Plasma hPG80 (Circulating Progastrin) as a Novel Prognostic Biomarker for early-stage breast cancer in a breast cancer cohort.

BACKGROUND: Recurrence and metastases are still frequent outcomes after initial tumour control in women diagnosed with breast cancer. Although therapies are selected based on tumour characteristics measured at baseline, prognostic biomarkers can identify those at risk of poor outcomes. Circulating progastrin or hPG80 was found to be associated with survival outcomes in renal and hepatocellular carcinomas and was a plausible prognostic biomarker for breast cancer. METHODS: Women with incident breast cancers from Calgary, Alberta, Canada enrolled in the Breast to Bone (B2B) study between 2010 to 2016 and provided blood samples prior to any treatment initiation. Plasma from these baseline samples were analysed for circulating progastrin or hPG80. Participant characteristics as well as tumour ones were evaluated for their association with hPG80 and survival outcomes (time to recurrence, recurrence - free survival, breast cancer specific survival and overall survival) in Cox proportional hazards regression models. RESULTS: The 464 participants with measurable hPG80 in this study had an average age of 57.03 years (standard deviation of 11.17 years) and were predominantly diagnosed with Stage I (52.2%) and Stage II (40.1%) disease. A total of 50 recurrences and 50 deaths were recorded as of June 2022. In Cox PH regression models adjusted for chemotherapy, radiation therapy, cancer stage and age at diagnosis, log hPG80 (pmol/L) significantly increased the risks for recurrence (Hazard Ratio (HR) = 1.330, 95% Confidence Interval (CI) = (0.995 - 1.777, p = 0.054)), recurrence-free survival (HR = 1.399, 95% CI = (1.106 - 1.770), p = 0.005) and overall survival (HR = 1.385, 95% CI = (1.046 - 1.834), = 0.023) but not for breast cancer specific survival (HR = 1.015, 95% CI = (0.684 - 1.505), p = 0.942). CONCLUSIONS: hPG80 levels measured at diagnosis were significantly associated with the risk of recurrence or death from any cause in women with breast cancer. Since the recurrence rates of breast cancer are still relatively high amongst women diagnosed at an early stage, identifying women at high risk of recurrence at their time of diagnosis is important. hPG80 is a promising new prognostic biomarker that could improve the identification of women at higher risk of poor outcomes.

ctDNA in Neuroendocrine Carcinoma of Gastroenteropancreatic Origin or of Unknown Primary: The CIRCAN-NEC Pilot Study.

INTRODUCTION: Gastroenteropancreatic neuroendocrine carcinomas (GEPNEC) are characterized by a heterogeneous molecular profile and a poor prognosis. Circulating tumour DNA (ctDNA) analysis may be useful for NEC management. This study aimed at describing ctDNA mutations, to assess their predictive value for response to chemotherapies, and their change according to disease progression. METHODS: The CIRCAN-NEC study included patients with GEPNEC or NEC from an unknown primary, scheduled to begin first- or second-line chemotherapy. Blood samples were collected prior to chemotherapy initiation, at first evaluation, and during disease progression. ctDNA was sequenced by next-generation sequencing (NGS). Molecular response was defined as a decrease of at least 30% of the mutant allele fraction. RESULTS: All 24 patients included received platinum-etoposide first-line chemotherapy; 19 received a FOLFIRI-based post-first-line regimen. Twenty-two patients had at least one driver mutation: TP53 (n = 21), RB1 (n = 2), KRAS (n = 4), and BRAF (n = 3). Ten (42%) had an "adenocarcinoma-like" profile. Five of 6 patients with matching ctDNA/tissue NGS harboured at least one concordant mutation (44% concordance at the gene level). The concordance rate between ctDNA mutation/immunohistochemistry profile was 64% (7/11) for TP53/p53+ and 14% (1/7) for RB1/pRb-. In this pilot study including few patients by subgroups, patients with KRAS (HR = 3.60, 95% CI [1.06-12.04]) and BRAF (HR = 4.25, 95% CI [1.11-16.40]) mutations had shorter progression-free survival (PFS) under platinum-etoposide, while the 2 patients with RB1 mutations had shorter PFS under FOLFIRI-based chemotherapy. Twenty-eight periods of treatment were assessed: 10 patients had a molecular response (7/10 had a morphological response), which was associated with longer PFS (HR = 0.37, 95% CI [0.15; 0.91]). CONCLUSION: This pilot study shows a high sensitivity of ctDNA assessment, which is encouraging for the future management of GEPNEC (tumour molecular diagnosis and evaluation of disease progression).

Modeling Approach to Predict the Impact of Inflammation on the Pharmacokinetics of CYP2C19 and CYP3A4 Substrates.

PURPOSE: For decades, inflammation has been considered a cause of pharmacokinetic variability, mainly in relation to the inhibitory effect of pro-inflammatory cytokines on the expression level and activity of cytochrome P450 (CYP). In vitro and clinical studies have shown that two major CYPs, CYP2C19 and CYP3A4, are both impaired. The objective of the present study was to quantify the impact of the inflammatory response on the activity of both CYPs in order to predict the pharmacokinetic profile of their substrates according to systemic C-reactive protein (CRP). METHODS: The relationships between CRP concentration and both CYPs activities were estimated and validated using clinical data first on midazolam then on voriconazole. Finally, clinical data on omeprazole were used to validate the findings. For each substrate, a physiologically based pharmacokinetics model was built using a bottom-up approach, and the relationships between CRP level and CYP activities were estimated by a top-down approach. After incorporating the respective relationships, we compared the predictions and observed drug concentrations. RESULTS: Changes in pharmacokinetic profiles and parameters induced by inflammation seem to be captured accurately by the models. CONCLUSIONS: These findings suggest that the pharmacokinetics of CYP2C19 and CYP3A4 substrates can be predicted depending on the CRP concentration.

p58(IPK)-mediated attenuation of the proapoptotic PERK-CHOP pathway allows malignant progression upon low glucose.

As solid tumors expand, oxygen and nutrients become limiting owing to inadequate vascularization and diffusion. How malignant cells cope with this potentially lethal metabolic stress remains poorly understood. We found that glucose shortage associated with malignant progression triggers apoptosis through the endoplasmic reticulum (ER) unfolded protein response (UPR). ER stress is in part caused by reduced glucose flux through the hexosamine pathway. Deletion of the proapoptotic UPR effector CHOP in a mouse model of K-ras(G12V)-induced lung cancer increases tumor incidence, strongly supporting the notion that ER stress serves as a barrier to malignancy. Overcoming this barrier requires the selective attenuation of the PERK-CHOP arm of the UPR by the molecular chaperone p58(IPK). Furthermore, p58(IPK)-mediated adaptive response enables cells to benefit from the protective features of chronic UPR. Altogether, these results show that ER stress activation and p58(IPK) expression control the fate of malignant cells facing glucose shortage.

Structure-activity relationship study: Mechanism of cyto-genotoxicity of Nitropyrazole-derived high energy density materials family.

Stringent toxicological tests have to be performed prior to the industrial development of alternative chemicals particularly high energy dense materials (HEDMs) such as explosives. The properties (e.g., power, stability) of these compounds are constantly being improved, the current axis of research being the nitration of nitrogen heterocycles leading to HEDMs such as nitropyrazole-derived molecules. However, except for 3,4,5-trinitropyrazole (3,4,5-TNP), which was shown to be highly toxic in mice, the toxicological impact of these HEDMs has so far not been investigated. Furthermore, as industrials are strongly advised to develop alternative safety testing assays to in vivo experiments, we herein focused on determining the cytotoxic and genotoxic effects of seven Nitropyrazole-derived HEDMs on three rodent cell lines (mouse embryonic BALB/3T3 clone A31 cells, Chinese hamster ovary cells CHO-K1 and mouse lymphoma L5178Y TK +/- clone (3.7.2C) cells), two human fibroblast lines (CRC05, PFS04062) and on the human hepatic HepaRG model (both in proliferative and differentiated cells). A stronger cytotoxic effect was observed for 1,3-dinitropyrazole (1, 3-DNP) and 3,4,5-TNP in all cell lines, though differentiated HepaRG cells clearly displayed fewer likely due to the metabolism and elimination of these molecules by their functional biotransformation pathways. At the mechanistic level, the sub-chronic cytotoxic and genotoxic effects were linked to ROS/RNS production (experimental assays), HA2.X and to transcriptomic data highlighting the increase in DNA repair mechanisms.

Toxicokinetics and tolerance of a high energy material 3,4,5-trinitropyrazole (TNP) in mice.

The high-energy compound 3,4,5-trinitropyrazole (TNP) was developed as an alternative to other less energetic and more sensitive explosive materials, in particular 1-methyl-2,4,6-trinitrobenzene (TNT). However, the level of toxicity of TNP remains understudied. Here using an in vivo CD1 mouse model, we mimicked an acute exposure (24 h) to TNP, given either orally or intravenously, and determined the maximum administrable doses (190 mg/kg and 11 mg/kg, respectively), as well as the lethal dose for 50% (LD50) of female or male mice (390 mg/kg for both) treated intravenously with TNP alone. Several metabolites including nitroso-dinitro-pyrazole, hydroxylamino-dinitro-pyrazole, hydroxyl-dinitro-pyrazole and amino-dinitro-pyrazole were identified in urine. TNP is quickly metabolized and eliminated via urine as two main amino-dinitro-pyrazole metabolites. A comparison of the transcriptomic effects of TNP and TNT after 10 days exposure enabled us to demonstrate no major induction of transcripts involved both in cell death mechanisms (apoptosis, necrosis, autophagy) and physiological pathways (glycolysis, ATP production). Finally, subchronic exposure to TNP was replicated in female mice, fed 16.8-52.8 mg/kg/day of TNP for one month, to study the impact on cellular functions. Although blood TNP levels remained high, a lower rate of TNP accumulation in the liver and lungs were observed than during an acute exposure. Conversely, cellular stress functions explored using the RT2 Profiler™ PCR Array Mouse Molecular Toxicology PathwayFinder remained unaltered after this chronic exposure. These findings demonstrate that TNP can be rapidly eliminated in vivo without accumulating in vital organs.

Deciphering the molecular mechanisms underlying the binding of the TWIST1/E12 complex to regulatory E-box sequences.

The TWIST1 bHLH transcription factor controls embryonic development and cancer processes. Although molecular and genetic analyses have provided a wealth of data on the role of bHLH transcription factors, very little is known on the molecular mechanisms underlying their binding affinity to the E-box sequence of the promoter. Here, we used an in silico model of the TWIST1/E12 (TE) heterocomplex and performed molecular dynamics (MD) simulations of its binding to specific (TE-box) and modified E-box sequences. We focused on (i) active E-box and inactive E-box sequences, on (ii) modified active E-box sequences, as well as on (iii) two box sequences with modified adjacent bases the AT- and TA-boxes. Our in silico models were supported by functional in vitro binding assays. This exploration highlighted the predominant role of protein side-chain residues, close to the heart of the complex, at anchoring the dimer to DNA sequences, and unveiled a shift towards adjacent ((-1) and (-1*)) bases and conserved bases of modified E-box sequences. In conclusion, our study provides proof of the predictive value of these MD simulations, which may contribute to the characterization of specific inhibitors by docking approaches, and their use in pharmacological therapies by blocking the tumoral TWIST1/E12 function in cancers.

Destabilization of the TWIST1/E12 complex dimerization following the R154P point-mutation of TWIST1: an in silico approach.

BACKGROUND: The bHLH transcription factor TWIST1 plays a key role in the embryonic development and in tumorigenesis. Some loss-of-function mutations of the TWIST1 gene have been shown to cause an autosomal dominant craniosynostosis, known as the Saethre-Chotzen syndrome (SCS). Although the functional impacts of many TWIST1 mutations have been experimentally reported, little is known on the molecular mechanisms underlying their loss-of-function. In a previous study, we highlighted the predictive value of in silico molecular dynamics (MD) simulations in deciphering the molecular function of TWIST1 residues. RESULTS: Here, since the substitution of the arginine 154 amino acid by a glycine residue (R154G) is responsible for the SCS phenotype and the substitution of arginine 154 by a proline experimentally decreases the dimerizing ability of TWIST1, we investigated the molecular impact of this point mutation using MD approaches. Consistently, MD simulations highlighted a clear decrease in the stability of the α-helix during the dimerization of the mutated R154P TWIST1/E12 dimer compared to the wild-type TE complex, which was further confirmed in vitro using immunoassays. CONCLUSIONS: Our study demonstrates that MD simulations provide a structural explanation for the loss-of-function associated with the SCS TWIST1 mutation and provides a proof of concept of the predictive value of these MD simulations. This in silico methodology could be used to determine reliable pharmacophore sites, leading to the application of docking approaches in order to identify specific inhibitors of TWIST1 complexes.

(E)-1,1,4,4-Tetramethyl-2-tetrazene (TMTZ): A Prospective Alternative to Hydrazines in Rocket Propulsion.

1,1,4,4-Tetramethyl-2-tetrazene (TMTZ) is considered as a prospective replacement for toxic hydrazines used in liquid rocket propulsion. The heat of formation of TMTZ was computed and measured, giving values well above those of the hydrazines commonly used in propulsion. This led to a predicted maximum Isp of 337 s for TMTZ/N2 O4 mixtures, which is a value comparable to that of monomethylhydrazine. We found that TMTZ has a vapor pressure well below that of liquid hydrazines, and it is far less toxic. Finally, an improved synthesis is proposed, which is compatible with existing industrial production facilities after minor changes. TMTZ is thus an attractive liquid propellant candidate, with a performance comparable to hydrazines but a lower vapor pressure and toxicity.

Pharmacokinetic interactions in mice between irinotecan and MBL-II-141, an ABCG2 inhibitor.

PURPOSE: The chromone derivative MBL-II-141, specifically designed to inhibit ABCG2, was previously demonstrated to combine strong inhibition potency, low toxicity and good efficiency in reversing resistance to irinotecan in a xenografted mouse model. Here, the pharmacokinetic interactions in mice between irinotecan, its active metabolite SN-38 and MBL-II-141 were characterized quantitatively in the blood and in the brain. METHODS: Compartmental models were used to fit the data. Goodness-of-fit was assessed by simulation-based diagnostic tools. RESULTS: Irinotecan increased the MBL-II-141 apparent clearance and Vss 1.5-fold, probably by increasing the MBL-II-141 unbound fraction. MBL-II-141 decreased the total apparent clearance of irinotecan by 23%, by decreasing its biliary clearance. MBL-II-141 increased 3-fold the brain accumulation of irinotecan, as a result of the rise of systemic exposure combined with the inhibition of ABCG2-mediated efflux at the blood-brain barrier. Finally, SN-38 exposure was increased by 1.16-fold under treatment with MBL-II-141, owing to the higher irinotecan exposure with increased metabolism towards the formation of SN-38. CONCLUSIONS: These results may help to anticipate the pharmacokinetic interactions between MBL-II-141 and other ABCG2 substrates. The irinotecan-MBL-II-141 interaction is also expected to occur in humans. Copyright © 2017 John Wiley & Sons, Ltd.

Quantification and kinetic study in plasma and tissues of (E)-1,1,4,4-tetramethyl-2-tetrazene, a liquid propellant and a transformation product of 1,1-dimethyl hydrazine.

(E)-1,1,4,4-tetramethyl-2-tetrazene (TMTZ) is formed from the oxidation of the unsymmetrical 1,1-dimethylhydrazine (UDMH) and is used as a storable liquid fuel which can be considered as a new potential propellant for space rocket propulsion. To better understand the toxicological behavior of the compound, an intraperitoneal administration of TMTZ was performed in mice to define its toxicokinetics and tissue distribution. A fully validated liquid chromatography coupled to tandem mass spectrometry (LC-MS/MS) assay was developed to determine TMTZ levels in biological samples. Determination of TMTZ was achieved using 50 µL of plasma or tissue solution. Precipitation with ammonium sulfate and acetonitrile was used for sample preparation. Liquid chromatography was performed on an Atlantis HILIC Silica column (Waters; 3 µm, 150 mm × 2.1 mm i.d.). Isocratic elution with a mixture of ammonium acetate buffer (pH 5, 100 mM)/water/acetonitrile (3:2:95, v/v/v) was used. The detection was conducted using an electrospray source in positive ion mode. TMTZ and (15)N2-TMTZ (internal standard) were quantitated in selected reaction monitoring mode using the transition m/z 117→72 and 119→74, respectively. Standard curves exhibited excellent linearity in the range of 10-500 ng/mL for plasma and 50-2000 ng/mL for all tissues (heart, liver, brain, kidney, and lung) analyzed, and acceptable precision and accuracy (<10 %) were obtained. The elimination rate constant strongly suggests that TMTZ was very quickly eliminated from the body. The results of tissue distribution experiments indicated that TMTZ underwent a rapid distribution into limited organs such as the liver, kidney, and brain.

Quantification of ß-aminopropionitrile, an inhibitor of lysyl oxidase activity, in plasma and tumor of mice by liquid chromatography tandem mass spectrometry.

Lysyl oxidase enzymes are reported to be involved in patho-physiological process such as tumorigenesis. ß-Aminopropionitrile (BAPN) is an irreversible inhibitor of lysyl oxidase activity, suggesting a potentially useful therapeutic of interest in oncology. This paper describes the first assay concerning the quantification of BAPN by mass spectrometry. A high-performance liquid chromatography tandem mass spectrometry (LC-MS/MS) assay was developed for the quantification of BAPN in plasma and tumor of mice. This method combines dansyl chloride (Dns) derivatization and extraction using a solid-phase extraction Oasis Max column. Deuterated BAPN was used as internal standard (IS). Separation was achieved using an C18 column HypersylGold, (ThermoElectron), 3.0 µm (100 × 2.1 mm i.d.). Gradient elution with water containing 0.1% acetic acid (A) and acetonitrile containing 0.1% acetic acid (B) was applied. Detection was performed with an electrospray ionization interface operating in negative ion mode. Selected reaction monitoring was used with ion transitions m/z 302 → 249 for BAPN-Dns and m/z 306 → 250 for the IS. The method was fully validated in plasma and was linear and sensitive in the range of 10-500 ng/mL. The lower limit of quantification in plasma was 2.5 ng/mL. This validated assay was successfully applied to a kinetic study of BAPN in mouse plasma and demonstrates that BAPN reaches the tumoral tissue.

Microchip for Immunomagnetic Sorting of Circulating Tumor Cells (CTCs).

Circulating tumor cells (CTCs) isolated directly from whole blood opens new perspectives for cancer monitoring and the development of personalized treatments. However, due to their rarity among the multitude of blood cells, it remains a challenge to recover them alive with high level of purity, i.e., with few remaining white blood cells, and in a time frame compatible with the clinical context. Microfluidic chips have emerged as promising tools to address these challenges. We propose a two-step workflow including a pre-enrichment step, performed by a size-based pre-enrichment system, and a purification step, performed by an immunomagnetic chip. Here, we describe the protocol for the fabrication of the immunomagnetic microchip, the preparation of the sample, and the procedure for injection into the microchip allowing the sorting of the CTCs.

Neuropilin 2 and soluble neuropilin 2 in neuroendocrine neoplasms.

Neuropilin 2 (NRP2), a transmembrane non-tyrosine kinase receptor, has been described as a potential critical player in the tumourigenesis of several solid cancers and particularly in neuroendocrine neoplasms (NENs). A soluble form of NRP2 (sNRP2) has been previously described and corresponds to a truncated splice isoform. Its prognostic value has never been studied in NEN. NRP2 expression was studied by immunochemistry on tissue microarrays (n = 437) and on circulating tumour cells (CTCs, n = 5 patients with neuroendocrine carcinoma, NEC). We described the levels of sNRP2 in 229 patients with NEN using the ELISA method to identify the factors associated with sNRP2 levels and to evaluate its prognostic role; 90 blood donors represented the healthy control group. NRP2 was found in 97% of neuroendocrine tumours (396/410) and in 74% of NEC (20/27). NRP2 was also expressed in CTC of all the studied patients. The receiver operating characteristic (ROC) analysis showed that sNRP2 had a weak capacity to discriminate between NEN patients and healthy controls (area under curve (AUC) = 0.601, P = 0.053). Abnormal sNRP2 levels were associated with inflammatory syndrome, bone and peritoneal metastases, and abnormal chromogranin A levels. Patients with high sNRP2 levels (sNRP2Q3-Q4) had significantly poorer overall survival in multivariate analysis (HR 0.16, 95% CI (0.04-0.67), P = 0.015). In conclusion, the present study found that sNRP2 and NRP2 could represent a new prognostic biomarker and a therapeutic target, respectively, particularly in aggressive NEN.

Intraperitoneal Nivolumab After Debulking Surgery and Hyperthermic Intraperitoneal Chemotherapy in Advanced Ovarian Cancer: A Phase I Study with Expansion Cohort.

PURPOSE: Cytoreductive surgery (CRS) and hyperthermic intraperitoneal chemotherapy (HIPEC) are expected to be synergistic with intraperitoneal (IP) immunotherapy by increasing tumor antigen expression and mutational load. We assessed the feasibility and safety of IP nivolumab following complete CRS and HIPEC in pretreated patients with recurrent ovarian cancer (ClinicalTrials.gov identifier: NCT03959761). PATIENTS AND METHODS: Patients received IP nivolumab (0.5, 1 and 3 mg/kg) using a 3+3 dose-escalation design, starting 5-7 days after CRS and HIPEC. Four IP Q2W nivolumab infusions were planned. The primary objective was to demonstrate the feasibility of IP nivolumab based on dose-limiting toxicity (DLT). Secondary objectives were to assess changes in tolerance of CRS and HIPEC. RESULTS: A total of 17 patients were enrolled including 10 patients in the dose-escalation and 7 patients in the expansion phase. No DLT was observed at any dose-level in the 9 evaluable patients. Six of the 17 patients (35%) did not complete all planned infusions: 4 (23.5%) due to peritoneal catheter complications, 2 (11.8%) due to early progression. No procedure-related deaths occurred. Eleven patients (65%) experienced serious adverse events (SAEs), mainly transitory grade 3-4 transaminases elevations (6/11), and surgery-related (9/11). Four SAEs were related to the peritoneal catheter and two to HIPEC. No SAEs/grade 3-4 adverse events related to IP nivolumab occurred. CONCLUSIONS: This is the first study demonstrating the feasibility of IP nivolumab in patients with relapsed advanced ovarian cancer; further investigation at 3 mg/kg is warranted.

Localization of putative binding sites for cyclic guanosine monophosphate and the anti-cancer drug 5-fluoro-2'-deoxyuridine-5'-monophosphate on ABCC11 in silico models.

BACKGROUND: The Multidrug Resistance Protein ABCC11/MRP8 is expressed in physiological barriers and tumor breast tissues in which it secretes various substrates including cGMP (cyclic guanosine monophosphate) and 5FdUMP (5-fluoro-2'-deoxyuridine-5'-monophosphate), the active metabolite of the anticancer drug 5-FluoroUracil (frequently included to anticancer therapy).Previously, we described that ABCC11 high levels are associated to the estrogen receptor (ER) expression level in breast tumors and in cell lines resistant to tamoxifen. Consequently, by lowering the intracellular concentration of anticancer drugs, ABCC11 likely promotes a multidrug resistance (MDR) phenotype and decreases efficiency of anticancer therapy of 5FdUMP. Since no experimental data about binding sites of ABCC11 substrate are available, we decided to in silico localize putative substrate interaction sites of the nucleotide derivatives. Taking advantage of molecular dynamics simulation, we also analysed their evolution under computational physiological conditions and during the time. RESULTS: Since ABCC11 crystal structure is not resolved yet, we used the X-ray structures of the mouse mdr3 (homologous to human ABCB1) and of the bacterial homolog Sav1866 to generate two independent ABCC11 homology models in inward- and outward-facing conformations. Based on docking analyses, two putative binding pockets, for cGMP and 5FdUMP, were localized in both inward- and outward-facing conformations. Furthermore, based on our 3D models, and available biochemical data from homologous transporters, we identified several residues, potentially critical in ABCC11 transport function. Additionally, molecular dynamics simulation on our inward-facing model revealed for the first time conformation changes assumed to occur during transport process. CONCLUSIONS: ABCC11 would present two binding sites for cGMP and for 5FdUMP. Substrates likely first bind at the intracellular side of the transmembrane segment while ABCC11 is open forward the cytoplasm (inward-facing conformation). Then, along with conformational changes, it would pass through ABCC11 and fix the second site (close to the extracellular side), until the protein open itself to the extracellular space and allow substrate release.

Direct Comparative Analysis of a Pharmacogenomics Panel with PacBio Hifi® Long-Read and Illumina Short-Read Sequencing.

BACKGROUND: Pharmacogenetics (PGx) aims to determine genetic signatures that can be used in clinical settings to individualize treatment for each patient, including anti-cancer drugs, anti-psychotics, and painkillers. Taken together, a better understanding of the impacts of genetic variants on the corresponding protein function or expression permits the prediction of the pharmacological response: responders, non-responders, and those with adverse drug reactions (ADRs). OBJECTIVE: This work provides a comparison between innovative long-read sequencing (LRS) and short-read sequencing (SRS) techniques. METHODS AND MATERIALS: The gene panel captured using PacBio HiFi® sequencing was tested on thirteen clinical samples on GENTYANE's platform. SRS, using a comprehensive pharmacogenetics panel, was performed in routine settings at the Civil Hospitals of Lyon. We focused on complex regions analysis, including copy number variations (CNVs), structural variants, repeated regions, and phasing-haplotyping for three key pharmacogenes: CYP2D6, UGT1A1, and NAT2. RESULTS: Variants and the corresponding expected star (*) alleles were reported. Although only 38.4% concordance was found for haplotype determination and 61.5% for diplotype, this did not affect the metabolism scoring. A better accuracy of LRS was obtained for the detection of the CYP2D6*5 haplotype in the presence of the duplicated wild-type CYP2D6*2 form. A total concordance was performed for UGT1A1 TA repeat detection. Direct phasing using the LRS approach allowed us to correct certain NAT2 profiles. CONCLUSIONS: Combining an optimized variant-calling pipeline and with direct phasing analysis, LRS is a robust technique for PGx analysis that can minimize the risk of mis-haplotyping.

Loss of Pla2r1 decreases cellular senescence and age-related alterations caused by aging and Western diets.

Cellular senescence is induced by many stresses including telomere shortening, DNA damage, oxidative, or metabolic stresses. Senescent cells are stably cell cycle arrested and they secrete many factors including cytokines and chemokines. Accumulation of senescent cells promotes many age-related alterations and diseases. In this study, we investigated the role of the pro-senescent phospholipase A2 receptor 1 (PLA2R1) in regulating some age-related alterations in old mice and in mice subjected to a Western diet, whereas aged wild-type mice displayed a decreased ability to regulate their glycemia during glucose and insulin tolerance tests, aged Pla2r1 knockout (KO) mice efficiently regulated their glycemia and displayed fewer signs of aging. Loss of Pla2r1 was also found protective against the deleterious effects of a Western diet. Moreover, these Pla2r1 KO mice were partially protected from diet-induced senescent cell accumulation, steatosis, and fibrosis. Together these results support that Pla2r1 drives several age-related alterations, especially in the liver, arising during aging or through a Western diet.

Paired Comparison of Routine Molecular Screening of Patient Samples with Advanced Non-Small Cell Lung Cancer in Circulating Cell-Free DNA Using Three Targeted Assays.

INTRODUCTION: Progressive advanced non-small cell lung cancer (NSCLC) accounts for about 80-85% of all lung cancers. Approximately 10-50% of patients with NSCLC harbor targetable activating mutations, such as in-frame deletions in Exon 19 (Ex19del) of EGFR. Currently, for patients with advanced NSCLC, testing for sensitizing mutations in EGFR is mandatory prior to the administration of tyrosine kinase inhibitors. PATIENTS AND METHODS: Plasma was collected from patients with NSCLC. We carried out targeted NGS using the Plasma-SeqSensei™ SOLID CANCER IVD kit on cfDNA (circulating free DNA). Clinical concordance for plasma detection of known oncogenic drivers was reported. In a subset of cases, validation was carried out using an orthogonal OncoBEAMTM EGFR V2 assay, as well as with our custom validated NGS assay. Somatic alterations were filtered, removing somatic mutations attributable to clonal hematopoiesis for our custom validated NGS assay. RESULTS: In the plasma samples, driver targetable mutations were studied, with a mutant allele frequency (MAF) ranging from 0.00% (negative detection) to 82.25%, using the targeted next-generation sequencing Plasma-SeqSensei™ SOLID CANCER IVD Kit. In comparison with the OncoBEAMTM EGFR V2 kit, the EGFR concordance is 89.16% (based on the common genomic regions). The sensitivity and specificity rates based on the genomic regions (EGFR exons 18, 19, 20, and 21) were 84.62% and 94.67%. Furthermore, the observed clinical genomic discordances were present in 25% of the samples: 5% in those linked to the lower of coverage of the OncoBEAMTM EGFR V2 kit, 7% in those induced by the sensitivity limit on the EGFR with the Plasma-SeqSensei™ SOLID CANCER IVD Kit, and 13% in the samples linked to the larger KRAS, PIK3CA, BRAF coverage of the Plasma-SeqSensei™ SOLID CANCER IVD kit. Most of these somatic alterations were cross validated in our orthogonal custom validated NGS assay, used in the routine management of patients. The concordance is 82.19% in the common genomic regions (EGFR exons 18, 19, 20, 21; KRAS exons 2, 3, 4; BRAF exons 11, 15; and PIK3CA exons 10, 21). The sensitivity and specificity rates were 89.38% and 76.12%, respectively. The 32% of genomic discordances were composed of 5% caused by the limit of coverage of the Plasma-SeqSensei™ SOLID CANCER IVD kit, 11% induced by the sensitivity limit of our custom validated NGS assay, and 16% linked to the additional oncodriver analysis, which is only covered by our custom validated NGS assay. CONCLUSIONS: The Plasma-SeqSensei™ SOLID CANCER IVD kit resulted in de novo detection of targetable oncogenic drivers and resistance alterations, with a high sensitivity and accuracy for low and high cfDNA inputs. Thus, this assay is a sensitive, robust, and accurate test.

A PK-PD model linking biomarker dynamics to progression-free survival in patients treated with everolimus and sorafenib combination therapy, EVESOR phase I trial.

PURPOSE: The objective was to develop a pharmacokinetic-pharmacodynamic (PK-PD) model linking everolimus and sorafenib exposure with biomarker dynamics and progression-free survival (PFS) based on data from EVESOR trial in patients with solid tumors treated with everolimus and sorafenib combination therapy and to simulate alternative dosing schedules for sorafenib. PATIENTS AND METHODS: Everolimus (5-10 mg once daily, qd) and sorafenib (200-400 mg twice daily, bid) were administered according to four different dosing schedules in 43 solid tumor patients. Rich PK and PD sampling for serum angiogenesis biomarkers was performed. Baseline activation of RAS/RAF/ERK (MAPK) pathway was assessed by quantification of mRNA specific gene panel in tumor biopsies. The PK-PD modeling was performed using NONMEM® software. RESULTS: An indirect response PK-PD model linking sorafenib plasma exposure with soluble vascular endothelial growth factor receptor 2 (sVEGFR2) dynamics was developed. Progression-free survival (PFS) was described by a parametric time-to-event model. Higher decreases in sVEGFR2 at day 21 and higher baseline activation of MAPK pathway were associated with longer PFS (p = 0.002 and p = 0.007, respectively). The simulated schedule sorafenib 200 mg bid 5 days-on/2 days-off + continuous everolimus 5 mg qd was associated with median PFS of 4.3 months (95% CI 1.6-14.4), whereas the median PFS in the EVESOR trial was 3.6 months (95% CI 2.7-4.2, n = 43). CONCLUSION: Sorafenib 200 mg bid 5 days-on/2 days-off + everolimus 5 mg qd continuous was selected for an additional arm of EVESOR trial to evaluate whether this simulated schedule is associated with higher clinical benefit. TRIAL REGISTRATION: ClinicalTrials.gov Identifier: NCT01932177.