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Abstract Colorectal cancer (CRC) is a global health concern, and the incidence of early onset (EO) CRC, has an upward trend. This study delves into the genomic landscape of EO-CRC, specifically focusing on pediatric (PED) and young adult (YA) patients, comparing them with adult (AD) CRC. In this retrospective monocentric investigation, we performed targeted next-generation sequencing to compare the mutational profile of 38 EO-CRCs patients (eight PED and 30 YA) to those of a ‘control group’ consisting of 56 AD-CRCs. Our findings reveal distinct molecular profiles in EO-CRC, notably in the WNT and PI3K-AKT pathways. In pediatrics, we observed a significantly higher frequency of RNF43 mutations, whereas APC mutations were more prevalent in adult cases. These observations suggest age-related differences in the activation of the WNT pathway. Pathway and copy number variation analysis reveal that AD-CRC and YA-CRC have more similarities than the pediatric patients. PED shows a peculiar profile with CDK6 amplification and the enrichment of lysine degradation pathway. These findings may open doors for personalized therapies, such as PI3K-AKT pathway inhibitors or CDK6 inhibitors for pediatric patients. Additionally, the distinct molecular signatures of EO-CRC underscore the need for age-specific treatment strategies and precision medicine. This study emphasizes the importance of comprehensive molecular investigations in EO-CRCs, which can potentially improve diagnostic accuracy, prognosis, and therapeutic decisions for these patients. Collaboration between the pediatric and adult oncology community is fundamental to improve oncological outcomes for this rare and challenging pediatric tumor.

Abstract In retrospective studies, metformin use has been associated with better clinical outcomes in diabetic patients with advanced, well-differentiated neuroendocrine tumors (WDNETs). However, prospective evidence of metformin safety and activity is lacking. Here, we conducted the first-in-human phase Ib MetNET2 trial to investigate the safety and antitumor activity of metformin in combination with the somatostatin analog lanreotide autogel (ATG) in both diabetic and non-diabetic patients with advanced WDNETs of the gastrointestinal (GI) or thoracic tract. Enrolled patients received lanreotide ATG 120 mg plus oral metformin, up to a maximum dosage of 2550 mg/day. We enrolled 20 patients, of whom 18 (90%) and 2 (10%) had WDNETs of the GI and thoracic tract, respectively. Fourteen patients (70%) were non-diabetic. With a 5% incidence of SAEs, the study met its primary objective of demonstrating treatment safety. With a median follow-up of 39 months (95% CI 28-NE), median PFS was 24 months (95% CI 16-NE), with 12-month and 24-month PFS probability of 75% (95% CI 58–97) and 49% (95% CI 31–77), respectively. We found no statistically significant PFS differences between diabetic and non-diabetic patients. Among exploratory analyses, the presence of tumor genomic alterations in DNA damage pathways was associated with trend towards worse PFS, whereas a precocious reduction of HOMA-IR index and plasma cholesterol concentration showed a trend towards an association with better PFS. In conclusion, metformin plus lanreotide ATG is a safe and well tolerated combination treatment that is associated with promising antitumor activity in both non-diabetic and diabetic patients with WDNETs, and that warrants further investigation in larger clinical trials.

BackgroundRetreatment with anti-EGFR monoclonal antibodies is a promising strategy in patients with RAS/BRAF wild-type (wt) metastatic colorectal cancer (mCRC) who achieved benefit from previous anti-EGFR exposure upon exclusion of mutations in RAS/BRAF genes according to circulating tumor DNA (ctDNA) analysis by means of liquid biopsy (LB). This treatment approach is now being investigated in the randomized phase II trial PARERE (NCT04787341). We here present preliminary findings of molecular screening.MethodsPatients with RAS/BRAFV600E wt mCRC according to tissue genotyping who benefited from previous anti-EGFR-based treatment (fluoropyrimidines, oxaliplatin, irinotecan, and antiangiogenics) and then experienced disease progression to EGFR targeting were eligible for screening in the PARERE trial. The next-generation sequencing (NGS) panel Oncomine™ was employed for ctDNA testing.ResultsA total of 218 patients underwent LB, and ctDNA sequencing was successful in 201 of them (92%). RAS/BRAFV600E mutations were found in 68 (34%) patients and were mainly subclonal (median variant allele fraction [VAF] for KRAS, NRAS, and BRAF mutant clones: 0.52%, 0.62%, and 0.12%, respectively; p = 0.01), with KRASQ61H being the most frequently detected (31%). Anti-EGFR-free intervals did not predict ctDNA molecular status (p = 0.12). Among the 133 patients with RAS/BRAFV600E wt tumors according to LB, 40 (30%) harbored a mutation in at least another gene potentially implied in anti-EGFR resistance, mainly with subclonal expression (median VAF, 0.56%). In detail, alterations in PIK3CA, FBXW7, GNAS, MAP2K, ERBB2, BRAF (class I and II non-BRAFV600E), SMAD, EGFR, AKT1, and CTNNB1 occurred in 13%, 8%, 7%, 3%, 2%, 2%, 1%, 1%, 1%, and 1% cases, respectively. Co-mutations were detected in 13 (33%) out of 40 patients.ConclusionsThis is the largest prospective cohort of mCRC patients screened with LB for anti-EGFR retreatment in a randomized study. ctDNA genotyping reveals that at least one out of three patients candidate for retreatment should be excluded from this therapy, and other potential drivers of anti-EGFR resistance are found in approximately one out of three patients with RAS/BRAFV600E wt ctDNA.

Durable remissions are observed in 10%–20% of treated patients with advanced metastatic melanoma but the factors associated with long-term complete clinical responses are largely unknown. Here, we report the molecular characteristics of tumor evolution during disease progression along a 9-year clinical course in a patient with advanced disseminated melanoma who received different treatments, including trametinib, ipilimumab, radiation, vemurafenib, surgical tumor debulking and a second ipilimumab course, ultimately achieving complete long-term disease remission.Longitudinal analyses of therapies-resistant metastatic tumors revealed the effects of different treatments on tumor’s microenvironment and immunogenicity, ultimately creating a milieu favorable to immunotherapy response. Monitoring of the temporal dynamics of T cells by analysis of the T cell receptor (TCR) repertoire in the tumor and peripheral blood during disease evolution indicated that T-cell clones with common TCR rearrangements, present at low levels at baseline, were maintained and expanded after immunotherapy, and that TCR diversity increased. Analysis of genetic, molecular, and cellular components of the tumor depicted a multistep process in which treatment with kinase inhibitors strongly conditioned the immune microenvironment creating an inflamed milieu converting cold into hot tumors, while ipilimumab impacted and increased the TCR repertoire, a requirement for tumor rejection.Since the optimal sequencing of treatment with antibodies targeting immune checkpoints and kinase inhibitors for advanced melanoma is still clinically debated, this case indicates that immunotherapy success is possible even after progression on targeted therapy.

This work aimed at evaluating the impact of human activity and geology on the surface water quality of the Ouled Mellouk dam (northwestern Algeria). Specifically, methodology aims to assess seasonal variations of several parameters (EC, TDS, pH, SO42−, Cl−, Ca2+, Na+, HCO3−, NO3−, PO43− and Mg2+, organic matter, suspended solids, BOD5, COD) from 2007 to 2013 to define the processes controlling the mineralization and pollution of the surface water. The results show high salinization recorded during the periods of heavy precipitation or flooding, due to leaching of dolomitic limestones and evaporitic deposits from the saliferous formations of the surrounding mountains. High concentrations of NO3−, NO2−, NH4+, and organic matter can instead be attributed to the use of fertilizers in agriculture. Moreover, the PCA application shows that salinization (49.92%), anthropogenic pollution (19.38%) and agricultural pollution (12%) are the most significant degradation factors, respectively. The saturation index of carbonates and gypsiferous minerals shows the carbonates precipitate before the gypsiferous minerals, which allowed the chemical elements coming from the dissolution of gypsum and halite to acquire significant contents. In addition, the evolution of biological oxygen demand and organic pollution shows slight pollution of the dam water. HIGHLIGHTS The study evaluated the impact of human activity and geology formations on the surface water quality of the Ouled Mellouk dam (Algeria).; The results show that saline geological formations, water–rock interaction process, and anthropogenic pollution are the significant degradation factors for the lake's surface water quality.; The lake's water is unsuitable for domestic use and irrigation without a proper treatment.;

Abstract Tumour DNA sequencing is essential for precision medicine since it guides therapeutic decisions but also fosters the identification of patients who may benefit from germline testing. Notwithstanding, the tumour-to-germline testing workflow presents a few caveats. The low sensitivity for indels at loci with sequences of identical bases (homopolymers) of ion semiconductor-based sequencing techniques represents a well-known limitation, but the prevalence of indels overlooked by these techniques in high-risk populations has not been investigated. In our study, we addressed this issue at the homopolymeric regions of BRCA1/2 in a retrospectively selected cohort of 157 patients affected with high-grade ovarian cancer and negative at tumour testing by ION Torrent sequencing. Variant allele frequency (VAF) of indels at each of the 29 investigated homopolymers was systematically revised with the IGV software. Thresholds to discriminate putative germline variants were defined by scaling the VAF to a normal distribution and calculating the outliers that exceeded the mean + 3 median-adjusted deviations of a control population. Sanger sequencing of the outliers confirmed the occurrence of only one of the five putative indels in both tumour and blood from a patient with a family history of breast cancer. Our results indicated that the prevalence of homopolymeric indels overlooked by ion semiconductor techniques is seemingly low. A careful evaluation of clinical and family history data would further help minimise this technique-bound limitation, highlighting cases in which a deeper look at these regions would be recommended.

Over the last few years, the Eastern Middle Cheliff plain has witnessed remarkable economic growth, particularly in the agricultural sector. However, the overexploitation of the alluvial aquifer, coupled with agricultural practices involving the use of fertilizers and pesticides, significantly contributed to the degradation of groundwater quality. The primary objective of this study is to comprehend the mechanisms governing the water chemistry of the Eastern Middle Cheliff alluvial aquifer. A total of 42 samples were collected and analyzed during the dry periods of the 2012 and 2017. The data processing and representation involved the utilization of analytical tools including Principal Component Analysis (PCA), Piper diagram, Stabler classification, binary diagrams, base exchange indices, saturation indices, and geostatistical methods. The interpretation of these data revealed the following findings: i) the groundwater in both the dry periods of 2012 and 2017 is characterized by the presence of two dominant hydro-chemical facies, namely calcium chloride and sodium chloride, as indicated by the Piper and Stabler diagrams; ii) the hydrochemical quality of the groundwater varies across different regions of the aquifer, fair to poor in the eastern and central regions, with the presence of certain contaminants resulting from the application of nitrates in agricultural activities. The quality is classified as very poor in the western region, primarily due to high salinity influenced by the lithology of the aquifer, the rise of deep salty water through the Cheliff fault due to the earthquake of the year 1980 and possibility by two climatic factors, namely evaporation and rainfall. Overall, this study provides new insights into the water chemistry dynamics of the Eastern Middle Cheliff alluvial aquifer, highlighting the varying hydrochemical facies and the impact of agricultural activities and climatic factors on groundwater quality in different areas of the aquifer.

BackgroundPapillary thyroid carcinoma (PTC) is the most common type of thyroid cancer (TC). Several genomic and transcriptomic studies explored the molecular landscape of follicular cell-derived TCs, and BRAFV600E, RAS mutations, and gene fusions are well-established drivers. DICER1 mutations were described in specific sets of TC patients but represent a rare event in adult TC patients.MethodsHere, we report the molecular characterization of 30 retrospective follicular cell-derived thyroid tumors, comprising PTCs (90%) and poorly differentiated TCs (10%), collected at our Institute. We performed DNA whole-exome sequencing using patient-matched control for somatic mutation calling, and targeted RNA-seq for gene fusion detection. Transcriptional profiles established in the same cohort by microarray were investigated using three signaling-related gene signatures derived from The Cancer Genome Atlas (TCGA).ResultsThe occurrence of BRAFV600E (44%), RAS mutations (13%), and gene fusions (13%) was confirmed in our cohort. In addition, in two patients lacking known drivers, mutations of the DICER1 gene (p.D1709N and p.D1810V) were identified. DICER1 mutations occur in two adult patients with follicular-pattern lesions, and in one of them a second concurrent DICER1 mutation (p.R459*) is also observed. Additional putative drivers include ROS1 gene (p.P2130A mutation), identified in a patient with a rare solid-trabecular subtype of PTC. Transcriptomics indicates that DICER1 tumors are RAS-like, whereas the ROS1-mutated tumor displays a borderline RAS-/BRAF-like subtype. We also provide an overview of DICER1 and ROS1 mutations in thyroid lesions by investigating the COSMIC database.ConclusionEven though small, our series recapitulates the genetic background of PTC. Furthermore, we identified DICER1 mutations, one of which is previously unreported in thyroid lesions. For these less common alterations and for patients with unknown drivers, we provide signaling information applying TCGA-derived classification.

Aim: Diagnostic laboratories are progressively introducing next-generation sequencing (NGS) technologies in the routine workflow to meet the increasing clinical need for comprehensive molecular characterization in cancer patients for diagnosis and precision medicine, including fusion-transcripts detection. Nevertheless, the low quality of messenger RNA (mRNA) extracted from formalin-fixed paraffin-embedded (FFPE) samples may affect the transition from traditional single-gene testing approaches [like fluorescence in situ hybridization (FISH), immunohistochemistry (IHC), or polymerase chain reaction (PCR)] to NGS. The present study is aimed at assessing the overall accuracy of RNA fusion transcripts detection by NGS analysis in FFPE samples in real-world diagnostics. Methods: Herein, NGS data from 190 soft tissue tumors (STTs) and carcinoma cases, discussed in the context of the institutional Molecular Tumor Board, are reported and analyzed by FusionPlex© Solid tumor kit through the manufacturer’s pipeline and by two well-known fast and accurate open-source tools [Arriba (ARR) and spliced transcripts alignment to reference (STAR)-fusion (SFU)]. Results: The combination of FusionPlex© Solid tumor with ArcherDX® Analysis suite (ADx) analysis package has been proven to be sensitive and specific in STT samples, while partial loss of sensitivity has been found in carcinoma specimens. Conclusions: Albeit ARR and SFU showed lower sensitivity, the use of additional fusion-detection tools can contribute to reinforcing or extending the output obtained by ADx, particularly in the case of low-quality input data. Overall, our results sustain the clinical use of NGS for the detection of fusion transcripts in FFPE material.

Introduction: The use of high-flow nasal oxygen (HFNO) is a simple method that can reduce intubation in patients with hypoxemic acute respiratory failure (ARF). Early and prolonged prone position has demonstrated benefits on mortality in mechanically ventilated patients and on intubation in awake patients with ARF. However, strategies to achieve adherence to awake prone positioning (APP) have not been previously described. Case and outcomes: We present six patients with ARF due to COVID-19 treated with HFNO and APP. The median (p25–75) of PaFiO2 upon admission was 121 (112–175). The average duration of APP on the first day was 16 h (SD 5 h). Duration (median p25–75) in APP for the following 20 days was 13 (10–18) h/day. Several strategies such as the presence of a health care team, recreational activities, adaptation of the circadian rhythm, oral nutritional support, and analgesics were used to improve prone tolerance. None of the patients suffered from delirium, all were ambulating on discharge from the ICU and none require intubation. Conclusion: The case series presented show the feasibility of prolonged use of HFNO and APP in patients with COVID-19 and severe persistent hypoxemia and described strategies to enhance adherence.

The proposed study aims to assess groundwater quality and suitability of the Upper and Middle Cheliff plains (northwest of Algeria) for irrigation and drinking. Here the groundwater is the main source for domestic, agricultural and industrial activities similarly to any other region of the world. The suitability for drinking and for irrigation was evaluated on the basis of water quality index, salinity risk, hardness risk, sodium risk, magnesium risk, permeability index, water infiltration rate, Kelly index and Wilcox and Richards diagrams. The aquifer system is mainly composed of alluvium (gravel, sand, silt, clay, …) from the Mio-Plio-Quaternary. The results of this study highlighted that the majority of the chemical elements analyzed exceed the WHO's drinking water standards and FAO's irrigation water standards. Based on the GroundWater Quality Index (GWQI) results, the Upper and Middle Cheliff groundwater plains shows Doubtful class in most of the plains. In addition, the GroundWater Quality Index for Irrigation (GWQII) shows the predominance of the Good/Permissible groundwater quality class in most of the plains. According to these results, drinking water can cause health problems (a danger) for the human consumption making necessary a proper treatment be able to use it. As for irrigation water, it does not present a danger for irrigating for the vast fields of the region, with the exception of sensitive crops such as: garlic, onion, beans and strawberry. The proposed approach demonstrated to be appropriate in assessing the groundwater quality for irrigation and drinking water supply since it can be easy applicable and suitable in humid, arid or semi-arid regions around the world. HIGHLIGHTS The study aims to assess groundwater quality of the Upper and Middle Cheliff plains (northwest of Algeria) for irrigation and drinking water supply.; Groundwater Quality Index for drinking shows a predominance of the Doubtful class in the plains.; Groundwater Quality Index for Irrigation shows a predominance of the Good/Permissible class in the plains.;

Abstract Background In patients with COVID-19-related acute respiratory failure (ARF), awake prone positioning (AW-PP) reduces the need for intubation in patients treated with high-flow nasal oxygen (HFNO). However, the effects of different exposure times on clinical outcomes remain unclear. We evaluated the effect of AW-PP on the risk of endotracheal intubation and in-hospital mortality in patients with COVID-19-related ARF treated with HFNO and analyzed the effects of different exposure times to AW-PP. Methods This multicenter prospective cohort study in six ICUs of 6 centers in Argentine consecutively included patients > 18 years of age with confirmed COVID-19-related ARF requiring HFNO from June 2020 to January 2021. In the primary analysis, the main exposure was awake prone positioning for at least 6 h/day, compared to non-prone positioning (NON-PP). In the sensitivity analysis, exposure was based on the number of hours receiving AW-PP. Inverse probability weighting–propensity score (IPW-PS) was used to adjust the conditional probability of treatment assignment. The primary outcome was endotracheal intubation (ETI); and the secondary outcome was hospital mortality. Results During the study period, 580 patients were screened and 335 were included; 187 (56%) tolerated AW-PP for [median (p25–75)] 12 (9–16) h/day and 148 (44%) served as controls. The IPW–propensity analysis showed standardized differences

In pseudomyxoma peritonei (PMP), KRAS and GNAS mutations are frequent. We hypothesized that these mutations may contribute to the suppression of antitumor immunity: KRAS may induce GMCSF expression, while GNAS may enhance the expression of cyclic adenosine monophosphate and A2AR signaling. This study aimed to explore possible mechanisms facilitated by KRAS and GNAS mutations for escaping immune surveillance. Additionally, we looked for new potential therapeutic and prognostic targets in this rare disease which is poorly characterized at the molecular level. GM-CSF, A2AR, CD73, CD39, and PD-L1 expression was investigated by immunohistochemistry in 40 PMPs characterized for GNAS and KRAS mutational status. Immune cell populations were studied by immunohistochemistry and nanostring nCounter®. Following the criteria of a prognostic nomogram reported for PMP, we stratified the patients into two different risk groups, with 28 “low-risk” and 12 “high-risk” patients. We observed the expression of GM-CSF (74%); CD39 (37%); CD73 (53%); A2AR (74%); and PD-L1 (16%) which was unrelated to GNAS or KRAS status. The tumor microenvironment showed the presence of CD4+ T cells (86%); CD8+ T cells (27%); CD20+ B (67%); CD15+ cells (86%); and CD163+ M2 macrophages (67%), while CD56+ NK cells were absent. CD163 expression (27%) in PMP tumor cells was associated with poor prognosis. GNAS mutation and A2AR expression were not associated with a specific immune transcriptional signature. However, the expression assay revealed 21 genes associated with prognosis. The “high-risk” patients exhibited worse progression-free survival (HR = 2.3, CI 95%: 1.1–5.1, p = 0.034) and significant downregulation of MET, IL8, PPARG, DTX4, HMGA1, ZIC2, WNT5B, and CCRL2. In conclusion, we documented the presence of immunosuppressive factors such as GM-CSF, A2AR, and PD-L1 in PMP. These factors were not associated with GNAS and KRAS status and could be explored as therapeutic molecular targets. Additionally, a set of potential prognostic biomarkers, including CD163 expression in tumor cells, deserve further investigation.