The Impact of Inadequate Exposure to Epidermal Growth Factor Receptor-Tyrosine Kinase Inhibitors on the Development of Resistance in Non-Small-Cell Lung Cancer Cells.

Epidermal growth factor receptor (EGFR)-mutant non-small-cell lung cancer (NSCLC) patients treated with EGFR-tyrosine kinase inhibitors (TKIs) inevitably develop resistance through several biological mechanisms. However, little is known on the molecular mechanisms underlying acquired resistance to suboptimal EGFR-TKI doses, due to pharmacodynamics leading to inadequate drug exposure. To evaluate the effects of suboptimal EGFR-TKI exposure on resistance in NSCLC, we obtained HCC827 and PC9 cell lines resistant to suboptimal fixed and intermittent doses of gefitinib and compared them to cells exposed to higher doses of the drug. We analyzed the differences in terms of EGFR signaling activation and the expression of epithelial-mesenchymal transition (EMT) markers, whole transcriptomes byRNA sequencing, and cell motility. We observed that the exposure to low doses of gefitinib more frequently induced a partial EMT associated with an induced migratory ability, and an enhanced transcription of cancer stem cell markers, particularly in the HCC827 gefitinib-resistant cells. Finally, the HCC827 gefitinib-resistant cells showed increased secretion of the EMT inducer transforming growth factor (TGF)-ß1, whose inhibition was able to partially restore gefitinib sensitivity. These data provide evidence that different levels of exposure to EGFR-TKIs in tumor masses might promote different mechanisms of acquired resistance.

Gut health benefits and associated systemic effects provided by functional components from the fermentation of natural matrices.

Recently, the role of the gut microbiota in metabolic health, immunity, behavioral balance, longevity, and intestine comfort has been the object of several studies from scientific communities. They were encouraged by a growing interest from food industries and consumers toward novel fermented ingredients and formulations with powerful biological effects, such as pre, pro, and postbiotic products. Depending on the selected strains, the operating conditions, the addition of suitable reagents or enzymes, the equipment, and the reactor configurations, functional compounds with high bioactivity, such as short-chain fatty acids, gamma-aminobutyric acid, bioactive peptides, and serotonin, can be enhanced and/or produced through fermentation of several vegetable matrices. Otherwise, their formation can also be promoted directly in the gut after the dietary intake of fermented foods: In this case, fermentation will aim to increase the content of precursor substances, such as indigestible fibers, polyphenols, some amino acids, and resistant starch, which can be potentially metabolized by endogenous gut microorganisms and converted in healthy molecules. This review provides an overview of the main functional components currently investigated in literature and the associated gut health benefits. The current state of the art about fermentation technology as a promising functionalization tool to promote the direct or indirect formation of gut-health-enhancing components was deepened, highlighting the importance of optimizing microorganism selection, system setups, and process conditions according to the target compound of interest. The collected data suggested the possibility of gaining novel functional food ingredients or products rich in functional molecules through fermentation without performing additional extraction and purification stages, which are needed when conventional culture broths are used.

Liquid Biopsy Testing for the Management of Patient with Non-Small Cell Lung Cancer Carrying a Rare Exon-20 EGFR Insertion.

Increasing evidence suggests that liquid biopsy might play a relevant role in the management of metastatic non-small cell lung cancer (NSCLC) patients. Here, we show how the Molecular Tumor Board (MTB) in our cancer center employed liquid biopsy to support therapeutic decisions in a patient with NSCLC carrying a rare EGFR mutation. A 44-year-old woman, never-smoker with an EGFR, ALK, and ROS1-negative lung adenocarcinoma and multiple brain metastases received systemic therapy and surgery before being referred to our Institute. The MTB suggested NGS testing of tumor biopsy that revealed a rare exon-20 EGFR insertion (p.His773dup; c.2315_2316insCCA) and EGFR amplification. The MTB recommended treatment with erlotinib and follow-up with liquid biopsy, by using both cell-free DNA (cfDNA) and circulating tumor cells (CTCs). An increase of EGFR mutation levels in cfDNA revealed resistance to treatment about 6 months before clinical progression. Extremely low levels of EGFR p.T790M were detected at progression. Based on preclinical data suggesting activity of osimertinib against EGFR exon-20 insertions, the MTB recommended treatment with brain and bone radiotherapy and osimertinib. A dramatic reduction of EGFR mutation levels in the cfDNA was observed after 4 weeks of treatment. The PET scan demonstrated a metabolic partial remission that was maintained for 9 months. This case supports the evidence that liquid biopsy can aid in the management of metastatic NSCLC. It also suggests that treatment with osimertinib might be a therapeutic option in patients with EGFR exon-20 insertions when a clinical trial is not available.

Effect of pH control during rice fermentation in preventing a gliadin P31-43 entrance in epithelial cells.

Coeliac disease is an increasingly recognised pathology, induced by the ingestion of gluten in genetically predisposed patients. Undigested gliadin peptide can induce adaptive and innate immune response that unleash the typical intestinal mucosal alterations. A growing attention is paid to alternative therapeutic approaches to the gluten-free diet: one of these approaches is the use of probiotics and/or postbiotics. We performed lactic fermentation of rice flour with and without pH control, using Lactobacillus paracasei CBA L74 as fermenting strain. We evaluated bacterial growth, lactic acid production during fermentation and gliadin peptide P31-43 entrance in CaCo-2 cells with and without pH control. When pH control was applied no differences were observed in terms of bacterial growth; on the contrary, lactic acid production was greater, as expected. Both samples could inhibit the P31-43 entrance in CaCo-2 cells but the effect was significantly greater for samples obtained when the pH control was applied.

Vascular Endothelial Growth Factor A Regulates the Secretion of Different Angiogenic Factors in Lung Cancer Cells.

Vascular endothelial growth factor A (VEGFA) is one of the main mediators of angiogenesis in non-small cell lung cancer (NSCLC). Recently, it has been described an autocrine feed-forward loop in NSCLC cells in which tumor-derived VEGFA promoted the secretion of VEGFA itself, amplifying the proangiogenic signal. In order to investigate the role of VEGFA in lung cancer progression, we assessed the effects of recombinant VEGFA on proliferation, migration, and secretion of other angiogenic factors in A549, H1975, and HCC827 NSCLC cell lines. We found that VEGFA did not affect NSCLC cell proliferation and migration. On the other hand, we demonstrated that VEGFA not only produced a strong and persistent increase of VEGFA itself but also significantly induced the secretion of a variety of angiogenic factors, including follistatin (FST), hepatocyte growth factor (HGF), angiopoietin-2 (ANGPT2), granulocyte-colony stimulating factor (G-CSF), interleukin (IL)-8, leptin (LEP), platelet/endothelial cell adhesion molecule 1 (PECAM-1), and platelet-derived growth factor bb (PDGF-BB). PI3K/AKT, RAS/ERK, and STAT3 signalling pathways were found to mediate the effects of VEGFA in NSCLC cell lines. We also observed that VEGFA regulation mainly occurred at post-transcriptional level and that NSCLC cells expressed different isoforms of VEGFA. Collectively, our data suggested that VEGFA contributes to lung cancer progression by inducing a network of angiogenic factors, which might offer potential for therapeutic intervention.

EGFR and MEK Blockade in Triple Negative Breast Cancer Cells.

Although evidence suggests that the RAF/MEK/ERK pathway plays an important role in triple negative breast cancer (TNBC), resistance to MEK inhibitors has been observed in TNBC cells. Different mechanisms have been hypothesized to be involved in this phenomenon, including receptor tyrosine kinase-dependent activation of the PI3K/AKT pathway. In this study, we analyzed the effects of the MEK1/2 inhibitor selumetinib in combination with the epidermal growth factor receptor (EGFR) tyrosine kinase inhibitor gefitinib in a panel of TNBC cell lines that showed different levels of sensitivity to single-agent selumetinib: SUM-149 and MDA-MB-231 cells resulted to be sensitive, whereas SUM-159, MDA-MB-468 and HCC70 cells were relatively resistant to the drug. Treatment of TNBC cells with selumetinib produced an increase of the phosphorylation of the EGFR both in selumetinib-sensitive SUM-149, MDA-MB-231 and in selumetinib-resistant MDA-MB-468 TNBC cells. The combination of selumetinib and gefitinib resulted in a synergistic growth inhibitory effect in all the TNBC cell lines, although the IC50 was not reached in SUM-159 and MDA-MB-468 cells. This effect was associated with an almost complete suppression of ERK1/2 activation and a reduction of selumetinib-induced AKT phosphorylation. In addition, in selumetinib-sensitive TNBC cells the combination of selumetinib and gefitinib induced a significant G0/G1 cell cycle arrest and apoptosis. Taken together, our data demonstrated that blockade of the EGFR might efficiently increase the antitumor activity of selumetinib in a subgroup of TNBC and that this phenomenon might be related to the effects of such combination on both ERK1/2 and AKT activation.

Mesenchymal stem cell-derived interleukin-6 and vascular endothelial growth factor promote breast cancer cell migration.

Several different cytokines and growth factors secreted by mesenchymal stem cells (MSCs) have been hypothesized to play a role in breast cancer progression. By using a small panel of breast cancer cell lines (MCF-7, T47D, and SK-Br-3 cells), we analyzed the role of interleukin-6 (IL-6) and vascular endothelial growth factor A (VEGF) in the cross-talk between MSCs and breast cancer cells. We performed migration assays in which breast cancer cells were allowed to migrate in response to conditioned medium from MSCs (MSCs-CM), in absence or in presence of the anti-VEGF antibody bevacizumab or an anti-IL-6 antibody, alone or in combination. We found that anti-VEGF and anti-IL-6 antibodies inhibited the migration of breast cancer cells and that the combination had an higher inhibitory effect. We next evaluated the effects of recombinant VEGF and IL-6 proteins on breast cancer cell growth and migration. IL-6 and VEGF had not significant effects on the proliferation of breast carcinoma cells. In contrast, both VEGF and IL-6 significantly increased the ability to migrate of MCF-7, T47D and SK-Br-3 cells, with the combination showing a greater effect as compared with treatment with a single protein. The combination of VEGF and IL-6 produced in breast cancer cells a more significant and more persistent activation of MAPK, AKT, and p38MAPK intracellular signaling pathways. These results suggest that MSC-secreted IL-6 and VEGF may act as paracrine factors to sustain breast cancer cell migration.

The EGFR Signaling Modulates in Mesenchymal Stem Cells the Expression of miRNAs Involved in the Interaction with Breast Cancer Cells.

We previously demonstrated that the epidermal growth factor receptor (EGFR) modulates in mesenchymal stem cells (MSCs) the expression of a number of genes coding for secreted proteins that promote breast cancer progression. However, the role of the EGFR in modulating in MSCs the expression of miRNAs potentially involved in the progression of breast cancer remains largely unexplored. Following small RNA-sequencing, we identified 36 miRNAs differentially expressed between MSCs untreated or treated with the EGFR ligand transforming growth factor α (TGFα), with a fold change (FC) < 0.56 or FC ≥ 1.90 (CI, 95%). KEGG analysis revealed a significant enrichment in signaling pathways involved in cancer development and progression. EGFR activation in MSCs downregulated the expression of different miRNAs, including miR-23c. EGFR signaling also reduced the secretion of miR-23c in conditioned medium from MSCs. Functional assays demonstrated that miR-23c acts as tumor suppressor in basal/claudin-low MDA-MB-231 and MDA-MB-468 cells, through the repression of IL-6R. MiR-23c downregulation promoted cell proliferation, migration and invasion of these breast cancer cell lines. Collectively, our data suggested that the EGFR signaling regulates in MSCs the expression of miRNAs that might be involved in breast cancer progression, providing novel information on the mechanisms that regulate the MSC-tumor cell cross-talk.

Role of the EGFR ligand/receptor system in the secretion of angiogenic factors in mesenchymal stem cells.

Increasing evidence suggests that bone marrow-derived mesenchymal stem cells (MSCs) are recruited into the stroma of developing tumors where they contribute to cancer progression. MSCs produce different growth factors that sustain tumor-associated neo-angiogenesis. Since the majority of carcinomas secrete ligands of the epidermal growth factor receptor (EGFR), we assessed the role of EGFR signaling in regulating the release of angiogenic factors in MSCs. Treatment of human primary MSCs and of the human osteoblastic cell line hFOB with transforming growth factor α (TGF-α), one of the main ligands of the EGFR, significantly induced activation of this receptor and of different intracellular signaling proteins, including the PI3K/AKT and the MEK/MAPK pathways. TGF-α induced a significant increase in the levels of secretion of vascular endothelial growth factor in both MSCs and hFOB. Conditioned medium from TGF-α treated MSCs showed an higher in vivo angiogenic effect as compared with medium from untreated cells. Treatment of MSCs with TGF-α also produced a significant increase in the secretion of other angiogenic growth factors such as angiopoietin-2, granulocyte-colony stimulating factor, hepatocyte growth factor, interleukin (IL)-6, IL-8, and platelet-derived growth factor-BB. Using selective MEK and PI3K inhibitors, we found that both MEK/MAPK and the PI3K/AKT signaling pathways mediate the ability of TGF-α to induce secretion of angiogenic factors in MSCs. Finally, stimulation with TGF-α increased the ability of MSCs to induce migration of MCF-7 breast cancer cells. These data suggest that EGFR signaling regulates the ability of MSCs to sustain cancer progression through the release of growth factors that promote neo-angiogenesis and tumor cell migration.

Promising Role of Circulating Tumor Cells in the Management of SCLC.

Small cell lung cancer is an aggressive disease for which few therapeutic options are currently available. Although patients initially respond to therapy, they rapidly relapse. Up to today, no biomarkers for guiding treatment of SCLC patients have been identified. SCLC patients rarely undergo surgery and often the available tissue samples are inadequate for biomarker analysis. Circulating tumor cells (CTCs) are rare cells in the peripheral blood that might be used as surrogates of tissue samples. Different methodological approaches have been developed for studies of CTCs in SCLC. In addition to CTC count, which might provide prognostic and predictive information, genomic and transcriptomic analyses allow the characterization of molecular profiles of CTCs and permit the study of tumor heterogeneity. The employment of CTC-derived xenografts offers complementary information to genomic analyses and CTC enumeration about the mechanisms involved in the sensitivity/resistance to treatments. Using these approaches, CTC analysis is providing relevant information on SCLC biology that might aid in the development of personalized therapeutic strategies for SCLC patients.

Morphological and Rheological Guided Design for the Microencapsulation Process of Lactobacillus paracasei CBA L74 in Calcium Alginate Microspheres.

The intestinal microbiota is a real ecosystem composed of several bacterial species and a very huge amount of strains that through their metabolic activities play a crucial role in the development and performance of the immune system and other functions. Microbiota modulation by probiotics establishes a new era into the pharmaceutical and healthcare market. Probiotics play, in fact, an important role in helping and sustaining human health, but in order to produce benefits, their viability must be preserved throughout the production process up to consumption, and in addition, their bioactivity required to be safeguarded while passing through the gastrointestinal tract. In this frame, encouraging results come from encapsulation strategies that have proven to be very promising in protecting bacteria and their viability. However, specific effort has to be dedicated to the design optimization of the encapsulation process and, in particular, to the processing parameters that affect capsules microstructure. Herein, focusing on calcium alginate microspheres, after a preliminary selection of their processing conditions based on size distribution, we implemented a micro-rheological analysis, by using the multiple-particle tracking technique, to correlate the inner microstructure to the selected process conditions and to the viability of the Lactobacillus paracasei CBA L74. It was assessed that the explored levels of cross-linking, although changing the microorganism constriction, did not affect its viability. The obtained results confirm how this technology is a promising and a valid strategy to protect the microorganism viability and ensure its stability during the production process.

Effects of the combined blockade of EGFR and ErbB-2 on signal transduction and regulation of cell cycle regulatory proteins in breast cancer cells.

Treatment of breast cancer cells with a combination of the EGFR-tyrosine kinase inhibitor (EGFR-TKI) gefitinib and the anti-ErbB-2 monoclonal antibody trastuzumab results in a synergistic antitumor effect. In this study, we addressed the mechanisms involved in this phenomenon. The activation of signaling pathways and the expression of cell cycle regulatory proteins were studied in SK-Br-3 and BT-474 breast cancer cells, following treatment with EGFR and/or ErbB-2 inhibitors. Treatment with the gefitinib/trastuzumab combination produced, as compared with a single agent, a more prolonged blockade of AKT and MAPK activation, a more pronounced accumulation of cells in the G0/G1 phase of the cell cycle, a more significant increase in the levels of p27(kip1) and of hypophosphorylated pRb2, and a decrease in the levels of Cyclin D1 and survivin. Similar findings were observed with the EGFR/ErbB-2 inhibitor lapatinib. Gefitinib, trastuzumab, and their combination increased the stability of p27(kip1), with the combination showing the highest effects. Blockade of both receptors with gefitinib/trastuzumab or lapatinib induced a significant increase in the levels of p27(kip1) mRNA and in the nuclear levels of the p27(kip1) transcription factor FKHRL-1. Inhibition of PI3K signaling also produced a significant raise in p27(kip1) mRNA. Finally, down-modulation of FKHRL-1 with siRNAs prevented the lapatinib-induced increase of p27(kip1) mRNA. The synergism deriving from EGFR and ErbB-2 blockade is mediated by several different alterations in the activation of signaling proteins and in the expression of cell cycle regulatory proteins, including transcriptional and posttranscriptional regulation of p27(kip1) expression.

Lactic fermentation of cereals aqueous mixture of oat and rice flours with and without glucose addition.

Studies of the ability of probiotics to ferment cereal flours are necessary to obtain products with enhanced nutritional value. In this study, Lactobacillus paracasei CBA-L74 was used to ferment cereal aqueous mixtures containing both oat (7.5% w/v) and rice flours (7.5% w/v), with and without glucose, to understand whether glucose addition could have any effect on growth and metabolism. Viability, pH, metabolites production during fermentation (24 h, 37 °C) and substrates reduction were analysed. The strain showed good growth in the cereal aqueous mixture both with and without glucose addition, but suspensions prepared with glucose showed the best results. A bacterial concentration of 7 log CFU mL-1, a pH value of 4.70 and lactic acid production of 1250 mg L-1 were achieved when fermentation was performed without glucose addition, while in the presence of glucose, a t24 bacterial growth of 8 log CFU mL-1 was reached, with a pH value of 3.11 and lactic acid production of 6050 mg L-1.

Next Generation Sequencing-Based Profiling of Cell Free DNA in Patients with Advanced Non-Small Cell Lung Cancer: Advantages and Pitfalls.

Lung cancer (LC) is the main cause of death for cancer worldwide and non-small cell lung cancer (NSCLC) represents the most common histology. The discovery of genomic alterations in driver genes that offer the possibility of therapeutic intervention has completely changed the approach to the diagnosis and therapy of advanced NSCLC patients, and tumor molecular profiling has become mandatory for the choice of the most appropriate therapeutic strategy. However, in approximately 30% of NSCLC patients tumor tissue is inadequate for biomarker analysis. The development of highly sensitive next generation sequencing (NGS) technologies for the analysis of circulating cell-free DNA (cfDNA) is emerging as a valuable alternative to assess tumor molecular landscape in case of tissue unavailability. Additionally, cfDNA NGS testing can better recapitulate NSCLC heterogeneity as compared with tissue testing. In this review we describe the main advantages and limits of using NGS-based cfDNA analysis to guide the therapeutic decision-making process in advanced NSCLC patients, to monitor the response to therapy and to identify mechanisms of resistance early. Therefore, we provide evidence that the implementation of cfDNA NGS testing in clinical research and in the clinical practice can significantly improve precision medicine approaches in patients with advanced NSCLC.

Targeted sequencing analysis of cell-free DNA from metastatic non-small-cell lung cancer patients: clinical and biological implications.

BACKGROUND: Sequencing artifacts, clonal hematopoietic mutations of indeterminate potential (CHIP) and tumor heterogeneity have been hypothesized to contribute to the low concordance between tissue and cell-free DNA (cfDNA) molecular profiling with targeted sequencing. METHODS: We analyzed by targeted sequencing cfDNA from 30 healthy individuals, and cfDNA and matched tumor samples from 30 EGFR-mutant and 77 EGFR wild-type metastatic non-small-cell lung cancer (mNSCLC) patients. Discordant cases were solved by droplet digital PCR (ddPCR). RESULTS: By testing cfDNA from healthy donors, we developed an algorithm to recognize sequencing artifacts. Applying this method to cfDNA from mNSCLC patients, EGFR mutations were detected with a good sensitivity (76.7%) and specificity (97.4%). In contrast, sensitivity and specificity for KRAS variants were 61.5% and 93.8%, respectively. All EGFR and KRAS variants detected in plasma but not in tissue were confirmed by ddPCR, thus excluding sequencing artifacts. In a fraction of cases, KRAS mutations found in plasma samples were confirmed in tumor tissue suggesting tumor heterogeneity. KRAS variants were found to be more likely sub-clonal as compared with EGFR mutations, and a correlation between clonal origin and frequency of detection in plasma was found. In a case with both EGFR and KRAS variants in cfDNA, we could demonstrate the presence of the KRAS variant in tumor tissue associated with lack of response to tyrosine kinase inhibitors (TKIs). CONCLUSIONS: Although sequencing artifacts can be identified in targeted sequencing of cfDNA, tumor heterogeneity and CHIP are likely to influence the concordance between plasma and tissue testing.

The potential of monitoring treatment response in non-small cell lung cancer using circulating tumour cells.

Introduction: Circulating tumor cell (CTC) counts represent an attractive strategy for monitoring response to therapy in patients with advanced non-small cell lung cancer (NSCLC). Changes in the CTCs number during the treatment have been proposed as a predictive biomarker of response to both chemotherapy and targeted therapies. Profiling of CTCs might also allow the assessment of the dynamics of predictive biomarkers such as EGFR, ALK, ROS1, and PD-L1, and provide relevant information in patients progressing on treatment with targeted agents including immunotherapeutics. Areas covered: A search of peer-reviewed literature in bibliographic databases was undertaken to discuss studies on CTCs and their predictive role in NSCLC. Expert opinion: To date, some challenges limit the clinical utility of CTCs in monitoring the response to treatment in NSCLC. The standardization of techniques for CTCs isolation and characterization and their validation on larger cohorts of patients might help to translate CTCs analysis in the clinic. However, studies on CTCs can provide information on molecular mechanisms involved in NSCLC progression and in the acquired resistance to treatments.

The role of the EGFR signaling in tumor microenvironment.

The epidermal growth factor receptor (EGFR) family comprehends four different tyrosine kinases (EGFR, ErbB-2, ErbB-3, and ErbB-4) that are activated following binding to epidermal growth factor (EGF)-like growth factors. It has been long established that the EGFR system is involved in tumorigenesis. These proteins are frequently expressed in human carcinomas and support proliferation and survival of cancer cells. However, activation of the EGFR in non-malignant cell populations of the neoplastic microenvironment might also play an important role in cancer progression. EGFR signaling regulates in tumor cells the synthesis and secretion of several different angiogenic growth factors, including vascular endothelial growth factor (VEGF), interleukin-8 (IL-8), and basic fibroblast growth factor (bFGF). Overexpression of ErbB-2 also leads to increased expression of angiogenic growth factors, whereas treatment with anti-EGFR or anti-ErbB-2 agents produces a significant reduction of the synthesis of these proteins by cancer cells. EGFR expression and function in tumor-associated endothelial cells has also been described. Therefore, EGFR signaling might regulate angiogenesis both directly and indirectly. In addition, activation of EGFR is involved in the pathogenesis of bone metastases. Within the bone marrow microenvironment, cancer cells stimulate the synthesis of osteoclastogenic factors by residing stromal cells, a phenomenon that leads to bone destruction. It has been shown that EGFR signaling regulates the ability of bone marrow stromal cells to produce osteoclastogenic factors and to sustain osteoclast activation. Taken together, these findings suggest that the EGFR system is an important mediator, within the tumor microenvironment, of autocrine and paracrine circuits that result in enhanced tumor growth.

Pharmacokinetic drug evaluation of palbociclib for the treatment of breast cancer.

INTRODUCTION: Cyclin-dependent kinases (CDKs) 4 and 6 regulate the transition from G0/G1-phase to S-phase of the cell cycle and have been identified as key drivers of proliferation in hormone receptor (HR)-positive breast cancer. The CDK4/6 inhibitor palbociclib in combination with endocrine therapy has been approved for treatment of HR-positive/HER2-negative breast cancer patients. Areas covered: In this article, we provide an update of the data on pharmacodynamics, pharmacokinetics, preclinical, and clinical studies of palbociclib in breast cancer. We performed a search of data on palbociclib in the PubMed and the clinicaltrials.gov databases, in the FDA website and in the ASCO and AACR proceedings. Expert opinion: In order to optimize the clinical outcome of HR-positive breast cancer patients treated with palbociclib, predictive biomarkers allowing patient selection are urgently needed. A recent study suggested that early dynamics of PIK3CA mutations in circulating tumor DNA might be a potential predictive biomarker for CDK4/6 inhibitors. Several clinical trials are ongoing with the aim to explore the activity of combinations of palbociclib with targeted agents and/or immunotherapy in the different subtypes of breast cancer in both metastatic and early phases of the disease. These combinations might allow improving the sensitivity and overcoming mechanisms of resistance.

RANTES and IL-6 cooperate in inducing a more aggressive phenotype in breast cancer cells.

Both the CC chemokine ligand 5 (CCL5/RANTES) and interleukin-6 (IL-6), released by mesenchymal stem cells (MSCs) as well as by neoplastic cells, promote breast cancer cell progression through autocrine and paracrine mechanisms. In order to assess the effects of the simultaneous overexpression of RANTES and IL-6 on the tumor cell phenotype, we overexpressed both proteins in MCF-7 and MDA-MB-231 human breast cancer cell lines. MCF-7 cells co-expressing RANTES and IL-6 had a greater ability to form colonies in soft agar, compared to cells overexpressing RANTES or IL-6. In addition, both MCF-7 and MDA-MB-231 clones co-expressing RANTES and IL-6 showed a significantly higher ability to migrate and to invade. The analysis of phosphorylated ERK1/2, AKT and STAT3 signal transduction proteins revealed that several signaling pathways are simultaneously activated in cells overexpressing both factors. Finally, the overexpression of RANTES and IL-6 in MCF-7 cells significantly increased the in vivo tumor growth. Collectively, our data suggest that the simultaneous expression of IL-6 and RANTES produces a more aggressive phenotype in breast cancer cells and provide evidence that IL-6 and RANTES might represent potential targets for novel therapeutic strategies aimed to block the tumor-stroma interaction.

Circulating programmed death ligand-1 (cPD-L1) in non-small-cell lung cancer (NSCLC).

BACKGROUND: This study aimed at investigating feasibility of programmed death ligand-1 (PD-L1) testing in plasma samples of advanced NSCLC patients receiving first-line treatment, assessing whether circulating (c)PD-L1 levels were modified by the therapy and whether baseline cPD-L1 levels were associated with patients' clinical responses and survival outcome. METHODS: Peripheral blood samples were collected from 16 healthy volunteers and 56 newly diagnosed NSCLC patients before and at 12th week during the course of first-line therapy. The level of PD-L1 was measured in plasma samples using the human (PD-L1/CD274) ELISA kit (CUSABIO, MD, USA). The Mann Whitney test or Fisher's test were used for comparisons. Survival analysis was performed using Kaplan Meyer method, providing median and p-value. RESULTS: Baseline median cPD-L1 was 42.21 pg/ml (range 12.00-143.49) in NSCLC patients and 37.81 pg/ml (range 9.73-90.21) in healthy control cohort (p = 0.78). Median cPD-L1 increased in patients treated with first-line chemotherapy (63.20 pg/ml vs 39.34 pg/ml; p = 0.002), with no changes in patients exposed to non-chemotherapy drugs (42.39 pg/ml vs 50.67 pg/ml; p = 0.398). Time to progression and overall survival were 4.4 vs 6.9 months (p = 0.062) and 8.8 vs 9.3 months (p = 0.216) in cPD-L1 positive vs cPD-L1 negative patients. Baseline cPD-L1 levels increased with the ascending number of metastatic sites, even if the association was not statistically significant (p = 0.063). CONCLUSIONS: This study showed that cPD-L1 testing is feasible, with chemotherapy influencing PD-L1 plasma levels. The possibility of using such test for predicting or monitoring the effect of immunotherapy or combination of chemotherapy and immunotherapy warrant further investigations.