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.

Platelet-derived growth factor-D enables liver myofibroblasts to promote tumor lymphangiogenesis in cholangiocarcinoma.

BACKGROUND & AIMS: In cholangiocarcinoma, early metastatic spread via lymphatic vessels often precludes curative therapies. Cholangiocarcinoma invasiveness is fostered by an extensive stromal reaction, enriched in cancer-associated fibroblasts (CAFs) and lymphatic endothelial cells (LECs). Cholangiocarcinoma cells recruit and activate CAFs by secreting PDGF-D. Herein, we investigated the role of PDGF-D and liver myofibroblasts in promoting lymphangiogenesis in cholangiocarcinoma. METHODS: Human cholangiocarcinoma specimens were immunostained for podoplanin (LEC marker), α-SMA (CAF marker), VEGF-A, VEGF-C, and their cognate receptors (VEGFR2, VEGFR3). VEGF-A and VEGF-C secretion was evaluated in human fibroblasts obtained from primary sclerosing cholangitis explants. Using human LECs incubated with conditioned medium from PDGF-D-stimulated fibroblasts we assessed migration, 3D vascular assembly, transendothelial electric resistance and transendothelial migration of cholangiocarcinoma cells (EGI-1). We then studied the effects of selective CAF depletion induced by the BH3 mimetic navitoclax on LEC density and lymph node metastases in vivo. RESULTS: In cholangiocarcinoma specimens, CAFs and LECs were closely adjacent. CAFs expressed VEGF-A and VEGF-C, while LECs expressed VEGFR2 and VEGFR3. Upon PDGF-D stimulation, fibroblasts secreted increased levels of VEGF-C and VEGF-A. Fibroblasts, stimulated by PDGF-D induced LEC recruitment and 3D assembly, increased LEC monolayer permeability, and promoted transendothelial EGI-1 migration. These effects were all suppressed by the PDGFRß inhibitor, imatinib. In the rat model of cholangiocarcinoma, navitoclax-induced CAF depletion, markedly reduced lymphatic vascularization and reduced lymph node metastases. CONCLUSION: PDGF-D stimulates VEGF-C and VEGF-A production by fibroblasts, resulting in expansion of the lymphatic vasculature and tumor cell intravasation. This critical process in the early metastasis of cholangiocarcinoma may be blocked by inducing CAF apoptosis or by inhibiting the PDGF-D-induced axis. LAY SUMMARY: Cholangiocarcinoma is a highly malignant cancer affecting the biliary tree, which is characterized by a rich stromal reaction involving a dense population of cancer-associated fibroblasts that promote early metastatic spread. Herein, we show that cholangiocarcinoma-derived PDGF-D stimulates fibroblasts to secrete vascular growth factors. Thus, targeting fibroblasts or PDGF-D-induced signals may represent an effective tool to block tumor-associated lymphangiogenesis and reduce the invasiveness of cholangiocarcinoma.

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.

Platelet-derived growth factor-D and Rho GTPases regulate recruitment of cancer-associated fibroblasts in cholangiocarcinoma.

UNLABELLED: Cholangiocarcinoma (CCA) is characterized by an abundant stromal reaction. Cancer-associated fibroblasts (CAFs) are pivotal in tumor growth and invasiveness and represent a potential therapeutic target. To understand the mechanisms leading to CAF recruitment in CCA, we studied (1) expression of epithelial-mesenchymal transition (EMT) in surgical CCA specimens and CCA cells, (2) lineage tracking of an enhanced green fluorescent protein (EGFP)-expressing human male CCA cell line (EGI-1) after xenotransplantation into severe-combined-immunodeficient mice, (3) expression of platelet-derived growth factors (PDGFs) and their receptors in vivo and in vitro, (4) secretion of PDGFs by CCA cells, (5) the role of PDGF-D in fibroblast recruitment in vitro, and (6) downstream effectors of PDGF-D signaling. CCA cells expressed several EMT biomarkers, but not alpha smooth muscle actin (α-SMA). Xenotransplanted CCA masses were surrounded and infiltrated by α-SMA-expressing CAFs, which were negative for EGFP and the human Y-probe, but positive for the murine Y-probe. CCA cells were strongly immunoreactive for PDGF-A and -D, whereas CAFs expressed PDGF receptor (PDGFR)ß. PDGF-D, a PDGFRß agonist, was exclusively secreted by cultured CCA cells. Fibroblast migration was potently induced by PDGF-D and CCA conditioned medium and was significantly inhibited by PDGFRß blockade with Imatinib and by silencing PDGF-D expression in CCA cells. In fibroblasts, PDGF-D activated the Rac1 and Cdc42 Rho GTPases and c-Jun N-terminal kinase (JNK). Selective inhibition of Rho GTPases (particularly Rac1) and of JNK strongly reduced PDGF-D-induced fibroblast migration. CONCLUSION: CCA cells express several mesenchymal markers, but do not transdifferentiate into CAFs. Instead, CCA cells recruit CAFs by secreting PDGF-D, which stimulates fibroblast migration through PDGFRß and Rho GTPase and JNK activation. Targeting tumor or stroma interactions with inhibitors of the PDGF-D pathway may offer a novel therapeutic approach.

Nuclear expression of S100A4 calcium-binding protein increases cholangiocarcinoma invasiveness and metastasization.

UNLABELLED: Cholangiocarcinoma (CCA) carries a severe prognosis because of its strong invasiveness and early metastasization. In several patients, otherwise eligible for surgical resection, micrometastasis are already present at the time of surgery. The mechanisms responsible for CCA invasiveness are unclear. S100A4, a member of the S100 family of small Ca(2+)-binding proteins, is expressed in mesenchymal cells, regulates cell motility in several cell types, and is expressed in some epithelial cancers. Thus, we aimed to study the role of S100A4 in CCA invasiveness and metastasization. The expression of S100A4 was studied by immunohistochemistry in 93 human liver samples of CCA patients undergoing surgical resection and correlated with metastases development (67 cases) and patient survival following surgery using log rank tests and multivariate analysis. S100A4 expression was studied in EGI-1 and TFK-1, human CCA cell lines with and without nuclear S100A4 expression, respectively. Metastatic properties of CCA cells were assessed by xenotransplantation in severe combined immunodeficiency (SCID) mice after transduction with lentiviral vectors encoding firefly luciferase gene. Proliferation, motility (wound healing), invasiveness (Boyden chamber), and metalloproteinases (MMPs) secretion were studied in CCA cells, with or without lentiviral silencing of S100A4. Nuclear expression of S100A4 by neoplastic ducts was a strong predictor of metastasization and reduced survival after resection (P < 0.01). EGI-1 CCA cells showed stronger metastatic properties than TFK-1 when xenotransplanted in SCID mice. S100A4-silenced EGI-1 cells showed significantly reduced motility, invasiveness, and MMP-9 secretion in vitro, without changes in cell proliferation. CONCLUSION: Nuclear S100A4 identifies a subset of CCA patients with a poor prognosis after surgical resection. Nuclear expression of S100A4 increases CCA cells invasiveness and metastasization, indicating that S100A4 may also represent a potential therapeutic target.

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.

Low P66shc with High SerpinB3 Levels Favors Necroptosis and Better Survival in Hepatocellular Carcinoma.

Cell proliferation and escape from apoptosis are important pathological features of hepatocellular carcinoma (HCC), one of the tumors with the highest mortality rate worldwide. The aim of the study was to evaluate the expression of the pro-apoptotic p66shc and the anti-apoptotic SerpinB3 in HCCs in relation to clinical outcome, cell fate and tumor growth. p66shc and SerpinB3 were evaluated in 67 HCC specimens and the results were correlated with overall survival. Proliferation and cell death markers were analyzed in hepatoma cells overexpressing SerpinB3, under different stress conditions. p66shc-/- mice and xenograft models were also used to assess the effects of p66shc and SerpinB3 on tumor growth. In patients with HCC, the best survival was observed in the subgroup with p66shc levels below median values and SerpinB3 levels above median values. Mice p66shc-/- showed high levels of SerpinB3, while in HepG2 cells overexpressing SerpinB3, p66shc expression was trivial. HepG2 overexpressing SerpinB3 cells were more prone to die after oxidizing treatments, such as diamide or high concentration H2O2. These cells injected in nude mice developed tumors five times smaller than those from control HepG2 cells. Tumors originating from HepG2 overexpressing SerpinB3 cells showed decreased activated Caspase-8, with concomitant increase of RIP3K and decreased levels of cleaved RIP3K, typical features of necroptosis. In conclusion, in patients affected by HCC, the pattern characterized by p66shc downregulation and elevated SerpinB3 levels was associated with markedly better survival. This pattern favored necroptosis in experimental high-stress conditions.

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.

A Multi-Center, Real-Life Experience on Liquid Biopsy Practice for EGFR Testing in Non-Small Cell Lung Cancer (NSCLC) Patients.

BACKGROUND: circulating tumor DNA (ctDNA) is a source of tumor genetic material for EGFR testing in NSCLC. Real-word data about liquid biopsy (LB) clinical practice are lacking. The aim of the study was to describe the LB practice for EGFR detection in North Eastern Italy. METHODS: we conducted a multi-regional survey on ctDNA testing practices in lung cancer patients. RESULTS: Median time from blood collection to plasma separation was 50 min (20-120 min), median time from plasma extraction to ctDNA analysis was 24 h (30 min-5 days) and median turnaround time was 24 h (6 h-5 days). Four hundred and seventy five patients and 654 samples were tested. One hundred and ninety-two patients were tested at diagnosis, with 16% EGFR mutation rate. Among the 283 patients tested at disease progression, 35% were T790M+. Main differences in LB results between 2017 and 2018 were the number of LBs performed for each patient at disease progression (2.88 vs. 1.2, respectively) and the percentage of T790M+ patients (61% vs. 26%).

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.

Hypoxia inducible factor-1alpha inactivation unveils a link between tumor cell metabolism and hypoxia-induced cell death.

Hypoxia and the acquisition of a glycolytic phenotype are intrinsic features of the tumor microenvironment. The hypoxia inducible factor-1alpha (HIF-1alpha) pathway is activated under hypoxic conditions and orchestrates a complex transcriptional program that enhances cell survival. Although the consequences of HIF-1alpha inactivation in cancer cells have been widely investigated, only a few studies have addressed the role of HIF-1alpha in the survival of cancer cells endowed with different glycolytic capacities. In this study, we investigated this aspect in ovarian cancer cells. Hypoxia-induced toxicity was increased in highly glycolytic cells compared with poorly glycolytic cells; it was also associated with a sharp decrease in intracellular ATP levels and was prevented by glucose supplementation. Stable HIF-1alpha silencing enhanced hypoxia-induced cell death in vitro due to a lack of cell cycle arrest. Tumors bearing attenuated HIF-1alpha levels had similar growth rates and vascularization as did controls, but tumors showed higher proliferation levels and increased necrosis. Moreover, tumors formed by HIF-1alpha deficient cells had higher levels of lactate and lower ATP concentrations than controls as shown by metabolic imaging. The findings that such metabolic properties can affect the survival of cancer cells under hypoxic conditions and that these properties contribute to the determination of the consequences of HIF-1alpha inactivation could have important implications on the understanding of the effects of anti-angiogenic and HIF-1alpha-targeting drugs in cancer.

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.

RNA interference: implications for cancer treatment.

RNA interference (RNAi) has emerged as one of the most important discoveries of the last years in the field of molecular biology. Following clarification of this highly conserved endogenous gene silencing mechanism, RNAi has largely been exploited as a powerful tool to uncover the function of specific genes and to understand the effects of selective gene silencing in mammalian cells both in vitro and in vivo. RNAi can be induced by direct introduction of chemically synthesized siRNAs into the cell or by the use of plasmid and viral vectors encoding for siRNA allowing a more stable RNA knockdown. Potential application of this technique both as a research tool and for therapeutic purposes has led to an extensive effort to overcome some critical constraints which may limit its successful application in vivo, including off-target and non-specific effects, as well as the relatively poor stability of siRNA. This review provides a brief overview of the RNAi mechanism and of its application in preclinical animal models of cancer.

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.

Morphological and genetic heterogeneity in multifocal lung adenocarcinoma: The case of a never-smoker woman.

Discrimination of multifocal primary lung cancers from lung metastases is crucial to allow for an appropriate clinical management. We report here a case of multifocal lung adenocarcinomas with different morphological and molecular patterns. Radical surgery of one lung nodule was performed at the time of diagnosis, and subsequently on two other lung nodules. At the time of distant relapse, biopsy was repeated for molecular characterization. The patient was treated with EGFR tyrosine kinase inhibitor according to the detection of EGFR exon 21 mutation in metastatic sample and in one of the three lung tumors, characterized by lower mutated allele frequency. The progression free survival was three months according to radiological criteria and the treatment was provided for six months, until clinical progression. Following the assessment of EGFR mutations by pyrosequencing, tumor samples were analyzed by a 30-gene next generation sequencing (NGS) panel, allowing to study intra- and inter-tumor heterogeneity and to confirm the three lung tumors as independent. Different molecular profiles of synchronous tumors and identical EGFR, PIK3CA and TP53 mutations in one of three primary lung tumors and the metachronous metastasis were identified. In conclusion, morphological and molecular characterization of multiple lung nodules by NGS may help to define synchronous and metachronous adenocarcinomas, thus affecting surgical indication and systemic treatment. Intratumor heterogeneity may be associated with differential sensitivity to targeted treatment.

Low-Dose Paclitaxel Reduces S100A4 Nuclear Import to Inhibit Invasion and Hematogenous Metastasis of Cholangiocarcinoma.

Nuclear expression of the calcium-binding protein S100A4 is a biomarker of increased invasiveness in cholangiocarcinoma, a primary liver cancer with scarce treatment opportunities and dismal prognosis. In this study, we provide evidence that targeting S100A4 nuclear import by low-dose paclitaxel, a microtubule-stabilizing agent, inhibits cholangiocarcinoma invasiveness and metastatic spread. Administration of low-dose paclitaxel to established (EGI-1) and primary (CCA-TV3) cholangiocarcinoma cell lines expressing nuclear S100A4 triggered a marked reduction in nuclear expression of S100A4 without modifying its cytoplasmic levels, an effect associated with a significant decrease in cell migration and invasiveness. While low-dose paclitaxel did not affect cellular proliferation, apoptosis, or cytoskeletal integrity, it significantly reduced SUMOylation of S100A4, a critical posttranslational modification that directs its trafficking to the nucleus. This effect of low-dose paclitaxel was reproduced by ginkolic acid, a specific SUMOylation inhibitor. Downregulation of nuclear S100A4 by low-dose paclitaxel was associated with a strong reduction in RhoA and Cdc42 GTPase activity, MT1-MMP expression, and MMP-9 secretion. In an SCID mouse xenograft model, low-dose metronomic paclitaxel treatment decreased lung dissemination of EGI-1 cells without significantly affecting their local tumor growth. In the tumor mass, nuclear S100A4 expression by cholangiocarcinoma cells was significantly reduced, whereas rates of proliferation and apoptosis were unchanged. Overall, our findings highlight nuclear S100A4 as a candidate therapeutic target in cholangiocarcinoma and establish a mechanistic rationale for the use of low-dose paclitaxel in blocking metastatic progression of cholangiocarcinoma. Cancer Res; 76(16); 4775-84. ©2016 AACR.