Liquid biopsy and non-small cell lung cancer: are we looking at the tip of the iceberg?

The possibility to analyse the tumour genetic material shed in the blood is undoubtedly one of the main achievements of translational research in the latest years. In the modern clinical management of advanced non-small cell lung cancer, molecular characterisation plays an essential role. In parallel, immunotherapy is widely employed, but reliable predictive markers are not available yet. Liquid biopsy has the potential to face the two issues and to increase its role in advanced NSCLC in the next future. The aim of this review is to summarise the main clinical applications of liquid biopsy in advanced non-small cell lung cancer, underlining both its potential and limitations from a clinically driven perspective.

Longitudinal liquid biopsy anticipates hyperprogression and early death in advanced non-small cell lung cancer patients treated with immune checkpoint inhibitors.

BACKGROUND: Immune checkpoint inhibitors (ICIs) have revolutionised treatment of advanced non-small cell lung cancer (aNSCLC), but a proportion of patients had no clinical benefit and even experienced detrimental effects. This study aims to characterise patients experiencing hyperprogression (HPD) and early death (ED) by longitudinal liquid biopsy. METHODS: aNSCLC receiving ICIs were prospectively enrolled. Plasma was collected at baseline (T1) and after 3/4 weeks of treatment, according to the treatment schedule (T2). Cell-free DNA (cfDNA) was quantified and analysed by NGS. cfDNA quantification and variant allele fraction (VAF) of tumour-associated genetic alterations were evaluated for their potential impact on outcome. The genetic alteration with the highest VAF (maxVAF) at baseline was considered as a reference. RESULTS: From March 2017 to August 2019, 171 patients were enrolled. Five cases matched criteria for HPD and 31 ED were recorded; one overlapped. Quantification of cfDNA at T2 and its absolute and relative variation (T2-T1) were significantly associated with the risk of ED (P = 0.012, P = 0.005, P = 0.009). MaxVAF relative change (T2-T1/T1) was significantly associated with the risk of HPD (P = 0.02). After identifying optimal cut-off values, a two-step risk assessment model was proposed. DISCUSSION: Liquid biopsy performed early during treatment has the potential to identify patients at high risk of ED and HPD.

Early assessment of KRAS mutation in cfDNA correlates with risk of progression and death in advanced non-small-cell lung cancer.

BACKGROUND: Liquid biopsy has the potential to monitor biological effects of treatment. KRAS represents the most commonly mutated oncogene in Caucasian non-small-cell lung cancer (NSCLC). The aim of this study was to explore association of dynamic plasma KRAS genotyping with outcome in advanced NSCLC patients. METHODS: Advanced NSCLC patients were prospectively enrolled. Plasma samples were collected at baseline (T1), after 3 or 4 weeks, according to treatment schedule (T2) and at first radiological restaging (T3). Patients carrying KRAS mutation in tissue were analysed in plasma with droplet digital PCR. Semi-quantitative index of fractional abundance of mutated allele (MAFA) was used. RESULTS: KRAS-mutated cohort included 58 patients, and overall 73 treatments (N = 39 chemotherapy and N = 34 immune checkpoint inhibitors) were followed with longitudinal liquid biopsy. Sensitivity of KRAS detection in plasma at baseline was 48.3% (95% confidence interval (CI): 35.0-61.8). KRAS mutation at T2 was associated with increased probability of experiencing progressive disease as best radiological response (adjusted odds ratio: 7.3; 95% CI: 2.1-25.0, p = 0.0016). Increased MAFA (T1-T2) predicted shorter progression-free survival (adjusted hazard ratio (HR): 2.1; 95% CI: 1.2-3.8, p = 0.0142) and overall survival (adjusted HR: 3.2; 95% CI: 1.2-8.4, p = 0.0168). CONCLUSIONS: Longitudinal analysis of plasma KRAS mutations correlated with outcome: its early assessment during treatment has great potentialities for monitoring treatment outcome in NSCLC patients.

LKB1 and Tumor Metabolism: The Interplay of Immune and Angiogenic Microenvironment in Lung Cancer.

Liver kinase B1 (LKB1) is a tumor suppressor gene whose inactivation is frequent in different tumor types, especially in lung adenocarcinoma (about 30% of cases). LKB1 has an essential role in the control of cellular redox homeostasis by regulating ROS production and detoxification. Loss of LKB1 makes the tumor cell more sensitive to oxidative stress and consequently to stress-inducing treatments, such as chemotherapy and radiotherapy. LKB1 loss triggers complex changes in tumor microenvironment, supporting a role in the regulation of angiogenesis and suggesting a potential role in the response to anti-angiogenic treatment. On the other hand, LKB1 deficiency can promote an immunosuppressive microenvironment and may be involved in primary resistance to anti-PD-1/anti-PD-L1, as it has been reported in lung cancer. The aim of this review is to discuss interactions of LKB1 with the tumor microenvironment and the potential applications of this knowledge in predicting response to treatment in lung cancer.

Detection of Loss of Heterozygosity in cfDNA of Advanced EGFR- or KRAS-Mutated Non-Small-Cell Lung Cancer Patients.

Liquid biopsy is currently approved for management of epidermal growth factor receptor (EGFR)-mutated non-small-cell lung cancer (NSCLC) patients. However, one unanswered question is whether the rate of cell-free DNA (cfDNA)-negative samples is due to technical limitations rather than to tumor genetic characteristics. Using four microsatellite markers that map specific chromosomal loci often lost in lung cancer, we conducted a pilot study to investigate whether other alterations, such as loss of heterozygosity (LOH), could be detected in EGFR-negative cfDNA. We analyzed EGFR-mutated NSCLC patients (n = 24) who were positive or negative for EGFR mutations in cfDNA and compared the results with a second cohort of 24 patients bearing KRAS-mutated cancer, which served as a representative control population not exposed to targeted therapy. The results showed that in EGFR-negative post-tyrosine-kinase-inhibitor (TKI) cfDNAs, LOH frequency was significantly higher than in both pre- and post-TKI EGFR-positive cfDNAs. By contrast, no association between KRAS status in cfDNA and number of LOH events was found. In conclusion, our study indicates the feasibility of detecting LOH events in cfDNA from advanced NSCLC and suggests LOH analysis as a new candidate molecular assay to integrate mutation-specific assays.

Non-Small Cell Lung Cancer Testing on Reference Specimens: An Italian Multicenter Experience.

INTRODUCTION: Biomarker testing is mandatory for the clinical management of patients with advanced non-small cell lung cancer (NSCLC). Myriads of technical platforms are now available for biomarker analysis with differences in terms of multiplexing capability, analytical sensitivity, and turnaround time (TAT). We evaluated the technical performance of the diagnostic workflows of 24 representative Italian institutions performing molecular tests on a series of artificial reference specimens built to mimic routine diagnostic samples. METHODS: Sample sets of eight slides from cell blocks of artificial reference specimens harboring exon 19 EGFR (epidermal growth factor receptor) p.E746_AT50del, exon 2 KRAS (Kirsten rat sarcoma viral oncogene homologue) p.G12C, ROS1 (c-ros oncogene 1)-unknown gene fusion, and MET (MET proto-oncogene, receptor tyrosine kinase) Δ exon 14 skipping were distributed to each participating institution. Two independent cell block specimens were validated by the University of Naples Federico II before shipment. Methodological and molecular data from reference specimens were annotated. RESULTS: Overall, a median DNA concentration of 3.3 ng/µL (range 0.1-10.0 ng/µL) and 13.4 ng/µL (range 2.0-45.8 ng/µL) were obtained with automated and manual technical procedures, respectively. RNA concentrations of 5.7 ng/µL (range 0.2-11.9 ng/µL) and 9.3 ng/µL (range 0.5-18.0 ng/µL) were also detected. KRAS exon 2 p.G12C, EGFR exon 19 p.E736_A750del hotspot mutations, and ROS1 aberrant transcripts were identified in all tested cases, whereas 15 out of 16 (93.7%) centers detected MET exon 14 skipping mutation. CONCLUSIONS: Optimized technical workflows are crucial in the decision-making strategy of patients with NSCLC. Artificial reference specimens enable optimization of diagnostic workflows for predictive molecular analysis in routine clinical practice.

Metabolic classification of non-small cell lung cancer patient-derived xenografts by a digital pathology approach: A pilot study.

Introduction: Genetically characterized patient-derived tumor xenografts (PDX) are a valuable resource to understand the biological complexity of cancer and to investigate new therapeutic approaches. Previous studies, however, lack information about metabolic features of PDXs, which may limit testing of metabolism targeting drugs. Methods: In this pilot study, we investigated by immunohistochemistry (IHC) expression of five essential metabolism-associated markers in a set of lung adenocarcinoma PDX samples previously established and characterized. We exploited digital pathology to quantify expression of the markers and correlated results with tumor cell proliferation, angiogenesis and time of PDX growth in mice. Results: Our results indicate that the majority of the analyzed PDX models rely on oxidative phosphorylation (OXPHOS) metabolism, either alone or in combination with glucose metabolism. Double IHC enabled us to describe spatial expression of the glycolysis-associated monocarboxylate transporter 4 (MCT4) marker and the OXPHOS-associated glutaminase (GLS) marker. GLS expression was associated with cell proliferation and with expression of liver-kinase B1 (LKB1), a tumor suppressor involved in the regulation of multiple metabolic pathways. Acetyl CoA carboxylase (ACC) was associated with the kinetics of PDX growth. Conclusion: Albeit limited by the small number of samples and markers analyzed, metabolic classification of existing collections of PDX by this mini panel will be useful to inform pre-clinical testing of metabolism-targeting drugs.

Diagnostic accuracy of circulating free DNA testing for the detection of KRAS mutations in non-small cell lung cancer: A systematic review and meta-analysis.

Kirsten rat sarcoma viral oncogene homolog (KRAS) gene encodes a GTPase that acts as a molecular switch for intracellular signal transduction, promoting cell growth and proliferation. Mutations in the KRAS gene represent important biomarkers for NSCLC targeted therapy. However, detection of KRAS mutations in tissues has shown some limitations. During the last years, analyses of circulating free DNA (cfDNA) has emerged as an alternative and minimally invasive, approach to investigate tumor molecular changes. Here, we assessed the diagnostic performance of cfDNA analysis, compared to tissues through a meta-analysis and systematic review of existing literature. From 561 candidate papers, we finally identified 40 studies, including 2,805 NSCLC patients. We extracted values relating to the number of true-positive, false-positive, false-negative, and true-negative. Pooled sensitivity, specificity, positive likelihood ratio, negative likelihood ratio, and diagnostic odds ratio, each with 95% CI, were calculated. A summary receiver operating characteristic curve and the area under curve (AUC) were used to evaluate the overall diagnostic performance. The pooled sensitivity was 0.71 (95% CI 0.68-0.74) and the specificity was 0.93 (95% CI 0.92-0.94). The diagnostic odds ratio was 35.24 (95% CI 24.88-49.91) and the area under the curve was 0.92 (SE = 0.094). These results provide evidence that detection of KRAS mutation using cfDNA testing is of adequate diagnostic accuracy thus offering to the clinicians a new promising screening test for NSCLC patients.

EGFR T790M testing through repeated liquid biopsy over time: a real-world multicentric retrospective experience.

Background: About 15% of non-small cell lung cancers (NSCLCs) harbor epidermal growth factor receptor (EGFR) mutations. Upfront treatment with first and second generation EGFR tyrosine kinase inhibitors (1-2gen TKIs) is superior to chemotherapy. The most frequent resistance mechanism to 1-2gen TKIs is EGFR T790M mutation, which is targeted by osimertinib. T790M mutation can be revealed by liquid biopsy (LB) or by tissue rebiopsy (TB). LB is easily feasible but less sensitive than TB. We focused on repeated LBs and analyzed clinical features associated with EGFR T790M detection. Methods: This is a retrospective multicenter observational study including EGFR-mutant NSCLC consecutive patients with disease progression (PD) after 1-2gen TKIs and with a first EGFR LB negative for T790M mutation, referred between 2016 and 2019. Aims of the study were to determine the prevalence of T790M mutation using LB in a real-life setting and the prevalence of T790M mutation by repeated LBs. We explored the association of T790M with clinical-pathological features and, through a survey, we evaluated the decision-making process behind LB request. Data on TBs were also collected. Results: One hundred and ten patients were included in the study, for a total of 326 LBs. Median number of LB per patient was 3.0. The T790M prevalence through LB was 34.5%. Over time, significantly more LBs were requested "at clinical and radiological PD" and "at radiological PD" compared to "arbitrarily". The probability of finding the T790M mutation for a patient across each subsequent LB did not significantly change. Liver and lymph node PD were significantly correlated to T790M positivity. Notably, "at PD" compared to "arbitrarily" LB request and liver, bone or lymph node PD were correlated to the detection of any EGFR mutation in cfDNA. TB was performed in 59.7% of patients with a T790M negative LB and 18.8% of them were T790M positive. In most cases, TB was not feasible due to anatomical reasons. In our study population, the overall T790M prevalence-detected with both LB and TB-was 42.7%. Conclusions: Repeated LB testing can be useful in a real-life scenario to detect EGFR T790M mutation.

Implementation of Next Generation Sequencing-Based Liquid Biopsy for Clinical Molecular Diagnostics in Non-Small Cell Lung Cancer (NSCLC) Patients.

Genetic screening of somatic mutations in circulating free DNA (cfDNA) opens up new opportunities for personalized medicine. In this study, we aim to illustrate the implementation of NGS-based liquid biopsy in clinical practice for the detection of somatic alterations in selected genes. Our work is particularly relevant for the diagnosis and treatment of NSCLC. Beginning in 2020, we implemented the use of Roche's Avenio ctDNA expanded panel in our diagnostic routine. In this study, we retrospectively review NGS-based clinical genetic tests performed in our laboratory, focusing on key analytical parameters. Avenio ctDNA kits demonstrated 100% sensitivity in detecting single nucleotide variants (SNVs) at >0.5% variant allele frequency (VAF), and high consistency in reproducibility. Since 2020, we performed cfDNA genotyping test in 86 NSCLC patients, and we successfully sequenced 96.5% (83/86) of samples. We observed consistency in sequencing performance based upon sequencing depth and on-target rate. At least one gene variant was identified in 52 samples (63%), and one or more actionable variants were detected in 21 out of 83 (25%) of analysed patients. We demonstrated the feasibility of implementing an NGS-based liquid biopsy assay for routine genetic characterization of metastatic NSCLC patients.

ESR1 Gene Mutation in Hormone Receptor-Positive HER2-Negative Metastatic Breast Cancer Patients: Concordance Between Tumor Tissue and Circulating Tumor DNA Analysis.

Endocrine therapy represents the cornerstone of treatment in hormone receptor-positive (HR+), HER2-negative metastatic breast cancer (mBC). The natural course of this disease is marked by endocrine resistance, mainly due to Estrogen Receptor 1 (ESR1) acquired mutations. The aim of this study is to evaluate the concordance between ESR1 status in metastatic tumor specimens and matched circulating tumor DNA (ctDNA). Forty-three patients with HR+, HER2-negative mBC underwent both a metastatic tumor biopsy and a liquid biopsy at the time of disease progression. DNA extracted from formalin fixed paraffin embedded (FFPE) tumor specimens and ctDNA from matched plasma were analyzed by droplet digital (dd)PCR for the main ESR1 mutations (Y537S, Y537C, Y537N, D538G, E380Q). We observed a total mutation rate of 21%. We found six mutations on tissue biopsy: Y537S (1), D538G (2), Y537N (1), E380Q (2). Three patients with no mutations in tumor tissue had mutations detected in ctDNA. The total concordance rate between ESR1 status on tumor tissue and plasma was 91%. Our results confirm the potential role of liquid biopsy as a non-invasive alternative to tissue biopsy for ESR1 mutation assessment in mBC patients.

Role of next generation sequencing-based liquid biopsy in advanced non-small cell lung cancer patients treated with immune checkpoint inhibitors: impact of STK11, KRAS and TP53 mutations and co-mutations on outcome.

BACKGROUND: Characterization of tumor-related genetic alterations is promising for the screening of new predictive markers in non-small cell lung cancer (NSCLC). Aim of the study was to evaluate prognostic and predictive role of most frequent tumor-associated genetic alterations detected in plasma before starting immune checkpoint inhibitors (ICIs). METHODS: Between January 2017 and October 2019, advanced NSCLC patients were prospectively screened with plasma next-generation sequencing (NGS) while included in two trials: VISION (NCT02864992), using Guardant360® test, and MAGIC (Monitoring Advanced NSCLC through plasma Genotyping during Immunotherapy: Clinical feasibility and application), using Myriapod NGS-IL 56G Assay. A control group of patients not receiving ICIs was analyzed. RESULTS: A total of 103 patients receiving ICIs were analyzed: median overall survival (OS) was 20.8 (95% CI: 16.7-24.9) months and median immune-related progression free disease (irPFS) 4.2 (95% CI: 2.3-6.1) months. TP53 mutations in plasma negatively affected OS both in patients treated with ICIs and in control group (P=0.001 and P=0.009), indicating a prognostic role. STK11 mutated patients (n=9) showed a trend for worse OS only if treated with ICIs. The presence of KRAS/STK11 co-mutation and KRAS/STK11/TP53 co-mutation affected OS only in patients treated with ICIs (HR =10.936, 95% CI: 2.337-51.164, P=0.002; HR =17.609, 95% CI: 3.777-82.089, P<0.001, respectively), indicating a predictive role. CONCLUSIONS: Plasma genotyping demonstrated prognostic value of TP53 mutations and predictive value of KRAS/STK11 and KRAS/STK11/TP53 co-mutations.

In situ Metabolic Profiling of Ovarian Cancer Tumor Xenografts: A Digital Pathology Approach.

Metabolic profiling of cancer is a rising interest in the field of biomarker development. One bottleneck of its clinical exploitation, however, is the lack of simple and quantitative techniques that enable to capture the key metabolic traits of tumor from archival samples. In fact, liquid chromatography associated with mass spectrometry is the gold-standard technique for the study of tumor metabolism because it has high levels of accuracy and precision. However, it requires freshly frozen samples, which are difficult to collect in large multi-centric clinical studies. For this reason, we propose here to investigate a set of established metabolism-associated protein markers by exploiting immunohistochemistry coupled with digital pathology. As case study, we quantified expression of MCT1, MCT4, GLS, PHGDH, FAS, and ACC in 17 patient-derived ovarian cancer xenografts and correlated it with survival. Among these markers, the glycolysis-associated marker MCT4 was negatively associated with survival of mice. The algorithm enabling a quantitative analysis of these metabolism-associated markers is an innovative research tool that can be exported to large sets of clinical samples and can remove the variability of individual interpretation of immunohistochemistry results.

Detection of Low-Frequency KRAS Mutations in cfDNA From EGFR-Mutated NSCLC Patients After First-Line EGFR Tyrosine Kinase Inhibitors.

BACKGROUND: Molecular profiling of advanced EGFR mutated NSCLC has recently demonstrated the co-existence of multiple genetic alterations. Specifically, co-existing KRAS-mutations in EGFR NSCLCs have been described, despite their prevalence at progression and their role in the response to EGFR tyrosine kinase inhibitors (TKIs) remain marginally explored. Aim of our study was to investigate the prevalence of co-existing KRAS mutations at the time of progressive disease and explore their impact on clinical outcome. MATERIALS AND METHODS: We retrospectively analyzed by digital droplet PCR prevalence of KRAS co-mutations in 106 plasma samples of EGFR mutated NSCLC patients, in progressive disease after EGFR TKI treatment as first-line therapy. RESULTS: KRAS co-mutations (codon 12 and 13) were identified in 3 patients (2.8% of analyzed samples), with low allelic frequency (<0.2%), and had a negative impact on clinical outcome to first-line EGFR TKI. CONCLUSION: Detection of KRAS mutations in cell-free DNA of EGFR mutant NSCLC patients at progression after first or second generation EGFR TKI is a rare event. Due to their low abundance, the negative impact of KRAS mutations on the response to EGFR TKI remains to be confirmed in larger studies.

Clinical Features and Progression Pattern of Acquired T790M-positive Compared With T790M-negative EGFR Mutant Non-small-cell Lung Cancer: Catching Tumor and Clinical Heterogeneity Over Time Through Liquid Biopsy.

BACKGROUND: Clinical-pathologic predictors of acquired T790M epidermal growth factor receptor (EGFR) mutation in Caucasian patients with non-small-cell lung cancer (NSCLC) progressing after first-/second-generation tyrosine kinase inhibitors (TKIs) is an open field for research. Similarly, the best time point for T790M detection by liquid or tissue biopsy after disease progression is currently matter of debate. PATIENTS AND METHODS: This is an observational study at 7 Italian centers enrolling patients with EGFR-mutant NSCLC progressing after first-/second-generation EGFR TKIs, between 2014 and 2018, aiming at comparing baseline clinical-pathologic features and progression patterns in acquired T790M-positive compared with T790M-negative cases. RESULTS: A total of 235 patients received first-line treatment with gefitinib (N = 126; 53%), erlotinib (N = 51; 22%), or afatinib (N = 58; 25%). In 120 (51%) cases, T790M was detected in liquid biopsy, tissue biopsy, or both. Age younger than 65 years (P = .037), the presence of common mutations (P = .004), and better response to first-line TKI (P = .023) were correlated with T790M positivity. T790M detection was associated with higher number of new progressing sites (P = .04), liver progression (P = .002), and a lower frequency of lung metastases (P = .027). When serial liquid biopsies were performed (N = 15), an oligoprogressive disease was correlated with a negative test outcome, whereas systemic progression was observed at the time of T790M positivity. CONCLUSION: This study on a Caucasian population showed that age, type of EGFR mutation at diagnosis, response to first-line treatment, and peculiar progression pattern are associated with T790M status. Serial liquid biopsy might be useful for treatment selection, especially when tissue rebiopsy is not feasible.

LKB1/AMPK Pathway and Drug Response in Cancer: A Therapeutic Perspective.

Inactivating mutations of the tumor suppressor gene Liver Kinase B1 (LKB1) are frequently detected in non-small-cell lung cancer (NSCLC) and cervical carcinoma. Moreover, LKB1 expression is epigenetically regulated in several tumor types. LKB1 has an established function in the control of cell metabolism and oxidative stress. Clinical and preclinical studies support a role of LKB1 as a central modifier of cellular response to different stress-inducing drugs, suggesting LKB1 pathway as a highly promising therapeutic target. Loss of LKB1-AMPK signaling confers sensitivity to energy depletion and to redox homeostasis impairment and has been associated with an improved outcome in advanced NSCLC patients treated with chemotherapy. In this review, we provide an overview of the interplay between LKB1 and its downstream targets in cancer and focus on potential therapeutic strategies whose outcome could depend from LKB1.

Involvement of NADPH Oxidase 1 in Liver Kinase B1-Mediated Effects on Tumor Angiogenesis and Growth.

The liver kinase B1 (LKB1) gene is a tumor suppressor with an established role in the control of cell metabolism and oxidative stress. However, whether dis-regulated oxidative stress promotes growth of LKB1-deficient tumors remains substantially unknown. Through in vitro studies, we observed that loss of LKB1 perturbed expression of several genes involved in reactive oxygen species (ROS) homeostasis. In particular, this analysis evidenced strongly up-modulated NADPH oxidase 1 (NOX1) transcript levels in tumor cells lacking LKB1. NOX1 accounted in part for enhanced cytotoxic effects of H2O2-induced oxidative stress in A549 LKB1-deficient tumor cells. Notably, genetic and pharmacologic inhibition of NOX1 activity reduced angiogenesis and growth of A549 tumors in mice. These results suggest that NOX1 inhibitors could counteract ROS production and the angiogenic switch in LKB1-deficient tumors.

Metformin Enhances Cisplatin-Induced Apoptosis and Prevents Resistance to Cisplatin in Co-mutated KRAS/LKB1 NSCLC.

INTRODUCTION: We hypothesized that activating KRAS mutations and inactivation of the liver kinase B1 (LKB1) oncosuppressor can cooperate to sustain NSCLC aggressiveness. We also hypothesized that the growth advantage of KRAS/LKB1 co-mutated tumors could be balanced by higher sensitivity to metabolic stress conditions, such as metformin treatment, thus revealing new strategies to target this aggressive NSCLC subtype. METHODS: We retrospectively determined the frequency and prognostic value of KRAS/LKB1 co-mutations in tissue specimens from NSCLC patients enrolled in the TAILOR trial. We generated stable LKB1 knockdown and LKB1-overexpressing isogenic H1299 and A549 cell variants, respectively, to test the in vitro efficacy of metformin. We also investigated the effect of metformin on cisplatin-resistant CD133+ cells in NSCLC patient-derived xenografts. RESULTS: We found a trend towards worse overall survival in patients with KRAS/LKB1 co-mutated tumors as compared to KRAS-mutated ones (hazard ratio: 2.02, 95% confidence interval: 0.94-4.35, p = 0.072). In preclinical experiments, metformin produced pro-apoptotic effects and enhanced cisplatin anticancer activity specifically in KRAS/LKB1 co-mutated patient-derived xenografts. Moreover, metformin prevented the development of acquired tumor resistance to 5 consecutive cycles of cisplatin treatment (75% response rate with metformin-cisplatin as compared to 0% response rate with cisplatin), while reducing CD133+ cells. CONCLUSIONS: LKB1 mutations, especially when combined with KRAS mutations, may define a specific and more aggressive NSCLC subtype. Metformin synergizes with cisplatin against KRAS/LKB1 co-mutated tumors, and may prevent or delay the onset of resistance to cisplatin by targeting CD133+ cancer stem cells. This study lays the foundations for combining metformin with standard platinum-based chemotherapy in the treatment of KRAS/LKB1 co-mutated NSCLC.

Therapeutic concentrations of digitoxin inhibit endothelial focal adhesion kinase and angiogenesis induced by different growth factors.

BACKGROUND AND PURPOSE: Cardiac glycosides are Na+ /K+ -ATPases inhibitors used to treat congestive heart failure and cardiac arrhythmias. Epidemiological studies indicate that patients on digitalis therapy are more protected from cancer. Evidence of a selective cytotoxicity against cancer cells has suggested their potential use as anticancer drugs. The effect on angiogenesis of clinically used cardiac glycosides has not been extensively explored. EXPERIMENTAL APPROACH: We studied the effect of digoxin, digitoxin and ouabain on early events of the angiogenic process in HUVECs. We determined HUVEC viability, proliferation, migration and differentiation into capillary tube-like structures. We also tested drug activity using an in vivo angiogenesis model. Activation of protein tyrosine kinase 2 (FAK) and signalling proteins associated with the Na+ /K+ -ATPase signalosome was determined by Western blotting. KEY RESULTS: Digitoxin and ouabain (1-100 nM) inhibited HUVEC migration, concentration-dependently, without affecting cell viability, while digoxin induced apoptosis at the same concentrations. Digitoxin antagonized growth factor-induced migration and tubularization at concentrations (1-25 nM) within its plasma therapeutic range. The anti-angiogenic effect of digitoxin was confirmed also by in vivo studies. Digitoxin induced Src, Akt and ERK1/2 phosphorylation but did not affect FAK autophosphorylation at Tyr397 . However, it significantly inhibited growth factor-induced FAK phosphorylation at Tyr576/577 . CONCLUSIONS AND IMPLICATIONS: Therapeutic concentrations of digitoxin inhibited angiogenesis and FAK activation by several pro-angiogenic stimuli. These novel findings suggest a potential repositioning of digitoxin as a broad-spectrum anti-angiogenic drug for diseases where pathological angiogenesis is involved.

LKB1 Expression Correlates with Increased Survival in Patients with Advanced Non-Small Cell Lung Cancer Treated with Chemotherapy and Bevacizumab.

Purpose: LKB1 is a key sensor of metabolic stress, including hypoxia and glucose deprivation, two features of the tumor microenvironment exacerbated by antiangiogenic therapy. We investigated the role of LKB1 as a potential predictive marker of sensitivity to bevacizumab in advanced non-small cell lung cancer (aNSCLC).Experimental design: We retrospectively analyzed LKB1 expression by IHC in 98 samples from 125 patients with aNSCLC, including 59 patients treated with chemotherapy and 39 treated with chemotherapy plus bevacizumab. IHC intensity was recoded in two classes (negative/weak vs. moderate/intense) and correlated with outcome according to treatment arm. Patient-derived tumor xenografts (PDXs) were used to investigate mechanisms involved in preclinical models.Results: In the whole study population (125), median OS and PFS were 11.7 [95% confidence interval (CI), 9.1-15.3] and 6.7 (95% CI, 5.7-7.2) months, respectively. Differential impact of the marker on outcome of the 98 patients was highlighted according to the treatment. Patients with negative/weak LKB1 status did not have a statistically significant benefit from bevacizumab added to chemotherapy (HR for patients treated with bevacizumab: 0.89; 95% CI, 0.51-1.56; P = 0.6803), whereas patients expressing moderate/intense LKB1 and receiving bevacizumab had significant lower risk of death compared with those not receiving bevacizumab (HR, 0.26; 95% CI, 0.10-0.64; P = 0.0035). Loss of LKB1 was associated with reduced AMPK activation in PDXs and increased tumor necrosis following bevacizumab administration, highlighting impaired control of the metabolic stress caused by this antiangiogenic drug.Conclusions: Our data hint at a possible predictive impact of LKB1 expression in patients with aNSCLC treated with chemotherapy plus bevacizumab. Clin Cancer Res; 23(13); 3316-24. ©2017 AACR.