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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. [ABSTRACT FROM AUTHOR]

PURPOSE: Current guidelines recommend molecular genotyping for patients newly diagnosed with metastatic nonsquamous (mNSq) non–small-cell lung cancer (NSCLC). The association between availability of molecular genotyping before first line (1L) therapy and overall survival (OS) is not known. METHODS: We conducted a real-world cohort study using electronic health records in patients newly diagnosed with mNSq NSCLC. Cox proportional-hazards multivariable regression models were constructed to examine the association between OS and test result availability before 1L therapy, adjusting for covariates. Additional analyses were conducted to assess the consistency and strength of the relationship. Multivariable logistic regression models were used to examine the association between concurrent tissue and plasma testing (v tissue alone) and result availability. RESULTS: Three hundred twenty-six patients were included, 80% (261/326) with results available before 1L (available testing group), and 20% (65/326) without results available (unavailable testing group). With 14.2-month median follow-up, patients in the available testing group had significantly longer OS relative to the unavailable testing group (adjusted hazard ratio, 0.43; 95% CI, 0.30 to 0.62; P <.0001). The adjusted odds of availability of results before 1L therapy was higher with concurrent tissue and plasma testing (v tissue testing alone; adjusted odds ratio, 2.06; 95% CI, 1.09 to 3.90; P =.026). CONCLUSION: Among patients with mNSq NSCLC in a real-world cohort, availability of molecular genotyping results before 1L therapy was associated with significantly better OS. Concurrent tissue and plasma testing was associated with a higher odds of availability of results before 1L therapy. These findings warrant renewed attention to the completion of molecular genotyping before 1L therapy. Availability of NGS results before 1L therapy for patients with mNSCLC is associated with better overall survival. [ABSTRACT FROM AUTHOR]

PURPOSE: As immune checkpoint inhibitors (ICI) become increasingly used in frontline settings, identifying early indicators of response is needed. Recent studies suggest a role for circulating tumor DNA (ctDNA) in monitoring response to ICI, but uncertainty exists in the generalizability of these studies. Here, the role of ctDNA for monitoring response to ICI is assessed through a standardized approach by assessing clinical trial data from five independent studies. PATIENTS AND METHODS: Patient-level clinical and ctDNA data were pooled and harmonized from 200 patients across five independent clinical trials investigating the treatment of patients with non–small-cell lung cancer with programmed cell death-1 (PD-1)/programmed death ligand-1 (PD-L1)–directed monotherapy or in combination with chemotherapy. CtDNA levels were measured using different ctDNA assays across the studies. Maximum variant allele frequencies were calculated using all somatic tumor-derived variants in each unique patient sample to correlate ctDNA changes with overall survival (OS) and progression-free survival (PFS). RESULTS: We observed strong associations between reductions in ctDNA levels from on-treatment liquid biopsies with improved OS (OS; hazard ratio, 2.28; 95% CI, 1.62 to 3.20; P <.001) and PFS (PFS; hazard ratio 1.76; 95% CI, 1.31 to 2.36; P <.001). Changes in the maximum variant allele frequencies ctDNA values showed strong association across different outcomes. CONCLUSION: In this pooled analysis of five independent clinical trials, consistent and robust associations between reductions in ctDNA and outcomes were found across multiple end points assessed in patients with non–small-cell lung cancer treated with an ICI. Additional tumor types, stages, and drug classes should be included in future analyses to further validate this. CtDNA may serve as an important tool in clinical development and an early indicator of treatment benefit. [ABSTRACT FROM AUTHOR]

We aim to determine the feasibility of a novel radiomic biomarker that can integrate with other established clinical prognostic factors to predict progression-free survival (PFS) in patients with non-small cell lung cancer (NSCLC) undergoing first-line immunotherapy. Our study includes 107 patients with stage 4 NSCLC treated with pembrolizumab-based therapy (monotherapy: 30%, combination chemotherapy: 70%). The ITK-SNAP software was used for 3D tumor volume segmentation from pre-therapy CT scans. Radiomic features (n = 102) were extracted using the CaPTk software. Impact of heterogeneity introduced by image physical dimensions (voxel spacing parameters) and acquisition parameters (contrast enhancement and CT reconstruction kernel) was mitigated by resampling the images to the minimum voxel spacing parameters and harmonization by a nested ComBat technique. This technique was initialized with radiomic features, clinical factors of age, sex, race, PD-L1 expression, ECOG status, body mass index (BMI), smoking status, recurrence event and months of progression-free survival, and image acquisition parameters as batch variables. Two phenotypes were identified using unsupervised hierarchical clustering of harmonized features. Prognostic factors, including PDL1 expression, ECOG status, BMI and smoking status, were combined with radiomic phenotypes in Cox regression models of PFS and Kaplan Meier (KM) curve-fitting. Cox model based on clinical factors had a c-statistic of 0.57, which increased to 0.63 upon addition of phenotypes derived from harmonized features. There were statistically significant differences in survival outcomes stratified by clinical covariates, as measured by the log-rank test (p = 0.034), which improved upon addition of phenotypes (p = 0.00022). We found that mitigation of heterogeneity by image resampling and nested ComBat harmonization improves prognostic value of phenotypes, resulting in better prediction of PFS when added to other prognostic variables. [ABSTRACT FROM AUTHOR]

Among non-small cell lung cancer (NSCLC) patients with therapeutically targetable tumor mutations in epidermal growth factor receptor (EGFR), not all patients respond to targeted therapy. Combining circulating-tumor DNA (ctDNA), clinical variables, and radiomic phenotypes may improve prediction of EGFR-targeted therapy outcomes for NSCLC. This single-center retrospective study included 40 EGFR-mutant advanced NSCLC patients treated with EGFR-targeted therapy. ctDNA data included number of mutations and detection of EGFR T790M. Clinical data included age, smoking status, and ECOG performance status. Baseline chest CT scans were analyzed to extract 429 radiomic features from each primary tumor. Unsupervised hierarchical clustering was used to group tumors into phenotypes. Kaplan–Meier (K–M) curves and Cox proportional hazards regression were modeled for progression-free survival (PFS) and overall survival (OS). Likelihood ratio test (LRT) was used to compare fit between models. Among 40 patients (73% women, median age 62 years), consensus clustering identified two radiomic phenotypes. For PFS, the model combining radiomic phenotypes with ctDNA and clinical variables had c-statistic of 0.77 and a better fit (LRT p = 0.01) than the model with clinical and ctDNA variables alone with a c-statistic of 0.73. For OS, adding radiomic phenotypes resulted in c-statistic of 0.83 versus 0.80 when using clinical and ctDNA variables (LRT p = 0.08). Both models showed separation of K–M curves dichotomized by median prognostic score (p < 0.005). Combining radiomic phenotypes, ctDNA, and clinical variables may enhance precision oncology approaches to managing advanced non-small cell lung cancer with EGFR mutations. [ABSTRACT FROM AUTHOR]

PURPOSE: Although the majority of patients with metastatic non–small-cell lung cancer (mNSCLC) lacking a detectable targetable mutation will receive pembrolizumab-based therapy in the frontline setting, predicting which patients will experience a durable clinical benefit (DCB) remains challenging. MATERIALS AND METHODS: Patients with mNSCLC receiving pembrolizumab monotherapy or in combination with chemotherapy underwent a 74-gene next-generation sequencing panel on blood samples obtained at baseline and at 9 weeks. The change in circulating tumor DNA levels on-therapy (molecular response) was quantified using a ratio calculation with response defined by a > 50% decrease in mean variant allele fraction. Patient response was assessed using RECIST 1.1; DCB was defined as complete or partial response or stable disease that lasted > 6 months. Progression-free survival and overall survival were recorded. RESULTS: Among 67 patients, 51 (76.1%) had > 1 variant detected at a variant allele fraction > 0.3% and thus were eligible for calculation of molecular response from paired baseline and 9-week samples. Molecular response values were significantly lower in patients with an objective radiologic response (log mean 1.25% v 27.7%, P <.001). Patients achieving a DCB had significantly lower molecular response values compared to patients with no durable benefit (log mean 3.5% v 49.4%, P <.001). Molecular responders had significantly longer progression-free survival (hazard ratio, 0.25; 95% CI, 0.13 to 0.50) and overall survival (hazard ratio, 0.27; 95% CI, 0.12 to 0.64) compared with molecular nonresponders. CONCLUSION: Molecular response assessment using circulating tumor DNA may serve as a noninvasive, on-therapy predictor of response to pembrolizumab-based therapy in addition to standard of care imaging in mNSCLC. This strategy requires validation in independent prospective studies. [ABSTRACT FROM AUTHOR]

• Evaluation of gene signatures to predict response to immunotherapy in NSCLC. • An EMT/Inflammation score was highly predictive of response and survival to ICB. • Identifies potential role of EMT in primary resistance to immunotherapy in NSCLC. Treatment of non-small cell lung cancer (NSCLC) with immune checkpoint blockade (ICB) has resulted in striking clinical responses, but only in a subset of patients. The goal of this study was to evaluate transcriptional signatures previously reported in the literature in an independent cohort of NSCLC patients receiving ICB. This retrospective study analyzed transcriptional profiles from pre-treatment tumor samples of 52 chemotherapy-refractory advanced NSCLC patients treated with anti-PD1/PD-L1 therapy. Gene signatures based on published reports were created and examined for their association with response to therapy and progression-free and overall survival (PFS, OS). Two signatures predicting response and outcomes were identified. One reflected the degree of immune infiltration and upregulation of interferon-gamma-induced genes. A second reflected the EMT status. Compared to those not responding to therapy, patients whose tumors responded to ICB had higher scores in an inflammatory gene signature (6.0 ± 2.9 vs -5.5 ± 3.4, p = 0.014) or a more epithelial phenotype (-1.7 ± 1.0 vs 2.1 ± 1.2, p = 0.016). Both signatures demonstrated a satisfactory predictive accuracy for response: AUC of 0.69 (95% CI: 0.54, 0.84) for the inflammatory and 0.70 (95% CI: 0.55, 0.85) for EMT signatures, respectively. A weighted score combining EMT and inflammatory signatures showed increased predictive value with AUC of 0.92 (95% CI: 0.85, 0.99). Kaplan-Meier curves for patients above and below the median combined score showed a significant separation for PFS and OS (all p < 0.01, log rank test). The EMT/Inflammation signature score may be useful in directing checkpoint inhibitor therapy in lung cancer and suggests that reversal of EMT might augment efficacy of ICB. [ABSTRACT FROM AUTHOR]

Highlights • First study to evaluate PEC enumeration and immunophenotyping in pleural fluid. • Detection of PECs in pleural effusions may be utilized to diagnose a MPE. • CellSearch® enables the non-invasive immunophenotyping of PECs from MPEs. • First study to demonstrate the prognostic value of the enumeration of PECs in MPEs. Abstract Objectives A malignant pleural effusion (MPE) is a common complication in non-small cell lung cancer (NSCLC) with important staging and prognostic information. Patients with MPEs are often candidates for advanced therapies, however, the current gold standard, cytological analysis of pleural fluid samples, has limited sensitivity. We aimed to demonstrate the feasibility of non-invasive enumeration and immunophenotyping of EpCAM-positive cells in pleural fluid samples for the diagnosis of a MPE in NSCLC patients. Materials and methods Pleural fluid specimens were prospectively collected from patients with NSCLC and the CellSearch® technology was utilized for the enumeration of pleural EpCAM-positive cells (PECs) and determination of PD-L1 expression on PECs from pleural fluid samples. The diagnostic performance of the enumeration of single PECs and PEC clusters was assessed using receiver operating characteristic (ROC) curves. The Kaplan-Meier method and Cox proportional hazards model was used to assess the impact of PECs and PEC clusters on overall survival (OS). Results 101 NSCLC patients were enrolled. The median number of PECs was significantly greater in the malignant (n = 84) versus non-malignant group (n = 17) (730 PECs/mL vs 1.0 PEC/mL, p < 0.001). The area under the ROC curve was 0.91. A cutoff value of 105 PECs/mL had a sensitivity and specificity of 73% and 100% for the diagnosis of a MPE, respectively. Among 69 patients with a pathology-confirmed MPE and tissue immunohistochemistry (IHC) results, 15 (22%) had greater than 50% PD-L1+ PECs. Overall concordance between tissue and PEC PD-L1 expression was 76%. Higher numbers of pleural effusion single PECs were associated with inferior overall survival (Cox adjusted HR 1.8, 95% CI: 1.02–3.05 p = 0.043). Conclusion Non-invasive measurement of PECs in NSCLC patients, using an automated, clinically available approach, may improve the diagnostic accuracy of a MPE, allow for immunophenotyping of PECs, and provide prognostic information. [ABSTRACT FROM AUTHOR]

Purpose: TP53 mutation (MT) in epidermal growth factor receptor (EGFR) -MT non–small cell lung cancer (NSCLC) is associated with poor response to targeted therapy; however, its impact on survival is not clearly established. Patients and Methods: We performed an analysis of patients with stage IV EGFR MT NSCLC with available gene sequencing data. Associations between baseline characteristics; molecular profile, including TP53 MT; and survival outcomes were assessed. Results: We identified 131 consecutive patients with EGFR MT; 81 (62%) had a TP53 MT, and 55 (42%) had other coexisting oncogenic MTs. Emergent EGFR T790M MT was observed in 42 patients (32%). Overall survival (OS) was longer for younger patients (P =.003), never smokers (P =.002), those with Eastern Cooperative Oncology Group performance status 0 to 1 (P =.004), and emergent T790M MT (P =.018). TP53 MT (P =.021) and other coexisting oncogenic MTs (P = 0.011) were associated with inferior OS. In a multivariable regression analysis adjusted for age, smoking, Eastern Cooperative Oncology Group performance status, and the presence of TP53 MT (P =.063) and other coexisting MTs (P =.064) did not achieve statistical significance. Patients with EGFR T790M /TP53 double MT had worse OS compared with patients with T790M MT alone (46.4 months v 82.9 months). In our series, five patients transformed to small-cell lung cancer (5.6%). All had TP53 MT. In four patients, allelic fraction of TP53 MT increased at the time of transformation. Conclusion: The presence of TP53 and other coexisting MTs in EGFR MT NSCLC were associated with inferior OS, including patients with emergent T790M MT. An increase in TP53 mutation allelic fraction may potentially be a useful clinical predictor of small-cell transformation. [ABSTRACT FROM AUTHOR]

Objectives Efficient use of nivolumab in non-small-cell lung cancer (NSCLC) has been limited by the lack of a definitive predictive biomarker. In patients with metastatic melanoma treated with ipilimumab, a pretreatment neutrophil-to-lymphocyte ratio (NLR) < 5 has been associated with improved survival. This retrospective cohort study aimed to determine whether the pretreatment NLR was associated with outcomes in NSCLC patients treated with nivolumab. Methods We reviewed the medical records of all patients with previously treated advanced NSCLC who received nivolumab between March 2015 and March 2016 outside of a clinical trial at the University of Pennsylvania. Patients were dichotomized according to pretreatment NLR < 5 vs. ≥5. Multivariable logistic regression and Cox proportional hazards models were used to assess the impact of pretreatment NLR on overall survival (OS), progression-free survival (PFS), and overall response rate (ORR). Results 175 patients were treated. Median age was 68 (range, 33–88); 54% were female. Twenty-five percent of patients had an Eastern Cooperative Oncology Group Performance Status (ECOG PS) ≥2; 46% had received ≥2 prior systemic therapies. In multivariate analyses, pretreatment neutrophil-to-lymphocyte ratio (NLR) ≥5 was independently associated with inferior OS (median 5.5 vs. 8.4 months; HR 2.07, 95% CI 1.3–3.3; p = 0.002) and inferior PFS (median 1.9 vs. 2.8 months; HR 1.43, 95% CI 1.02–2.0; p = 0.04). Conclusions In a cohort of patients with NSCLC treated with nivolumab in routine practice, pretreatment NLR ≥ 5 was associated with inferior outcomes. It is unclear whether this marker is predictive or prognostic. Prospective studies are warranted to determine the utility of NLR in the context of other biomarkers of programmed death-1 (PD-1) therapy. [ABSTRACT FROM AUTHOR]