy in patients with non-small cell lung cancer remains unknown. In this study, serum samples were collected in two independent, prospective, multicenter cohorts before the initiation of ICB. Twenty cytokines were quantified, and cutoff values were determined by receiver operating characteristic analyses to predict non-durable benefit. The associations of each dichotomized cytokine status with survival outcomes were assessed. In the discovery cohort (atezolizumab cohort; N = 81), there were significant differences in progression-free survival (PFS) in accordance with the levels of IL-6 (log-rank test, P = 0.0014), IL-15 (P = 0.00011), MCP-1 (P = 0.013), MIP-1ß (P = 0.0035), and PDGF-AB/BB (P = 0.016). Of these, levels of IL-6 and IL-15 were also significantly prognostic in the validation cohort (nivolumab cohort, N = 139) for PFS (log-rank test, P = 0.011 for IL-6 and P = 0.00065 for IL-15) and overall survival (OS; P = 3.3E-6 for IL-6 and P = 0.0022 for IL-15). In the merged cohort, IL-6high and IL-15high were identified as independent unfavorable prognostic factors for PFS and OS. The combined IL-6 and IL-15 status stratified patient survival outcomes into three distinct groups for both PFS and OS. In conclusion, combined assessment of circulating IL-6 and IL-15 levels at baseline provides valuable information to stratify the clinical outcome of patients with non-small cell lung cancer treated with ICB. Further studies are required to decipher the mechanistic basis of this finding.

HOXA9 gene promotor methylation and copy number variation of SOX2 and HV2 genes in cell free DNA: A potential diagnostic panel for non-small cell lung cancer.

BACKGROUND: Most cases of lung cancer are diagnosed at advanced stage. Detection of genetic and epigenetic markers in cell-free DNA (cfDNA) is a promising tool for the diagnosis of lung cancer at an early stage. The aim of this study was to identify non-invasive diagnostic markers in cell free DNA (cfDNA) for non-small cell lung cancer (NSCLC) as it is the most common type of lung cancer. METHODS: We investigated the cfDNA HOXA9 gene promotor methylation by pyrosequencing. Copy number variation of SOX2 and HV2 genes were detected by real-time PCR in cfDNA extracted from plasma samples of 25 newly diagnosed NSCLC patients and 25 age and sex matched controls. RESULTS: Methylation level of HOXA9 was significantly higher in NSCLC patients than controls (p > 0.001). SOX2 showed significantly higher CNV and HV2 showed lower CNV in patients than controls (p > 0.001, p = 0.001 respectively). Receiver Operating Characteristic (ROC) curve analysis for HOXA9 methylation, SOX2 CNV and HV2 CNV showed a discrimination power of 79.4%, 80% and 77.5% respectively and the area under the curve for the combined analysis of the three genes was 0.958 with 88% sensitivity and 100% specificity. CONCLUSIONS: In this study, we suggest a potentially diagnostic panel that may help in detection of lung cancer with high sensitivity and specificity using cell free DNA. This Panel included HOXA9 gene methylation and the CNV of SOX2 and HV2 genes.