Recurrence Of Non-small Cell Lung Cancer Research Articles

Residual ctDNA after treatment predicts early relapse in patients with early-stage NSCLC.

8517 Background: Liquid biopsies based on circulating tumor DNA (ctDNA) analysis are being investigated for detection of residual disease and recurrence. Conclusive evidence for utility of ctDNA in early-stage non-small cell lung cancer (NSCLC) is awaited. Due to low ctDNA levels in early-stage disease or post-treatment, effective methods require high analytical sensitivity to detect mutant allele fractions (MAF) below 0.01%. Methods: We analysed 363 plasma samples from 88 patients with NSCLC recruited to the LUng cancer CIrculating tumour DNA (LUCID) study, with disease stage I (49%), II (28%) and III (23%). 62% were adenocarcinomas. Plasma was collected before and after treatment, and at 3, 6 and 9 months after surgery (N = 69) or chemoradiotherapy (N = 19). Additional plasma was collected at disease relapse for 17 patients. Median follow-up was 3 years, and 40 patients progressed or died of any cause. We employed the RaDaR™ assay, a highly sensitive personalized assay using deep sequencing of up to 48 tumor-specific variants. Variants identified by tumor exome analysis were tested by deep sequencing of tumor tissue and buffy coat DNA to verify somatic mutations and exclude clonal hematopoiesis. The RaDaR assay demonstrated 90% sensitivity at 0.001% MAF in analytical validation studies. Results: ctDNA was detected in 26% of samples, at median MAF of 0.047% (range: 0.0007% to > 2%), and prior to treatment in 87%, 77% and 24% for disease stage III, II and I respectively. For 62 patients, plasma was collected at a landmark timepoint, between 2 weeks and 4 months after initial treatment. ctDNA detection at the landmark timepoint was strongly predictive of clinical disease relapse, with Hazard Ratio of 20.7 (CI: 7.7-55.5, p-value < 0.0001). All 11 cases with ctDNA detected at landmark had disease progression, a median of 121 days after detection, and these included all 8 patients that relapsed within 300 days of treatment. Across 27 patients whose disease progressed during the study, ctDNA was detected at any timepoint post-treatment in 17 cases, with a median lead time of 203 days, and up to 741 days prior to clinical progression. ctDNA was detected post-treatment, in 13 of the 15 patients that progressed and had ctDNA detected prior to treatment. Conclusions: Our results support an emerging paradigm shift, by demonstrating that liquid biopsies can reliably detect recurrence of NSCLC at a preclinical stage, many months before clinical progression, thereby offering the opportunity for earlier therapeutic intervention. Clinical trial information: NCT04153526.

Circulating CD15+ LOX-1+ PMN-MDSCs are a potential biomarker for the early diagnosis of non-small-cell lung cancer.

Non-small-cell lung cancer (NSCLC) is the most common clinical lung cancer. Polymorphonuclear-myeloid derived suppressor cells (PMN-MDSCs), which are the major population of MDSCs, are involved in NSCLC progression. Recently, it was found that lectin-type oxidized LDL receptor 1 (LOX-1) could identify human PMN-MDSCs. However, the role of CD15+ LOX-1+ PMN-MDSCs in NSCLC early diagnosis has not been revealed. Here, we tried to confirm the application of the newly identified CD15+ LOX-1+ PMN-MDSCs in the early diagnosis of NSCLC. Flow cytometry (FCM) was used to detect the proportion of CD15+ LOX-1+ PMN-MDSCs in the peripheral blood (PB) of healthy controls (HC) and NSCLC patients. The correlation of CD15+ LOX-1+ PMN-MDSC frequency with levels of cytokeratin 19-fragments (CYFRA21-1), carcinoembryonic antigen (CEA), and carbohydrate antigen 125 (CA125) was analysed. Receiver operating characteristic (ROC) curve was used to estimate the diagnostic efficacy of CD15+ LOX-1+ PMN-MDSCs for NSCLC. Additionally, the association of CD15+ LOX-1+ PMN-MDSC frequency with NSCLC prognosis/recurrence after surgery was explored. The proportion of CD15+ LOX-1+ PMN-MDSCs increased in PB of NSCLC patients. CD15+ LOX-1+ PMN-MDSC proportion was positively correlated with levels of CEA, CA125 and CYFRA21-1. Detection of PMN-MDSC percentage in PB owed high sensitivity and specificity for NSCLC diagnosis. The proportion of CD15+ LOX-1+ PMN-MDSCs decreased in patients after surgery. The frequency of CD15+ LOX-1+ PMN-MDSCs was lower in NSCLC patients without recurrence compared to those with recurrence after surgery. Circulating CD15+ LOX-1+ PMN-MDSCs are a potential diagnostic marker for NSCLC, and are associated with NSCLC prognosis and recurrence after surgery.

Preoperative inflammation-based scores predict early recurrence after lung cancer resection.

BackgroundInflammation in the tumor microenvironment is hypothesized to have a major role in cancer invasiveness, progression, and metastases. The purpose of this study was to evaluate the prognostic value of preoperative inflammation-based scores in terms of estimating the timing of recurrence by hazard curves in a cohort of operable, early-stage non-small cell lung cancer (NSCLC) patients.MethodsA total of 387 patients with NSCLC who underwent complete pulmonary resection from 2010 to 2019 had their C-reactive protein-to-albumin ratio (CAR), neutrophil-to-lymphocyte ratio (NLR), platelet-to-lymphocyte ratio (PLR), Glasgow prognostic score (GPS), modified GPS, systemic immune-inflammation index (SII), and advance lung cancer inflammation index (ALI) measurements taken before surgery. Hazard curves indicating changes in hazards over time were evaluated.ResultsMedian follow-up was 39.2 months. In total, 105 patients (27.1%) experienced recurrence. The resulting hazard curve with elevated CAR, SII, GPS, and mGPS, values displayed an initial high peak during the first year. Multivariate analyses showed that an elevated CAR [hazard ratio (HR), 1.987; 95% confidence interval (CI), 1.202–3.284] independently predicted the recurrence-free survival. Even in stage I disease, patients with elevated CAR and SII values showed an earlier peak of recurrence, which was about 12 to 16 months earlier than those with low values.ConclusionsEven after complete resection of stage I NSCLC, patients with elevated CAR and SII values retain a high risk of early recurrence. Preoperative inflammation-based scores can be an objective, simple, and cost-effective measurement for predicting early recurrence of NSCLC.

Quantification of BIM mRNA in circulating tumor cells of osimertinib-treated patients with EGFR mutation-positive lung cancer

The response rate for osimertinib is high among patients with untreated epidermal growth factor receptor (EGFR) mutation-positive non-small cell lung cancer (NSCLC). However, there exist no biomarkers to predict the efficacy of the same. This study investigated whether BIM-γ mRNA expression in circulating tumor cells (CTCs) predicts poor outcomes for osimertinib treatment in patients with EGFR mutation-positive NSCLC. Patients with advanced EGFR-tyrosine kinase inhibitor-untreated NSCLC or post-operative recurrence with EGFR-sensitive mutations (exon 19 deletion or L858R mutation) were included. Informed consent was obtained from all participants. The candidate biomarker BIM-γ was measured in CTCs after blood collection (10mL of whole blood) at baseline. CTCs were collected with the ClearCell FX system, and quantitative real-time PCR was performed. Relative expression of BIM-γ mRNA from CTCs, as normalized to the reference gene (GAPDH mRNA), was calculated using the KCL22cell line for calibration. We enrolled 30 EGFR mutation-positive NSCLC patients treated with osimertinib during the period from April 2018 through December 2019. All the patients had an EGFR mutation at the primary site: exon 19 deletion in 15 cases and L858R in 15 cases. Median CTC count at baseline was 12 (range 3-127)/7.5mL, and median BIM-γ mRNA expression was 0.073 (range 0-1.37). Furthermore, the response rate to osimertinib was worse in patients with high than in those with low BIM-γ mRNA expression (n=15 each) (26.6% vs. 73.3%, respectively; p=0.011). Progression-free survival did not significantly differ between groups (p=0.13). BIM-γ mRNA overexpression in CTCs from EGFR mutation-positive NSCLC patients is a potential a biomarker for poor response to osimertinib. UMIN:00032055.

GOLM1 predicts poor prognosis of patients with NSCLC and is associated with the proliferation and chemo-sensitivity of cisplatin in NSCLC cells: bioinformatics analysis and laboratory validation.

Lung cancer is one of the deadliest malignant tumors with non-small cell lung cancer (NSCLC) being the most prevalent type. Patients with NSCLC usually were diagnosed at the advance clinical stages, and these patients often had high rate of tumor-recurrence, thus leading to poor prognosis. Yet, the molecular mechanisms underlying NSCLC progression and recurrence are largely unknown. This study aimed to identify potential hub genes associated with the pathophysiology of NSCLC by bioinformatics analysis and laboratory validation. The GSE51852, GSE52248 and GSE75037 datasets were downloaded from the Gene Expression Omnibus database. The overlapping differentially expressed genes (DEGs) were analyzed by GEO2R tool. Gene Ontology (GO) and KEGG pathway enrichment analysis were performed on these overlapping DEGs. The protein-protein interaction network was constructed to identify hub genes from DEGs. The expression and survival analysis of these hub genes were performed by using the integrated bioinformatics tools. Finally, the effects of GOLM1 on the proliferation and chemo-sensitivity of NSCLC cells were determined by in vitro functional assays. A total of 197 overlapping DEGs (37 up-regulated and 160 down-regulated) were identified from the microarray datasets. Furthermore, the PPI network with 89 nodes and 768 edges was constructed and 17 hub genes were identified from PPI network by using MCODE analysis. The survival analysis revealed that the expression of 5 hub genes (FGF2, GOLM1, GPC3, IL6 and SPP1) were significantly correlated with overall survival of patients with lung cancer. Furthermore, the in vitro functional studies showed that GOLM1 overexpression promoted the NSCLC cell proliferation and colony formation; while GOLM1 knockdown exerted the opposite effects. Importantly, GOLM1 overexpression reduced the chemo-sensitivity of cisplatin in NSCLC cells by attenuating the inhibitory effects of cisplatin on the cell proliferation and colony formation. In conclusion, the present study showed that 5 hub genes including FGF2, GOLM1, GPC3, IL6 and SPP1 were deregulated in NSCLC tissues and may predict the prognosis of patients with NSCLC. GOLM1 may play an important role in regulating the cell proliferation and chemo-sensitivity of cisplatin in NSCLC.

Mechanisms of resistance to chemotherapy in non-small cell lung cancer.

Non-small cell lung cancer (NSCLC), which represents 80-85% of lung cancer cases, is one of the leading causes of human death worldwide. The majority of patients undergo an intensive and invasive treatment regimen, which may include radiotherapy, chemotherapy, targeted therapy, immunotherapy, or a combination of these, depending on disease stage and performance status. Despite advances in therapeutic regimens, the 5-year survival of NSCLC is approximately 20-30%, largely due to diagnosis at advanced stages. Conventional chemotherapy is still the standard treatment option for patients with NSCLC, especially those with advanced disease. However, the emergence of resistance to chemotherapeutic agents (chemoresistance) poses a significant obstacle to the management of patients with NSCLC. Therefore, to develop efficacious chemotherapeutic approaches for NSCLC, it is necessary to understand the mechanisms underlying chemoresistance. Several mechanisms are known to mediate chemoresistance. These include altered cellular targets for chemotherapy, decreased cellular drug concentrations, blockade of chemotherapy-induced cell cycle arrest and apoptosis, acquisition of epithelial-mesenchymal transition and cancer stem cell-like phenotypes, deregulated expression of microRNAs, epigenetic modulation, and the interaction with tumor microenvironments. In this review, we summarize the mechanisms underlying chemoresistance and tumor recurrence in NSCLC and discuss potential strategies to avoid or overcome chemoresistance.

Circulating tumor cells can predict the prognosis of patients with non-small cell lung cancer after resection: a retrospective study.

BackgroundThe development of metastasis is the primary cause of death in patients with non-small cell lung cancer (NSCLC). However, identifying those NSCLC patients who will have loco-regional or distant disease recurrence after surgery is still challenging. Circulating tumor cells (CTCs) can accurately reflect the impact of micro-metastasis of tumor cells in circulating blood on patients’ treatment and prognosis. The aim of the present study was to explore the value of preoperative CTC concentration in predicting postoperative metastasis and recurrence risk in patients with NSCLC.MethodsThis study enrolled 347 patients with stage I–IIIA NSCLC. The CTCs were isolated using folate receptor (FR) positivity from peripheral blood samples before surgery, and then enriched and analyzed. Patients were divided into two groups for retrospective survival analysis based on the geometric mean of CTC concentration. The primary study endpoint was recurrence-free survival. Spearman’s correlation was used to evaluate the relationship between CTC concentration and clinical characteristics of NSCLC patients. A nomogram based on the multivariate Cox regression model was developed to predict recurrence and metastasis in the NSCLC patients. The performance of the nomogram was evaluated using the concordance index, calibration curve, and Hosmer-Lemeshow test.ResultsThe median follow-up time was 38 months. Preoperative CTC concentration was not significantly related to tumor-node-metastasis staging (P>0.05) and was an independent prognostic factor for NSCLC patients [hazard ratio (HR), 5.489; 95% confidence interval (CI): 2.660–11.326, P<0.001]. The nomogram based on preoperative CTC concentration had a concordance index value of 0.82. Validation revealed that the nomogram possessed excellent predictive ability and calibration.ConclusionsPreoperative CTC concentration is an independent and sensitive biomarker of prognosis in patients with NSCLC. Our nomogram based on preoperative CTC concentration is an effective and non-invasive tool for predicting the recurrence and metastasis of NSCLC.

Can the Japanese National Clinical Database risk calculator predict long-term survival of patients who undergo palliative segmentectomy for primary lung cancer?

ObjectivesSelection criteria for palliative limited surgery in patients with non-small cell lung cancer (NSCLC) can vary by institution or surgeon. We retrospectively reviewed outcomes of poor-risk patients who underwent palliative segmentectomy (PS), using the National Clinical Database Risk Calculator (RC).MethodsWe retrospectively analyzed medical records of patients with NSCLC tumors ≥ 20 mm and consolidation/tumor ratios ≥ 0.5 on computed tomography, who underwent PS from January 2009 to March 2016. Median follow-up time was 47 months (range 2–102 months).ResultsWe enrolled 67 patients (median age: 73.0 years), of whom 54 received thoracoscopic surgery and 28 received medial lymph-node dissection. The RC’s mean predictive probability rate for perioperative mortality or severe complications was 7.1%. Of the 67 patients, 24 patients (43.0%) suffered post-surgical complications, including 2 (3%) who died in hospital; 17 eventually suffered NSCLC recurrences and/or metastases, 11 eventually died from NSCLC, and 17 died from other diseases. Five-year overall survival (OS) was 59.4%. When the patients were divided into high-risk (HR) and low-risk (LR) groups based on the RC, 5-year OS was significantly less in the HR group (43.9%) than in the LR group (82.2%; P < 0.05).ConclusionThe RC, which was developed primarily to determine perioperative risk, can predict long-term prognosis for compromised patients who undergo PS.

Combined prognostic value of the SUVmax derived from FDG-PET and the lymphocyte-monocyte ratio in patients with stage IIIB-IV non-small cell lung cancer receiving chemotherapy

BackgroundWe evaluated the prognostic potential of tumor 18F-fluorodeoxyglucose (FDG) uptake derived from positron emission tomography (PET) and known inflammatory hematological markers, both individually and in combination, for chemosensitivity and survival in patients with stage IIIB-IV non-small cell lung cancer (NSCLC) receiving first-line chemotherapy.MethodsA total of 149 patients with stage IIIB and IV NSCLC (based on TNM 7th edition) were retrospectively reviewed. Maximum standardized uptake value (SUVmax) were used to quantitatively assess FDG uptake. The lymphocyte-monocyte ratio (LMR), neutrophil-lymphocyte ratio (NLR) and platelet-lymphocyte ratio (PLR) were selected as hematological markers. Receiver operating characteristic (ROC) curves were constructed for the determination of optimal cut-off values to predict chemotherapeutic response.ResultsPatients with SUVmax > 11.6 or LMR ≤3.73 exhibited a significantly lower objective response rate (ORR) to chemotherapy (p < 0.001 and p < 0.001). Through multivariable logistic regression analysis, both the SUVmax and LMR were identified as independent predictive factors for chemotherapeutic response (p = 0.001 and p < 0.001). Furthermore, a multivariable Cox proportional hazard model identified a high SUVmax (> 11.6) and low LMR (≤3.73) as independent predictors of poor PFS (p < 0.001 and p = 0.025) and OS (p < 0.001 and p = 0.032). A novel score system was constructed based on the SUVmax and LMR (SUV_LMR score), and patients were stratified into three subgroups. The patients with a score of 0 had a significantly higher ORR (88.9%) than did those with a score of 1 (59.6%) and score of 2 (25.0%) (p < 0.001). Moreover, multivariable Cox analysis further identified the SUV_LMR score as an independent prognostic factor for PFS (p < 0.001) and OS (p < 0.001).ConclusionsPre-treatment SUVmax and LMR were not only predictive factors for chemotherapeutic response but also independent prognostic factors of survival in stage IIIB-IV NSCLC. Moreover, the SUV_LMR score, which is based on primary tumor metabolic activity and the systemic inflammatory response, might provide a promising tool to predict chemosensitivity, recurrence and survival of advanced NSCLC.

Clinical utility of the C-reactive protein:albumin ratio in non-small cell lung cancer patients treated with nivolumab.

BackgroundNivolumab is a second‐line chemotherapy for non‐small cell lung cancer (NSCLC). This study explored the impact of clinical biomarkers such as neutrophil:lymphocyte ratio (NLR), C‐reactive protein:albumin ratio (CAR), and modified Glasgow prognostic score on the efficacy and outcome of nivolumab monotherapy in previously treated NSCLC patients.MethodsWe retrospectively analyzed advanced or postoperative recurrence of NSCLC in 113 patients in two Japanese facilities from January 2015 to December 2019. Optimal cutoff values of NLR and CAR were assessed by the area under the receiver operating characteristic curves predicting death events to conduct regression analysis. Baseline values and values collected eight weeks after nivolumab treatment were measured to investigate time‐series changes of these markers.ResultsThe patients showed median overall survival (OS) and progression‐free survival (PFS) of 14.0 months and 2.3 months, respectively, with both being significantly longer in patients with partial response (PR) than in patients with progressive disease (PD). Optimal cutoff levels for NLR and CAR were 5.8 and 0.83, with significant decrease in CAR (P = 0.002) from baseline levels in PR patients and significant increase in PD patients. Baseline CAR ≥0.83 was significantly associated with one‐year mortality events and overall survival (OS), and multivariate analysis showed significant association of age ≤70 years, an Eastern Cooperative Oncology Group performance status score of 2 or 3, and a baseline CAR ≥0.83 with inferior OS.ConclusionsFor second‐line nivolumab therapy, evaluation of baseline CAR and subsequent changes in CAR may be predictive of therapeutic response to nivolumab and long‐term survival in NSCLC patients.Key points Significant findings of the study The baseline value of C‐reactive protein:albumin ratio was significantly associated with one‐year mortality and overall survival in non‐small cell lung cancer patients treated with nivolumab. What this study adds Time‐series change of C‐reactive protein:albumin ratio may be useful for predicting the treatment efficacy in patients treated with nivolumab.

Thermal treatment decreases resistance to osimertinib in non-small cell lung cancer through the EGFR/PI3K/AKT pathway.

Osimertinib (OSI) resistance commonly occurs during the treatment of non-small-cell lung cancer (NSCLC). This study aims to investigate whether the thermal effects of radiofrequency ablation (RFA) can increase the sensitivity of OSI-resistant NSCLC to OSI treatment and whether OSI effectively inhibits the recurrence of OSI-resistant NSCLC following RFA treatment and improve survival of NSCLC patients. In vitro, OSI-resistant NCI-H1975 (NCI-H1975/OSIR) cells and thermotolerant NCI-H1975/OSIR (NCI-H1975/OSIR-a-h) cells were established using human NSCLC cell line NCI-H1975. Cell viability, apoptosis, sensitivity to OSI, threonine-methionine amino acid substitution at position 790 (T790M) mutation levels, and protein expression of epidermal growth factor receptor (EGFR), phosphatidylinositol 3-kinase (PI3K), protein kinase B (AKT), hypoxia-inducible factor-1 alpha (HIF-1a) were detected using different methods. In vivo, a nude mouse model of metastatic human lung cancer was developed and subjected to RFA treatment. The tumor growth, apoptosis, sensitivity to OSI, expression of EGFR/PI3K/AKT/HIF-1a, and CD34 levels were detected in the micrometastases of the transition zone (TZ) around the central ablation zone, and the reference zone (RZ) far from central ablation zone. NCI-H1975/OSIR and thermotolerant NCI-H1975/OSIR cell models were successfully established. Thermotolerant NCI-H1975/OSIR cells show higher sensitivity to OSI than NCI-H1975/OSIR cells and NCI-H1975 cells. OSI treatment can inhibit the EGFR/PI3K/AKT pathway and induce apoptosis in both NCI-H1975 cells and thermotolerant NCI-H1975/OSIR cells, but not in NCI-H1975/OSIR cells. In vivo, RFA treatment increases sensitivity to OSI in NCI-H1975/OSIR cell micrometastases in the TZ but not in the RZ. OSI intervention effectively inhibits the over-proliferation of micrometastases and activation of the EGFR/PI3K/AKT pathway, and induces apoptosis of micrometastases in the TZ, but shows little effects on the micrometastases in the RZ. The thermal effects can increase the sensitivity of OSI-resistant NSCLC cells to OSI through the EGFR/PI3K/AKT/HIF-1a signaling pathway, indicating that RFA combined with OSI might be a clinically effective and comprehensive therapy for the treatment of OSI-resistant NSCLC.

Tie2-expressing monocytes as a novel angiogenesis-related cellular biomarker for non-small cell lung cancer.

To investigate the clinical value of Tie2-expressing monocytes (TEMs) in the early diagnosis of lung cancer and assess its correlation with angiogenesis, a total of 184 patients with non-small cell lung cancer (NSCLC), 101 patients with benign pulmonary disease (BPD), and 77 healthy controls were enrolled in our study. The distribution of TEMs in lung tissue was determined by immunofluorescence staining. Lung microvascular density was assessed by immunohistochemical staining. Receiver-operating characteristic (ROC) curve analysis was performed to assess the diagnostic value of TEM frequency. Patients with NSCLC were followed up for 26 months. We found that the TEM frequency in peripheral blood monocytes of patients with NSCLC was significantly greater than that in patients with BPD and healthy controls. TEM frequency showed a correlation with NSCLC recurrence. The majority of TEMs in tumor tissues were localized around blood vessels; tumoral TEM frequency showed a positive correlation with microvascular density. High percentage of TEMs in the peripheral blood was associated with poor overall survival. ROC curve analysis revealed the potential diagnostic value of circulating TEM frequency in NSCLC. Thus, we believe that TEM frequency is related to angiogenesis in tumor tissues and may serve as a diagnostic marker for NSCLC.

Caspase-3 knockout attenuates radiation-induced tumor repopulation via impairing the ATM/p53/Cox-2/PGE2 pathway in non-small cell lung cancer.

Radiotherapy is an effective treatment for non-small cell lung cancer (NSCLC). However, irradiated, dying tumor cells generate potent growth stimulatory signals during radiotherapy that promote the repopulation of adjacent surviving tumor cells to cause tumor recurrence. We investigated the function of caspase-3 in NSCLC repopulation after radiotherapy. We found that radiotherapy induced a DNA damage response (DDR), activated caspase-3, and promoted tumor repopulation in NSCLC cells. Unexpectedly, caspase-3 knockout attenuated the ataxia-telangiectasia mutated (ATM)/p53-initiated DDR by decreasing nuclear migration of endonuclease G (EndoG), thereby reducing the growth-promoting effect of irradiated, dying tumor cells. We also identified p53 as a regulator of the Cox-2/PGE2 axis and its involvement in caspase-3-induced tumor repopulation after radiotherapy. In addition, injection of caspase-3 knockout NSCLC cells impaired tumor growth in a nude mouse model. Our findings reveal that caspase-3 promotes tumor repopulation in NSCLC cells by activating DDR and the downstream Cox-2/PGE2 axis. Thus, caspase-3-induced ATM/p53/Cox-2/PGE2 signaling pathway could provide potential therapeutic targets to reduce NSCLC recurrence after radiotherapy.

Response Rate And Local Recurrence After Concurrent Immune Checkpoint Therapy And Radiation For Non-Small Cell Lung Cancer And Melanoma Brain Metastases

Growing evidence suggests that immune checkpoint therapy (ICT) and radiation (RT) may be synergistic in the treatment of brain metastases (BrM), but the optimal timing of therapy to maximize synergy remains unclear. We evaluated the impact of treatment timing on response rate and local recurrence in patients with non-small cell lung cancer (NSCLC) or melanoma BrM. We reviewed the records of BrM patients who received ICT and brain-directed RT between 2007-2016 at our institution. ICT consisted of ipilimumab, pembrolizumab, and/or nivolumab; RT included stereotactic radiosurgery/radiotherapy (SRS/T) or whole brain RT (WBRT). We evaluated response rate and local recurrence on a per-metastasis basis (unidimensional RANO-BM). Individual metastases were included only if they underwent RT within 90 days of ICT, to minimize selection biases while analyzing the impact of treatment timing on outcomes. Within this cohort, concurrent treatment was defined as receiving RT on the same day as a dose of ICT or in between doses of an ICT course; all other treatment was non-concurrent (but still within 90 days). Multivariable Cox proportional hazards models were used to assess time to response and local recurrence on a per-metastasis basis, adjusting for confounding variables and using a sandwich estimator to account for intra-patient correlation. The final cohort included 110 patients treated for 340 BrM, with 102 BrM treated concurrently and 238 BrM treated non-concurrently. In total, 68% had melanoma and 32% had NSCLC; 59% of BrM were treated with SRS/T and 41% with WBRT; and 44% received ipilimumab, 46% received anti-PD-1 therapy, and 9% received both simultaneously. Median radiographic follow-up after RT was 6.9 months (IQR 3.5-13.3 months). Rates of any response (complete or partial) were higher with concurrent treatment (70% vs 47%, p<0.001), with correspondingly lower rates of progressive disease (5% vs 26%). On multivariable analysis, time to response was improved with concurrent treatment (HR 1.78, 95% CI 1.18-2.66, p = 0.005), and local recurrence was also decreased with concurrent treatment (HR 0.46, 95% CI 0.26-0.82, p = 0.009). Only 1/103 BrM with a complete response later progressed. In a sensitivity analysis comparing concurrent treatment (n = 102 BrM) to treatment that was non-concurrent but within 30 days (n = 110 BrM), there remained improved rates of any response with concurrent treatment (70% vs 53%, p<0.001). Concurrent brain-directed RT and ICT improves response rates and decreases local recurrence of NSCLC and melanoma BrM, even when compared to treatment that was non-concurrent but within 90 days. Further study of this combination is warranted, particularly in patients in whom rapid treatment response is needed.

The association of non-small cell lung cancer recurrence with allogenic blood transfusion after surgical resection: A propensity score analysis of 1,803 patients.

Conflicting evidence underlies the controversial role of allogenic blood transfusion in recurrence of non-small cell lung cancer (NSCLC). Insufficient sample size and failure to measure effects of important confounders in previous studies contribute to the conflicting findings. To overcome these limitations, we applied robust statistics and weighted covariates in a large study cohort. Cox regression analyses were used to estimate the recurrence and survival in patients with NSCLC disease stages I through III who were transfused for a haemoglobin level less than 8.0g/dL within seven days after surgical resection. Inverse probability of treatment weighting (IPTW) was used to balance covariates in the sequential cohort of patients receiving an incremental amount of blood. We applied restricted cubic spline functions to characterise dose-response effects of transfusion amount on recurrence and mortality. A total of 209 (11.6%) of 1803 patients received transfusions. Over a median of 42 months after surgery (interquartile range 24.9-71.9), patients who received blood had a greater risk of early recurrence (IPTW-adjusted HR: 1.81, 95% CI: 1.59-2.06, P<0.001) and all-cause mortality (IPTW-adjusted hazard ratio, HR: 2.38, 95% CI: 1.97-2.87, P<0.001). A non-linear dose-response occurred between transfusion amount and recurrence or mortality. The greater risk of disease recurrence and early mortality after surgical resection in NSCLC patients who receive blood transfusion supports use of clinical strategies to reduce exposure. Further studies are needed to identify benchmarks to guide evidence-based practices.

Association of Mps one binder kinase activator 1 (MOB1) expression with poor disease-free survival in individuals with non-small cell lung cancer.

BackgroundMps one binder kinase activator 1 (MOB1) is a core component of the Hippo signaling pathway and has been implicated as a tumor suppressor. Here, we evaluated the possible relationship of MOB1 expression in non‐small cell lung cancer (NSCLC) to prognosis.MethodsWe retrospectively analyzed 205 lung adenocarcinoma patients treated at Kyushu University Hospital between November 2007 and October 2012. MOB1 expression in tumor cells of surgical specimens was evaluated by immunohistochemistry. Invasive activity of NSCLC cell lines in vitro was measured with a transwell assay.ResultsExpression of MOB1 was classified as high in 105 of the 205 (51.2%) tumor specimens, and such high expression was significantly associated with poor disease‐free survival (P = 0.0161). Among the various clinicopathologic parameters examined, high MOB1 expression was significantly associated only with intratumoral vascular invasion (P = 0.0005). Multivariate analysis also identified high MOB1 expression as a significant independent risk factor for disease‐free survival (P = 0.0319). The invasiveness of H1299 cells in vitro was increased or attenuated by overexpression or knockdown of MOB1, respectively.ConclusionsOur results suggest that MOB1 might promote early recurrence of NSCLC by increasing vascular invasion by tumor cells.Key pointsSignificant findings of the studyWe found that high MOB1 expression in surgical specimens of lung adenocarcinoma was associated with poor disease‐free survival and with intratumoral vascular invasion. MOB1 expression also promoted the invasiveness of NSCLC cells in vitro.What this study addsOur results thus suggest that high MOB1 expression is a risk factor for early postoperative recurrence in lung adenocarcinoma.

Abstract 408: Reduction of tumor-infiltrating B cells linked to recurrence of NSCLC tumors

Abstract Introduction: Lung cancer is the leading cause of cancer related mortality worldwide. Non-small cell lung cancer (NSCLC), accounting for approximately 80-85% of all lung cancer cases, is characterized by a poor response to chemotherapy and a low survival rate. Early stage NSCLC patients are typically treated with complete surgical resection of the tumor, but nevertheless 30-50% of these patients end up developing recurrence within 5 years of surgery. While the presence of tumor-infiltrating lymphocytes (TILs) has been shown to be significantly correlated with a positive clinical prognosis in multiple types of cancer the role of tumor-infiltrating B cells (TIBs) remains more controversial. Experimental Procedures: To perform a comprehensive immunoprofiling of NSCLC FFPE tumor samples we used MultiOmyx™, a proprietary immunofluorescent multiplexing assay. Using a 15-marker panel we have analyzed the proportion of T cell subtypes, B cells, Granulocytes, M1/M2-type tumor-associated macrophages, as well as the expression of PD-1, PD-L1, LAG-3, TIM-3, ICOS, and OX40 in 12 samples from patients with early stage (stage I-II) NSCLC (6 non-recurrent and 6 recurrent). Results: Using the MultiOmyx image analysis suite based on NeoGenomics' proprietary deep-learning algorithms, we compared the number and overall density of immune cell populations between non-recurrent and recurrent NSCLC samples and found recurrent samples to have a highly significant decrease in B cells (166.8 cells/mm2 to 26.2 cells/mm2; p-value 0.006). In addition to a decrease in the overall density of B cells, we also observed a decrease in the tumor-infiltration rate, measured as the % of cells found inside the tumor area versus in the stroma of the tumor samples. This demonstrates that not only are there fewer B cells in the recurrent samples, but the B cells present are also less likely to infiltrate into the tumor area. While research on tumor immunity has been highly focused on T cells, the specific role of B cells in this area has been somewhat overlooked. While the release of antibodies and interleukin-10 might be involved in TIB-mediated tumor immunity, interactions between TIBs and TILs as well as the PD-1/PD-L1 cascade has been documented more recently. Indeed, in our study we did observe a significant positive correlation between the densities of B cells and cytotoxic lymphocytes, as well as B cells and PD-L1, although interestingly this was only observed in the recurrent group of patients. We are currently in the process of performing further multiplexing analyses on these samples to examine the mechanism behind these correlations and their potential significance for NSCLC recurrence. Conclusion: Identifying early stage NSCLC patients at high risk of recurrence following surgical resection can help guide a more accurate patient selection and treatment plan for this group. We present data to suggest that B-cell density and tumor-infiltration rate represents novel biomarkers for NSCLC patients at risk for recurrence. Citation Format: Anna Juncker-Jensen, Nicholas Stavrou, Mate Nagy, Erinn Parnell, Judy Kuo, Eric Leones, Kathy Pham, Flora Sahafi. Reduction of tumor-infiltrating B cells linked to recurrence of NSCLC tumors [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 408.

CircRNA CDR1as/miR-641/HOXA9 pathway regulated stemness contributes to cisplatin resistance in non-small cell lung cancer (NSCLC)

BackgroundCisplatin (DDP) is the first-line chemotherapeutic drug for non-small cell lung cancer (NSCLC), and long-term DDP stimulation increased resistance of NSCLC cells to this drug by enriching cancer stem cells (CSCs), which contributed to recurrence and worse prognosis of NSCLC, but the molecular mechanisms are still not fully delineated.MethodsReal-Time qPCR and Western Blot analysis were conducted to examine gene expressions at mRNA and protein levels, respectively. Dual-luciferase reporter gene system was used to validate the targeting sites among circRNA CDR1as, miR-641 and HOXA9 mRNA. Cell growth was evaluated by CCK-8 assay, trypan blue staining assay and colony formation assay. The Annexin V-FITC/PI double staining method was employed to measure cell apoptosis ratio. Spheroid formation and flow cytometer assay was used to evaluate cell stemness. Xenograft mice models were established to measure tumorgenicity in vivo, and Ki67 expressions in mice tumor tissues were examined by immunohistochemistry (IHC).ResultsHere we identified a novel circRNA CDR1as/miR-641/Homeobox protein Hox-A9 (HOXA9) pathway regulated stemness and DDP chemoresistance in NSCLC. Mechanistically, circRNA CDR1as and HOXA9 were high-expressed, while miR-641 was low-expressed in DDP-resistant NSCLC cells, instead of their corresponding parental DDP-sensitive NSCLC cells. Additionally, we validated that circRNA CDR1as positively regulated HOXA9 in NSCLC cells by serving as an RNA sponge for miR-641, and knock-down of circRNA CDR1as increased the sensitivity of DDP-resistant NSCLC cells, which were reversed by downregulating miR-641 and upregulating HOXA9. Consistently, overexpression of circRNA CDR1as increased drug resistance of DDP-sensitive NSCLC cells by regulating miR-641/HOXA9 axis. In addition, the expression levels of stemness signatures (SOX2, OCT4 and Nanog) were higher in DDP-resistant NSCLC cells, which also tended to form spheres and enrich CD44+CD166+ population compared to their parental DDP-sensitive NSCLC cells, suggesting that CSCs were enriched in DDP-resistant NSCLC cells. Notably, knock-down of circRNA CDR1as inhibited stemness of DDP-resistant NSCLC cells by inhibiting HOXA9 through upregulating miR-641.ConclusionsTaken together, this study identified that circRNA CDR1as regulated stemness and DDP chemoresistance in NSCLC cells by targeting miR-641/HOXA9 axis.

Detection of residual disease and recurrence in early-stage non-small cell lung cancer (NSCLC) patients using sensitive personalized ctDNA sequencing assays.

e15560 Background: Detection of residual circulating tumour DNA (ctDNA) in patient plasma following curative intervention for localized non-small cell lung cancer (NSCLC) could identify patients who are at higher risk of relapse. These patients may benefit from adjuvant treatment, even if they have no macroscopic disease identified by radiographic imaging, which is the current standard of care. Here we evaluate the performance of the Inivata personalized sequencing assays to detect ctDNA in a cohort of 90 patients with early-stage NSCLC undergoing treatment with curative intent. Methods: The Inivata assay uses a highly sensitive next-generation sequencing platform, to identify tumor-specific variants from exome sequencing of tumor tissue and to track up to 48 patient-specific mutations in plasma specimens by multiplex PCR and ultra-high-depth next-generation sequencing. Samples from 90 patients with Stage I-III NSCLC who underwent radical treatment with curative intent, either surgery or radiotherapy ± chemotherapy, were collected as part of the LUng cancer - CIrculating tumor DNA (LUCID) study. Results: 350 plasma samples from 90 patients were analyzed using the Inivata assay, including samples collected before and after treatment and at subsequent follow-up visits. ctDNA was detected in pre-treatment samples in 38% of 32 patients (12/32) with Stage I NSCLC and in 90% of 21 patients (19/21) with Stage II/III disease, at allele fractions ranging from 6 parts per million (ppm, equivalent to 0.0006%) to over 20,000 ppm (equivalent to 2%). In plasma samples collected post-treatment, ctDNA was detected in close to 50% of cases. Conclusions: These findings highlight the Inivata assay is a sensitive method for detection of residual ctDNA and recurrence in early stage NSCLC. Initial detection rates ranged from 38% in Stage I disease to 90% for patients with Stage II/III disease prior to treatment, including detection of ctDNA to levels as low as a few parts per million. ctDNA was detected in at least one post-treatment timepoint in close to 50% patients. Together with additional data to be presented from the full 90 patient cohort, this suggests a possible route to improving treatment and designs of adjuvant trials for early stage NSCLC by detection of residual disease post-treatment and monitoring for early detection of relapse.

Circulating tumor DNA (ctDNA) analysis predicts recurrence following surgery in patients with stage I-IIIA non-small-cell lung cancer (NSCLC): Results of GASTO1035 and GASTO1018.

9023 Background: Circulating tumor DNA can be detected in the plasma and serum of patients with solid tumors and has emerged as a noninvasive biomarker for dynamically monitoring tumor. Postsurgical ctDNA analysis of early-stage NSCLC may identify patients at high risk of recurrence and facilitate early intervention and personalized cancer therapy. Methods: These studies recruited 123 patients with newly diagnosed resectable stage I-IIIA NSCLC. Preoperative and postoperative plasma and postoperative tissue samples were subjected to next-generation sequencing (Nanjing Shihe Jiyin Biotechnology Inc.) using a 425 cancer-related genes panel. Peripheral blood samples were collected before surgery, postoperatively within 1 month, and every 3-6 months for up to 3 years. Plasma samples with at least 1 variants detected in tissue samples were defined as ctDNA positive. Results: After 4 exclusions, 119 eligible patients were enrolled from June 2016 to February 2019. Presurgical ctDNA was detectable in 31 of 117 (26.5%) patients and was associated with inferior recurrence-free survival (HR, 3.90, 95% CI, 1.44-10.58, P = 0.004). Similarly, ctDNA was detected in 13 of 116 (11.2%) of the first postsurgical samples and was associated with shorter RFS (HR, 3.54, 95% CI, 1.22-10.23, P = 0.002). During surveillance after surgery, ctDNA-positive patients (38/119, 31.9%) were more than 9 times more likely to experience disease recurrence than ctDNA-negative patients (HR, 9.17, 95% CI, 2.60-32.42, P &lt;0.001). Serial ctDNA detection preceded radiologic disease recurrence by a median lead time of 4.23 months (95% CI, 0.91-7.54 months). We also observed a positive correlation between the ctDNA detection rate and the disease stage. Conclusions: These results suggest that detection of ctDNA before and after surgery is associated with the identification of a high risk of disease recurrence of resectable NSCLC. Perioperative ctDNA analyses identify disease recurrence earlier than standard-of-care radiologic imaging, and thus could facilitate personalized cancer treatment at early time points. Clinical trial information: NCT03465241 and NCT03172156.