Exosomes multiplex profiling, a promising strategy for early diagnosis of laryngeal cancer.

BACKGROUND: Exosomes are nanosized vesicles released from all cells into surrounding biofluids, including cancer cells, and represent a very promising direction in terms of minimally invasive approaches to early disease detection. They carry tumor-specific biological contents such as DNA, RNA, proteins, lipids, and sugars, as well as surface molecules that are able to pinpoint the cellular source. By the above criteria, exosomes may be stratified according to the presence of tissue and disease-specific signatures and, due to their stability in such biofluids as plasma and serum, they represent an indispensable source of vital clinical insights from liquid biopsies, even at the earliest stages of cancer. Therefore, our work aimed to isolate and characterize LCa patients' derived exosomes from serum by Flow Cytometry in order to define a specific epitope signature exploitable for early diagnosis. METHODS: Circulating exosomes were collected from serum collected from 30 LCa patients and 20 healthy volunteers by the use of antibody affinity method exploiting CD63 specific surface marker. Membrane epitopes were then characterized by Flow cytometry multiplex analysis and compared between LCa Patients and Healthy donors. Clinical data were also matched to obtain statistical correlation. RESULTS: A distinct overexpression of CD1c, CD2, CD3, CD4, CD11c, CD14, CD20, CD44, CD56, CD105, CD146, and CD209 was identified in LCa patients compared to healthy controls, correlating positively with tumor presence. Conversely, CD24, CD31, and CD40, though not overexpressed in tumor samples, showed a significant correlation with nodal involvement in LCa patients (p < 0.01). CONCLUSION: This approach could allow us to set up a cost-effective and less invasive liquid biopsy protocol from a simple blood collection in order to early diagnose LCa and improve patients' outcomes and quality of life.

A Combination of Microarray-Based Profiling and Biocomputational Analysis Identified miR331-3p and hsa-let-7d-5p as Potential Biomarkers of Ulcerative Colitis Progression to Colorectal Cancer.

Ulcerative colitis (UC), an inflammatory bowel disease (IBD), may increase the risk of colorectal cancer (CRC) by activating chronic proinflammatory pathways. The goal of this study was to find serum prediction biomarkers in UC to CRC development by combining low-density miRNA microarray and biocomputational approaches. The UC and CRC miRNA expression profiles were compared by low-density miRNA microarray, finding five upregulated miRNAs specific to UC progression to CRC (hsa-let-7d-5p, hsa-miR-16-5p, hsa-miR-145-5p, hsa-miR-223-5p, and hsa-miR-331-3p). The circRNA/miRNA/mRNA competitive endogenous RNA (ceRNA) network analysis showed that the candidate miRNAs were connected to well-known colitis-associated CRC ACVR2A, SOCS1, IGF2BP1, FAM126A, and CCDC85C mRNAs, and circ-SHPRH circRNA. SST and SCARA5 genes regulated by hsa-let-7d-5p, hsa-miR-145-5p, and hsa-miR-331-3p were linked to a poor survival prognosis in a CRC patient dataset from The Cancer Genome Atlas (TCGA). Lastly, our mRNA and miRNA candidates were validated by comparing their expression to differentially expressed mRNAs and miRNAs from colitis-associated CRC tissue databases. A high level of hsa-miR-331-3p and a parallel reduction in SOCS1 mRNA were found in tissue and serum. We propose hsa-miR-331-3p and possibly hsa-let-7d-5p as novel serum biomarkers for predicting UC progression to CRC. More clinical sample analysis is required for further validation.

Immune cell infiltration and inflammatory landscape in primary brain tumours.

BACKGROUND: Primary malignant brain tumours are more than one-third of all brain tumours and despite the molecular investigation to identify cancer driver mutations, the current therapeutic options available are challenging due to high intratumour heterogeneity. In addition, an immunosuppressive and inflammatory tumour microenvironment strengthens cancer progression. Therefore, we defined an immune and inflammatory profiling of meningioma and glial tumours to elucidate the role of the immune infiltration in these cancer types. METHODS: Using tissue microarrays of 158 brain tumour samples, we assessed CD3, CD4, CD8, CD20, CD138, Granzyme B (GzmB), 5-Lipoxygenase (5-LOX), Programmed Death-Ligand 1 (PD-L1), O-6-Methylguanine-DNA Methyltransferase (MGMT) and Transglutaminase 2 (TG2) expression by immunohistochemistry (IHC). IHC results were correlated using a Spearman correlation matrix. Transcript expression, correlation, and overall survival (OS) analyses were evaluated using public datasets available on GEPIA2 in Glioblastoma (GBM) and Lower Grade Glioma (LGG) cohorts. RESULTS: Seven out of ten markers showed a significantly different IHC expression in at least one of the evaluated cohorts whereas CD3, CD4 and 5-LOX were differentially expressed between GBMs and astrocytomas. Correlation matrix analysis revealed that 5-LOX and GzmB expression were associated in both meningiomas and GBMs, whereas 5-LOX expression was significantly and positively correlated to TG2 in both meningioma and astrocytoma cohorts. These findings were confirmed with the correlation analysis of TCGA-GBM and LGG datasets. Profiling of mRNA levels indicated a significant increase in CD3 (CD3D, CD3E), and CD138 (SDC1) expression in GBM compared to control tissues. CD4 and 5-LOX (ALOX5) mRNA levels were significantly more expressed in tumour samples than in normal tissues in both GBM and LGG. In GBM cohort, GzmB (GZMB), SDC1 and MGMT gene expression predicted a poor overall survival (OS). Moreover, in LGG cohort, an increased expression of CD3 (CD3D, CD3E, CD3G), CD8 (CD8A), GZMB, CD20 (MS4A1), SDC1, PD-L1, ALOX5, and TG2 (TGM2) genes was associated with worse OS. CONCLUSIONS: Our data have revealed that there is a positive and significant correlation between the expression of 5-LOX and GzmB, both at RNA and protein level. Further evaluation is needed to understand the interplay of 5-LOX and immune infiltration in glioma progression.

Fluorescent nanodiamonds as innovative delivery systems for MiR-34a replacement in breast cancer.

Nanodiamonds are innovative nanocrystalline carbon particles able to deliver chemically conjugated miRNAs. In oncology, the use of miRNA-based therapies may represent an advantage, based on their ability to simultaneously target multiple intracellular oncogenic targets. Here, nanodiamonds were tested and optimized to deliver miR-34a, a miRNA playing a key role in inhibiting tumor development and progression in many cancers. The physical-chemical properties of nanodiamonds were investigated suggesting electrical stability and uniformity of structure and size. Moreover, we evaluated nanodiamond cytotoxicity on two breast cancer cell models and confirmed their excellent biocompatibility. Subsequently, nanodiamonds were conjugated with miR-34a, using the chemical crosslinker polyethyleneimine; real-time PCR analysis revealed a higher level of miR-34a in cancer cells treated with the different formulations of nanodiamonds than with commercial transfectant. A significant and early nanodiamond-miR-34a uptake was recorded by FACS and fluorescence microscopy analysis in MCF7 and MDA-MB-231 cells. Moreover, nanodiamond-miR-34a significantly inhibited both cell proliferation and migration. Finally, a remarkable anti-tumor effect of miR-34a-conjugated nanodiamonds was observed in both heterotopic and orthotopic murine xenograft models. In conclusion, this study provides a rationale for the development of new therapeutic strategies based on use of miR-34a delivered by nanodiamonds to improve the clinical treatment of neoplasms.

Whey Improves In Vitro Endothelial Mitochondrial Function and Metabolic Redox Status in Diabetic State.

Endothelial dysfunction plays a critical role in the progression of type 2 diabetes mellitus (T2DM), leading to cardiovascular complications. Current preventive antioxidant strategies to reduce oxidative stress and improve mitochondrial function in T2DM highlight dietary interventions as a promising approach, stimulating the deepening of knowledge of food sources rich in bioactive components. Whey (WH), a dairy by-product with a considerable content of bioactive compounds (betaines and acylcarnitines), modulates cancer cell metabolism by acting on mitochondrial energy metabolism. Here, we aimed at covering the lack of knowledge on the possible effect of WH on the mitochondrial function in T2DM. The results showed that WH improved human endothelial cell (TeloHAEC) function during the in vitro diabetic condition mimicked by treating cells with palmitic acid (PA) (0.1 mM) and high glucose (HG) (30 mM). Of note, WH protected endothelial cells from PA+HG-induced cytotoxicity (p < 0.01) and prevented cell cycle arrest, apoptotic cell death, redox imbalance, and metabolic alteration (p < 0.01). Moreover, WH counteracted mitochondrial injury and restored SIRT3 levels (p < 0.01). The SiRNA-mediated suppression of SIRT3 abolished the protective effects exerted by WH on the mitochondrial and metabolic impairment caused by PA+HG. These in vitro results reveal the efficacy of whey as a redox and metabolic modulator in the diabetic state and pave the way for future studies to consider whey as the source of dietary bioactive molecules with health benefits in preventive strategies against chronic diseases.

Characterization of KRAS Mutational Regression in Oligometastatic Patients.

Background: We previously reported rare regressive genetic trajectories of KRAS pathogenic mutations as a specific hallmark of the genuine oligometastatic status in colorectal cancer (CRC). Methods: Survival and prognostic impact of disease extent in 140 metastatic CRC patients were evaluated through the Kaplan-Meyer curves and the Log-Rank test. KRAS mutations were assessed through the Illumina NovaSeq 6000 platform and TruSight™ Oncology 500 kit. HLA typing was carried out by PCR with sequence-specific oligonucleotides. Lymphocyte densities in tumors were expressed as cells per square millimeter. NKs isolated and CD8+ from NK-depleted PBMCs were characterized through flow cytometry. CD107a externalization was evaluated as NKs/CD8 cytotoxicity toward human colon cancer cells HT29, SW620, HCT116, and LS174T carrying different KRAS mutations. Results: The oligometastatic status was a strong and independent variable for survival (HR: 0.08 vs. polymetastatic disease; 95% CI: 0.02-0.26; p < 0.001). Eighteen oligometastatic patients were selected. Twelve were alive at the last follow-up, and 9 were characterized. Genetic regression of KRAS was observed in 3 patients: patient (PAT)2, PAT5, and PAT8. PAT2 and PAT5 presented the highest levels of GrzB+ lymphocytes in the tumor cores of the metastases (120 ± 11.2 and 132 ± 12.2 cells/mm2, respectively). Six out of 9 patients (67%), including PAT2 and PAT5, expressed HLA-C7. Twopatients (PAT2 and PAT5) presented high CD3+/CD8+-dependent cytotoxicity against HLA-C7+ SW620 cells (p.G12V-mutated cells), which was consistent with their observed mutational regression (p.G12V/p.G13D in primary→p.G13D in metastatic tumor). Conclusions: We provide evidence that CD3+/CD8+ lymphocytes from oligometastatic CRC patients display differential cytotoxicity against human colon cancer cells carrying KRAS mutations. This could provide an interesting basis for monitoring oligometastatic disease and developing future adoptive immunotherapies.

Polydatin Incorporated in Polycaprolactone Nanofibers Improves Osteogenic Differentiation.

Polycaprolactone nanofibers are used as scaffolds in the field of tissue engineering for tissue regeneration or drug delivery. Polycaprolactone (PCL) is a biodegradable hydrophobic polyester used to obtain implantable nanostructures, which are clinically applicable due to their biological safety. Polydatin (PD), a glycosidic precursor of resveratrol, is known for its antioxidant, antitumor, antiosteoporotic, and bone regeneration activities. We aimed to use the osteogenic capacity of polydatin to create a biomimetic innovative and patented scaffold consisting of PCL-PD for bone tissue engineering. Both osteosarcoma cells (Saos-2) and mesenchymal stem cells (MSCs) were used to test the in vitro cytocompatibility of the PD-PCL scaffold. Reverse-phase (RP) HPLC was used to evaluate the timing release of PD from the PCL-PD nanofibers and the MTT assay, scanning electron microscopy, and alkaline phosphatase (ALP) activity were used to evaluate the proliferation, adhesion, and cellular differentiation in both osteosarcoma and human mesenchymal stem cells (MSCs) seeded on PD-PCL nanofibers. The proliferation of osteosarcoma cells (Saos-2) on the PD-PCL scaffold decreased when compared to cells grown on PLC nanofibers, whereas the proliferation of MSCs was comparable in both PCL and PD-PCL nanofibers. Noteworthy, after 14 days, the ALP activity was higher in both Saos-2 cells and MSCs cultivated on PD-PCL than on empty scaffolds. Moreover, the same cells showed a spindle-shaped morphology after 14 days when grown on PD-PCL as shown by SEM. In conclusion, we provide evidence that nanofibers appropriately coated with PD support the adhesion and promote the osteogenic differentiation of both human osteosarcoma cells and MSCs.

MiR-423-5p prevents MALAT1-mediated proliferation and metastasis in prostate cancer.

BACKGROUND: The long non-coding RNA (lncRNA), MALAT1, plays a key role in the development of different cancers, and its expression is associated with worse prognosis in patients. However, its mechanism of action and its regulation are not well known in prostate cancer (PCa). A general mechanism of action of lncRNAs is their interaction with other epigenetic regulators including microRNAs (miRNAs). METHODS: Using lentiviral stable miRNA transfection together with cell biology functional assays and gene expression/target analysis, we investigated the interaction between MALAT1 and miR-423-5p, defined as a target with in silico prediction analysis, in PCa. RESULTS: Through bioinformatic analysis of data available from TCGA, we have found that MALAT1 expression correlates with high Gleason grade, metastasis occurrence, and reduced survival in PCa patients. These findings were validated on a TMA of PCa showing a significant correlation between MALAT1 expression with both stage and grading. We report that, in PCa cells, MALAT1 expression and activity is regulated by miR-423-5p that binds MALAT1, downregulates its expression and inhibits its activity in promoting proliferation, migration, and invasion. Using NanoString analysis, we unraveled downstream cell pathways that were affected by miR-423-5p expression and MALAT1 downregulation and identified several alterations in genes that are involved in metastatic response and angiogenic pathways. In addition, we showed that the overexpression of miR-423-5p increases survival and decreases metastases formation in a xenograft mouse model. CONCLUSIONS: We provide evidence on the role of MALAT1 in PCa tumorigenesis and progression. Also, we identify a direct interaction between miR-423-5p and MALAT1, which results in the suppression of MALAT1 action in PCa.

A Proteomic Approach Reveals That miR-423-5p Modulates Glucidic and Amino Acid Metabolism in Prostate Cancer Cells.

Recently, we have demonstrated that miR-423-5p modulates the growth and metastases of prostate cancer (PCa) cells both in vitro and in vivo. Here, we have studied the effects of miR-423-5p on the proteomic profile in order to identify its intracellular targets and the affected pathways. Applying a quantitative proteomic approach, we analyzed the effects on the protein expression profile of miR-423-5p-transduced PCa cells. Moreover, a computational analysis of predicted targets of miR-423-5p was carried out by using several target prediction tools. Proteomic analysis showed that 63 proteins were differentially expressed in miR-423-5-p-transfected LNCaP cells if compared to controls. Pathway enrichment analysis revealed that stable overexpression of miR-423-5p in LNCaP PCa cells induced inhibition of glycolysis and the metabolism of several amino acids and a parallel downregulation of proteins involved in transcription and hypoxia, the immune response through Th17-derived cytokines, inflammation via amphorin signaling, and ion transport. Moreover, upregulated proteins were related to the S phase of cell cycle, chromatin modifications, apoptosis, blood coagulation, and calcium transport. We identified seven proteins commonly represented in miR-423-5p targets and differentially expressed proteins (DEPs) and analyzed their expression and influence on the survival of PCa patients from publicly accessible datasets. Overall, our findings suggest that miR-423-5p induces alterations in glucose and amino acid metabolism in PCa cells paralleled by modulation of several tumor-associated processes.

Distinctive Role of the Systemic Inflammatory Profile in Non-Small-Cell Lung Cancer Younger and Elderly Patients Treated with a PD-1 Immune Checkpoint Blockade: A Real-World Retrospective Multi-Institutional Analysis.

An immune checkpoint blockade with mAbs to PD-1 and PD-L1 is an expanding therapeutic option for mNSCLC patients. This treatment strategy is based on the use of mAbs able to restore the anti-tumor activity of intratumoral T cells inhibited by PD-1 binding to PD-L1/2 on tumor and inflammatory cells. It has been speculated that a chronic status of systemic inflammation as well as the immunosenescence physiologically occurring in elderly patients may affect the efficacy of the treatment and the occurrence of irAEs. We performed a multi-institutional retrospective study aimed at evaluating the effects of these mAbs (nivolumab or atezolizumab) in 117 mNSCLC patients younger (90 cases) and older (27 cases) than 75 years in correlation with multiple inflammatory parameters (NLR, CRP, ESR, LDH and PCT). No differences were observed when the cohorts were compared in terms of the frequency of PFS, OS, inflammatory markers and immune-related adverse events (irAEs). Similarly, the occurrence of irAEs was strictly correlated with a prolonged OS survival in both groups. On the contrary, a negative correlation between the high baseline levels of inflammatory markers and OS could be demonstrated in the younger cohort only. Overall, PD-1/PD-L1-blocking mAbs were equally effective in young and elderly mNSCLC patients; however, the detrimental influence of a systemic inflammation at the baseline was only observed in young patients, suggesting different aging-related inflammation immunoregulative effects.

Polydatin Induces Differentiation and Radiation Sensitivity in Human Osteosarcoma Cells and Parallel Secretion through Lipid Metabolite Secretion.

Osteosarcoma is a bone cancer characterized by the production of osteoid tissue and immature bone from mesenchymal cells. Osteosarcoma mainly affects long bones (femur is most frequently site) and occur in children and young adults with greater incidence. Here, we investigated the role accomplished by polydatin, a natural antioxidative compound, in promoting osteogenic differentiation alone or after radiation therapy on osteosarcoma cells. In vitro, polydatin significantly induced cell cycle arrest in S-phase and enhanced bone alkaline phosphatase activity. Moreover, the differentiation process was paralleled by the activation of Wnt-ß-catenin pathway. In combination with radiotherapy, the pretreatment with polydatin promoted a radiosensitizing effect on osteosarcoma cancer cells as demonstrated by the upregulation of osteogenic markers and reduced clonogenic survival of tumor cells. Additionally, we analyzed, by mass spectrometry, the secretion of sphingolipid, ceramides, and their metabolites in osteosarcoma cells treated with polydatin. Overall, our results demonstrate that polydatin, through the secretion of sphingolipids and ceramide, induced osteogenic differentiation, alone and in the presence of ionizing therapy. Future investigations are needed to validate the use of polydatin in clinical practice as a potentiating agent of radiotherapy-induced anticancer effects.

Inflammatory Markers and Procalcitonin Predict the Outcome of Metastatic Non-Small-Cell-Lung-Cancer Patients Receiving PD-1/PD-L1 Immune-Checkpoint Blockade.

Peripheral-immune-checkpoint blockade (P-ICB) with mAbs to PD-1 (nivolumab and pembrolizumab) or PD-L1 (atezolizumab, durvalumab, avelumab) alone or combination with chemotherapy represents a novel active treatment for mNSCLC patients. However, this therapy can be associated to immune-related adverse events (irAEs) and high cost. Therefore, finding reliable biomarkers of response and irAEs is strongly encouraged to accurately select patients who may potentially benefit from the immuno-oncological treatment. This is a retrospective multi-institutional analysis performed on ninety-five mNSCLC patients who received real-world salvage therapy with nivolumab or atezolizumab between December 2015 and April 2020. The outcome of these patients in term of PFS and OS was evaluated in comparison with different serum levels of C-reactive protein (CRP), Erythrocyte Sedimention Rate (ESR) and Procalcitonin (PCT) by performing Kaplan-Meier and Log-rank test and multivariate analysis. We found that high baseline levels of CRP, ESR, and PCT were strongly predictive of poor outcome (P <0.05) with the worse prognosis detected in those patients with a baseline levels of both ESR and PCT over the pre-established cut off (median OS recorded in patients with no marker over the cut off vs. those with just one marker over the cut off vs. those with both markers over the cut off: 40 ± 59 vs. 15.5 ± 5.5 vs. 5.5 ± 1.6 months, respectively; P <0.0001). Our results suggest the predictive value of systemic inflammation and suggest a potential role of PCT in predicting a poor outcome in mNSCLC receiving PD-1/PD-L1 blocking mAbs. This finding also suggests a potential role of subclinical bacterial infections in defining the response to PD-1/PD-L1 blocking mAbs that deserves further and more specific investigations.

Assessment of Total, PTEN-, and AR-V7+ Circulating Tumor Cell Count by Flow Cytometry in Patients with Metastatic Castration-Resistant Prostate Cancer Receiving Enzalutamide.

INTRODUCTION: Metastatic castration-resistant prostate cancer (mCRPC) is a deadly disease. Enzalutamide is an oral second-generation anti-androgen that is active in mCRPC. Circulating tumor cells (CTC) count correlates with overall survival (OS) in mCRPC, whereas detection of the androgen-receptor splice variant 7 (AR-V7) in CTC predicts poor response to oral second-generation anti-androgens. Also, loss of PTEN (phosphatase and tensin homolog) in CTC is a biomarker of poor prognosis in mCRPC. PATIENTS AND METHODS: In this translational study, we employed flow cytometry to assess total, PTEN-, and AR-V7+ CTC count per 7.5 mL of whole blood in a prospective cohort of patients with mCRPC receiving enzalutamide. RESULTS: CTCs were assessed in a total of 45 men with mCRPC at baseline and at 12 weeks. Overall, CTC, PTEN- CTC, and AR-V7+ CTC detection rate was high, at baseline, with 84.4%, 71.1%, and 51.1% of samples showing at least 1 cell/7.5-mL blood, respectively, and after 3 months, with 93.3%, 64.4%, and 77.7% of samples showing at least 1 cell/7.5-mL blood, respectively. Median radiographic progression-free survival (rPFS) and OS were 6 (95% confidence interval [CI], 5.6-9) and 14.3 (95% CI, 12.8-20.3) months, respectively. Median (interquartile range) total CTC count at baseline was 5 (3; 8), whereas median (interquartile range) PTEN- CTC count was 2 (0; 4) and median (interquartile range) AR-V7+ CTC count was 1 (0; 3). At baseline, ≥ 5 versus < 5 total CTC count was associated with worse rPFS (hazard ratio [HR], 2.35; 95% CI, 1.14-4.84; P= .021) and OS (HR, 3.08; 95% CI, 1.45-6.54; P = .003), whereas ≥ 2 versus < 2 PTEN- CTC count was associated with worse rPFS (HR, 3.96; 95% CI, 1.8-8.72; P= .001) and OS (HR, 2.36; 95% CI, 1.12-5; P= .025). Finally, ≥ 1 versus < 1 AR-V7+ CTC count was also associated with worse rPFS (HR, 5.05; 95% CI, 2.4-10.64; P< .001) and OS (HR, 2.25; 95% CI, 1.1-4.58; P= .026). CONCLUSIONS: Despite multiple limitations, including the small sample size, our preliminary study suggests that assessment of CTC via flow cytometry may provide potentially useful prognostic and predictive information in advanced prostate cancer. Further studies are warranted. Micro-Abstract: In this study, men with metastatic castration-resistant prostate cancer, scheduled to start enzalutamide, were assessed for circulating tumor cell count and molecular characterization (total, PTEN-, and AR-V7+ circulating tumor cell count) by the use of flow cytometry. We found that flow cytometry could be used to enumerate circulating tumor cells, but also to assess molecular biomarkers on their surface.

Aflibercept or bevacizumab in combination with FOLFIRI as second-line treatment of mRAS metastatic colorectal cancer patients: the ARBITRATION study protocol.

BACKGROUND: The intensive study of predictive factors has strongly ameliorated the therapeutic flow-chart of metastatic colorectal cancer (mCRC) by allowing the selection of patients who benefit from specific therapies. For instance, in mRAS (mutated RAS) mCRC patients, anti-EGFR drugs (cetuximab and panitumumab) are not recommended; in this group of patients, the use of anti-angiogenic drugs (bevacizumab and aflibercept) is predominant. However, at progression to standard bevacizumab-based first-line chemotherapy, still to date, there are no studies to guide oncologists in the choice of the best second-line anti-angiogenic drug (bevacizumab beyond progression versus aflibercept). METHODS: ARBITRATION is a prospective, observational study assessing efficacy differences between second-line fluorouracil/irinotecan (FOLFIRI)/bevacizumab versus FOLFIRI/aflibercept at progression to fluoropyrimidines, oxaliplatin and bevacizumab in mRAS mCRC patients. A test power of 80%, a median survival of 9 months from second-line treatment start and a hazard ratio of 0.67 between the two schedules were the basis for statistical design. The final sample will be 220 patients (110 per treatment). The significance will be verified with a two-tailed log-rank test with an alpha value of the I-type error of 5%. Time-to-outcome will be described by Kaplan-Meier curves and prognostic factors studied through multivariable analyses based on the Cox model. Secondary objectives include safety, responses' duration and progression-free survival. A translational research will be conducted to measure several angiogenic proteins in patients' serum before starting the therapy in order to evidence any angiogenic factor patterns related to outcome. DISCUSSION: We present a large, prospective, observational study aiming to answer two scientific questions: (1) outcome differences between second-line treatments with FOLFIRI/bevacizumab beyond progression versus FOLFIRI/aflibercept in mRAS mCRC patients, (2) angiogenic factors' patterns that could associate with efficacy and help oncologists to apply best the therapeutic anti-angiogenic strategies. TRIAL REGISTRATION: The ARBITRATION trial (version 0.0, 13 April 2020) has been registered into the clinicaltrials.gov registry on 20 May 2020 with identifier NCT04397601.

KRAS Mutational Regression Is Associated With Oligo-Metastatic Status and Good Prognosis in Metastatic Colorectal Cancer.

BACKGROUND: We previously reported that loss of KRAS mutations ("regressive" mutational trajectories) from primary tumors to metastases associated with the oligo-metastatic status in colorectal cancer (CRC). The present study was undertaken in order to analyze the mutational trajectories of KRAS in a well-characterized cohort of CRC patients who developed poly- or oligo-metastatic disease. MATERIAL AND METHODS: Patients were treated and followed-up according to European Society of Medical Oncology guidelines. Primary CRC FFPE tissue and metastatic circulating-free DNA were extracted using the QIAamp DNA specific kits (Qiagen, Hilden, Germany). Samples were sequenced with the Oncomine Solid Tumour DNA kit (Thermo Fisher Scientific, Waltham, MA, USA). Plasma collection for liquid biopsy was done from 1 to 14 days before starting first-line chemotherapy. Analysis of the prognostic power of KRAS evolutionary trajectories was done with uni- and multivariate analyses. RESULTS: One-hundred-fourteen patients were enrolled. Sixty-three patients presented with mutated KRAS (mutKRAS) and 51 with wild-type KRAS (wtKRAS). KRAS mutational concordance was high (70.1%).Two divergent subsets were identified: mutKRAS in primary tumors and wtKRAS in metastatic ones (regressive: mutKRAS → wtKRAS in 8.8% of patients), and vice versa (progressive: wtKRAS → mutKRAS in 21.1% of patients). An association between KRAS regressive trajectory and the oligo-metastatic status (P <0.0001) was found. At multivariate analysis, regressive and progressive mutational trajectories emerged as independent prognostic factors for survival, with Hazard Ratios of 0.22 (CI 95%: 0.08-0.61; median survival: not reached) and 2.70 (CI 95%: 1.11-6.56, median survival: 12.1 months), respectively. CONCLUSIONS: Our data provide evidence that the evolutionary trajectories of KRAS can have a strong clinical prognostic role and that they can be involved in discriminating between poly-metastatic aggressive vs oligo-metastatic indolent CRC.

The Role of microRNAs in Development of Colitis-Associated Colorectal Cancer.

Colorectal cancer (CRC) is the third most deadly cancer worldwide, and inflammatory bowel disease (IBD) is one of the critical factors in CRC carcinogenesis. IBD is responsible for an unphysiological and sustained chronic inflammation environment favoring the transformation. MicroRNAs (miRNAs) belong to a class of highly conserved short single-stranded segments (18-25 nucleotides) non-coding RNA and have been extensively discussed in both CRC and IBD. However, the role of miRNAs in the development of colitis-associated CRC (CAC) is less clear. The aim of this review is to summarize the major upregulated (miR-18a, miR-19a, miR-21, miR-31, miR-155 and miR-214) and downregulated (miR-124, miR-193a-3p and miR-139-5p) miRNAs in CAC, and their roles in genes' expression modulation in chronic colonic-inflammation-induced carcinogenesis, including programmed cell-death pathways. These miRNAs dysregulation could be applied for early CAC diagnosis, to predict therapy efficacy and for precision treatment.

LncRNAs and Immunity: Coding the Immune System with Noncoding Oligonucleotides.

Long noncoding RNAs (lncRNAs) represent key regulators of gene transcription during the inflammatory response. Recent findings showed lncRNAs to be dysregulated in human diseases, such as inflammatory bowel disease, diabetes, allergies, asthma, and cancer. These noncoding RNAs are crucial for immune mechanism, as they are involved in differentiation, cell migration and in the production of inflammatory mediators through regulating protein-protein interactions or their ability to assemble with RNA and DNA. The last interaction can occur in cis or trans and is responsible for all the possible lncRNAs biological effects. Our proposal is to provide an overview on lncRNAs roles and functions related to immunity and immune mediated diseases, since these elucidations could be beneficial to untangle the complex bond between them.

Evolution of Mutational Landscape and Tumor Immune-Microenvironment in Liver Oligo-Metastatic Colorectal Cancer.

Genetic dynamics underlying cancer progression are largely unknown and several genes involved in highly prevalent illnesses (e.g., hypertension, obesity, and diabetes) strongly concur to cancer phenotype heterogeneity. To study genotype-phenotype relationships contributing to the mutational evolution of colorectal cancer (CRC) with a focus on liver metastases, we performed genome profiling on tumor tissues of CRC patients with liver metastatic disease and no co-morbidities. We studied 523 cancer-related genes and tumor-immune microenvironment characteristics in primary and matched metastatic tissues. We observed a loss of KRAS and SMAD4 alterations and a high granzyme-B+ T-cell infiltration when the disease did not progress. Conversely, gain in KRAS, PIK3CA and SMAD4 alterations and scarce granzyme-B+ T-cells infiltration were observed when the tumor evolved towards a poly-metastatic spread. These findings provide novel insights into the identification of tumor oligo-metastatic status, indicating that some genes are on a boundary line between these two clinical settings (oligo- vs. poly-metastatic CRC). We speculate that the identification of these genes and modification of their evolution could be a new approach for anti-cancer therapeutic strategies.