Clinical utility of circulating tumor DNA sequencing with a large panel: a National Center for Precision Medicine (PRISM) study.

BACKGROUND: Circulating tumor DNA (ctDNA) sequencing is a promising approach for tailoring therapy in patients with cancer. We report hereby the results from a prospective study where we investigated the impact of comprehensive molecular profiling of ctDNA in patients with advanced solid tumors. PATIENTS AND METHODS: Genomic analysis was performed using the FoundationOne Liquid CDx Assay [324 genes, tumor mutational burden (TMB), microsatellite instability status]. Each individual genomic report was reviewed and discussed weekly by a multidisciplinary tumor board (MTB). Actionable targets were classified by ESMO Scale for Clinical Actionability of Molecular Targets (ESCAT) tier leading to molecular-based treatment suggestions wherever it was possible. RESULTS: Between December 2020 and November 2021, 1772 patients with metastatic solid tumors underwent molecular profiling. Median time to assay results was 12 days. Results were contributive for 1658 patients (94%). At least one actionable target was detected in 1059 patients (64%) with a total of 1825 actionable alterations including alteration of the DNA damage repair response pathway (n = 336, 18%), high TMB (>16 mutations/Mb; n = 243, 13%), PIK3CA mutations (n = 150, 8%), ERBB family pathway alterations (n = 127, 7%), PTEN alterations (n = 95, 5%), FGFR alterations (n = 67, 4%) and MET activations (n = 13, 0.7%). The MTB recommended a matched therapy for 597 patients (56%) with a total of 819 therapeutic orientations: clinical trials (n = 639, 78%), off-label/compassionate use (n = 81, 10%), approved drug (n = 51, 6%), and early access program (n = 48, 6%). In total, 122 patients (21%) were treated. Among the assessable patients (n = 107), 4 (4%) had complete response, 35 (33%) had partial response, 27 (25%) had stable disease, and 41 (38%) a progressive disease as best response. The median progression-free survival and median overall survival were 4.7 months (95% confidence interval 2.7-6.7 months) and 8.3 months (95% confidence interval 4.7-11.9 months) respectively. CONCLUSIONS: ctDNA sequencing with a large panel is an efficient approach to match patients with advanced cancer with targeted therapies.

Osimertinib benefit in EGFR-mutant NSCLC patients with T790M-mutation detected by circulating tumour DNA.

Background: Approximately 50% of epidermal growth factor receptor (EGFR) mutant non-small cell lung cancer (NSCLC) patients treated with EGFR tyrosine kinase inhibitors (TKIs) will acquire resistance by the T790M mutation. Osimertinib is the standard of care in this situation. The present study assesses the efficacy of osimertinib when T790M status is determined in circulating cell-free tumour DNA (ctDNA) from blood samples in progressing advanced EGFR-mutant NSCLC patients. Material and methods: ctDNA T790M mutational status was assessed by Inivata InVision™ (eTAm-Seq™) assay in 48 EGFR-mutant advanced NSCLC patients with acquired resistance to EGFR TKIs without a tissue biopsy between April 2015 and April 2016. Progressing T790M-positive NSCLC patients received osimertinib (80 mg daily). The objectives were to assess the response rate to osimertinib according to Response Evaluation Criteria in Solid Tumours (RECIST) 1.1, the progression-free survival (PFS) on osimertinib, and the percentage of T790M positive in ctDNA. Results: The ctDNA T790M mutation was detected in 50% of NSCLC patients. Among assessable patients, osimertinib gave a partial response rate of 62.5% and a stable disease rate of 37.5%. All responses were confirmed responses. After median follow up of 8 months, median PFS by RECIST criteria was not achieved (95% CI: 4-NA), with 6- and 12-months PFS of 66.7% and 52%, respectively. Conclusion(s): ctDNA from liquid biopsy can be used as a surrogate marker for T790M in tumour tissue.

Correlation between the effects of retinoic acid and dexamethasone on liver tyrosine aminotransferase.

A single dose of 50 microg of trans-retinoic acid administered to rats significantly raised the level of hepatic tyrosine after a few hours. This effect was compared with that of dexamethasone and a possible correlation between these effectors was also investigated. An equal increase in enzyme activity level caused by retinoic acid was observed in adrenalectomized rats, leading to the suggestion that the effect of retinoic acid on liver tyrosine aminotransferase is independent of glucocorticoids. However, the study of the binding activity of the liver nuclear glucocorticoid receptors vs dexamethasone showed that this activity is favoured by retinoic acid, whereas no variation was evidenced for retinoic acid receptors caused by dexamethasone. In the adrenalectomized rat, the synergistic effect produced by the association of retinoic acid and dexamethasone at the lowest doses used led us to conclude that retinoic acid is an efficient effector of liver tyrosine aminotransferase. It probably affects tyrosine aminotransferase activity in a dependent and an independent way, regulated respectively by the glucorticoid status and by the provision of retinoic acid.

Polyamines and ripening of photoreceptor outer segments in chicken embryos.

Polyamines and their related monoacetyl derivatives were studied in rod outer segment (ROS) and cone outer segment (COS) of photoreceptor cells from chick embryo retina during eye development (7th-18th days). Putrescine was found to be necessary, in the second phase of retinogenesis, to sustain both ROS and COS differentiation and, after acetylation, gamma-aminobutyric acid synthesis. On the other hand, spermidine and even more spermine intervene in the third phase of development when photoreceptors mature. Moreover, the presence of N1-acetylspermidine already at the 7th day indicates that in the outer segment of photoreceptor cells too, as in the whole retina, putrescine synthesis comes about by two pathways. One pathway involves ornithine decarboxylase; the other, spermidine/spermine N1-acetyltransferase and FAD-dependent polyamine oxidase activities that convert spermidine to putrescine via N1-acetylspermidine. These different biosynthetic pathways are probably also decisive in permitting gamma-aminobutyric acid synthesis, which is very important in the ripening process of neural retina.

Micellar electrokinetic chromatography of polyamines and monoacetylpolyamines.

A selective procedure for qualitative and quantitative analysis of ten polyamines by micellar electrokinetic chromatography (MEKC) was developed. Benzoylated polyamines and acetylpolyamines in micellar phase of SDS (10 mM) were separated at 25 degrees C by 20 mM borate buffer pH 8.5, containing 8% ethanol, with an applied voltage of 25 kV (5 microA) and then detected at 198 nm. The experimental factors and operational parameters were optimized by performing analysis at different surfactant concentrations, pH, voltage and temperature with and without ethanol. The repeatibility of migration times and peak heights is a peculiarity of the method here described.

Development and validation of a fast and sensitive chromatographic assay for all-trans-retinol and tocopherols in human serum and plasma using liquid-liquid extraction.

A sensitive HPLC assay for all-trans-retinol, alpha-tocopherol, and gamma-tocopherols in human serum and plasma is reported. Sample preparation is performed in one step and involves precipitation of proteins and extraction of lipids with two volumes of an ethanol-chloroform mixture (3:1, v/v) without I.S. addition. After removal of the precipitated protein, 20 microl aliquots of the supernatant (equivalent to 6.7 microl of serum or plasma) were injected into the HPLC system and analyzed using fluorometric detection. RP-HPLC was performed using a C(18) S3 ODS2 column with a methanol-water step gradient (97:3 to 100) at 1.0 ml/min. The quantification limit expressed as nanograms of analyte per milliliter of serum or plasma was approximately 30 ng for all-trans-retinol, 300 ng for alpha-tocopherol and 250 ng for gamma- and delta-tocopherol. The method was validated and applied to human serum and plasma from a total of 120 subjects. This procedure requires a small volume of serum or plasma and can therefore be a valuable tool for measuring low concentrations of these vitamins in preterm infants with sensitivity, precision and accuracy.

Effects of doxorubicin on mouse heart catalase.

The behaviour of heart and liver catalase was studied 4 days after the administration of different doxorubicin doses to CD 1 mice. The antiblastic increased the specific activity of the heart enzyme with a clear dose-response relationship (+27% after 5 mg/kg i.p.; +61% after 7 mg/kg; + 108% after 10 mg/kg; + 147% after 15 mg/kg). This did not occur in the liver where, on the contrary, a significant reduction of catalase (-31%) was noticed after the highest dose. Analyses by gel filtration excluded the possibility that doxorubicin induces major changes in the molecular properties of heart catalase. In vivo experiments with aminotriazole, which blocks catalase irreversibly, indicated that doxorubicin stimulates the synthesis of cardiac catalase. These findings are discussed with reference to the possible mechanisms of anthracycline cardiotoxicity. The catalase elevation could represent a reaction by the heart to free radicals generated by doxorubicin.

[The newborn infant of the diabetic mother: the clinical findings in 431 subjects]. / Il neonato di madre diabetica: rilievi clinici su 431 soggetti.

431 newborns of diabetic mothers (NDM) were studied between the years 1980 and 1990. They were divided into two groups: a first group of 227 patients born from mother with gestational diabetes mellitus; a second group of 204 patients born from mother with pregestational diabetes mellitus. The first and the second group were subdivided into two subgroups: the A, referring to the period 1980-85, and the B, referring to the period 1986-90. Subgroups B were characterized by a better metabolic control of pregnancies. Our protocol consisted of anamnestic study, clinical, metabolic, instrumental examinations and clinical follow-up for a period ranging from 1 to 10 years. The study of our data suggests that embryo-feto-neonatal mortality doesn't show significantly difference between the first and the second group and between subgroups A and B. The percentage of preterm infants was significantly more elevated in the second group, the percentage of full-term newborns was significantly higher in the first group. The incidence of cesarean sections was significantly higher in subgroups B. Mean birthweight was more elevated in the first group and the incidence of macrosomia was higher in subgroups A. The incidence of asphyxia, hypotonia, seizures, transient cardiomegaly, transient hypoglycaemia and hypocalcemia, was higher in subgroups A, without significant difference between the first and the second group. Congenital anomalies were heterogeneous and there were no significant difference between the first and the second group. Only the incidence of congenital cardiopathies was significantly more elevated in the second group. At follow-up neurologic sequelae were mainly characterized by behavioural anomalies.

Retinol dehydrogenase from bovine retinal rod outer segments. Kinetic mechanism of the solubilized enzyme.

Retinol dehydrogenase solubilized by Lubrol 12A9 from bovine retinal rod outer segments forms mixed micelles of Stokes radius 8.5 nm. The kinetic properties of the solubilized retinol dehydrogenase were examined and retinaldehyde reduction and retinol oxidation were seen to proceed at pH 8.3 by a sequential Ordered Bi Bi mechanism. This conclusion was supported by bisubstrate initial velocity studies, dead-end and product inhibition. The kinetic mechanism of retinol dehydrogenase is not altered by the effect of Lubrol until a concentration of 2 mM is reached, at which the detergent lowers the values of the Michaelis and dissociation constants. The catalytic rate of the retinol dehydrogenase is significantly lowered by detergent in the range of pH 3 to 9.

Retinol and retinyl esters in pigment epithelium of rats with inherited retinal degeneration.

A comparative study of the retinol and retinyl ester concentrations was performed in the retinal pigment epithelium of the normal and affected rats. Our findings indicate that in dystrophic rat retinol content increases, whereas the amount of retinyl esters is always lower than normal. An hypothesis can be made on the deficiency of enzymic activities which regulate retinol retinol and retinyl esters levels in the pigment epithelium.

Binding of 11-cis retinaldehyde to the partially purified cellular retinaldehyde binding protein from bovine retinal pigment epithelium.

11-cis retinaldehyde binding analysis was performed on a bovine retinal pigment epithelium preparation of cellular retinaldehyde binding protein (CRALBP), whose purity degree was estimated as 75%. Equilibrium binding studies were carried out measuring the replacement of tritium-labeled with unlabeled 11-cis retinaldehyde at 25 degrees C. Analysis of the experimental data both by a direct curve-fitting procedure utilizing a non linear least square regression analysis and by a conventional Scatchard plot revealed a single non-interacting binding site with an apparent equilibrium constant of 0.9 X 10(-7) M. A binding stoichiometry of approximately 1 mol of 11-cis retinaldehyde/mol of binding protein can be calculated from the experimental data. Competition studies carried out in the presence of unlabeled 'trans' and 'cis' isomers of vitamin A derivatives confirm the high degree of specificity of the 11-cis retinaldehyde binding.

Isolation procedure for bovine retinal rod outer segments.

An isolation procedure to obtain rod outer segments from cattle retinas is reported. Centrifugation of homogenates in discontinuous and continuous sucrose density gradients yields purified photoreceptor cell outer segments. Assay of the final preparation for rhodopsin content gives a ratio of 2.4 for DO280nm/DO498nm.

Xanthine oxidase catalyzes the synthesis of retinoic acid.

Milk xanthine oxidase (xanthine: oxygen oxidoreductase; XO; EC 1.1.3.22) was found to catalyze the conversion of retinaldehyde to retinoic acid. The ability of XO to synthesize all trans-retinoic acid efficiently was assessed by its turnover number of 31.56 min-1, determined at pH 7.0 with 1 nM XO and all trans-retinaldehyde varying between 0.05 to 2 microM. The determination of both retinoid and purine content in milk was also considered in order to correlate their concentrations with kinetic parameters of retinaldehyde oxidase activity. The velocity of the reaction was dependent on the isomeric form of the substrate, the all trans- and 9-cis-forms being the preferred substrates rather than 13-cis-retinaldehyde. The enzyme was able to oxidize retinaldehyde in the presence of oxygen with NAD or without NAD addition. In this latter condition the catalytic efficiency of the enzyme was higher. The synthesis of retinoic acid was inhibited 87% and 54% by 4 microM and 2 microM allopurinol respectively and inhibited 48% by 10 microM xanthine in enzyme assays performed at 2 microM all trans-retinaldehyde. The Ki value determined for xanthine as an inhibitor of retinaldehyde oxidase activity was 4 microM.

Rapid and simultaneous high-performance liquid chromatography assay of polyamines and monoacetylpolyamines in biological specimens.

A rapid, resolutive and reproducible reversed-phase high-performance liquid chromatography (RP-HPLC) method was developed for polyamines and acetylpolyamines by adopting pre-column derivatization with benzoyl chloride. In a single run lasting less than 15 min ten polyamines were separated as well as traces of benzoic acid, methylbenzoate and benzoic anhydride. These contaminants, produced during the derivatization reaction, were almost all eliminated by washing steps envisaged in the same procedure. This simple and sensitive method can be applied to routine determination of polyamines in biological samples. A fine application of this procedure to the determination of endogenous content of polyamines in chick embryo retina was reported.

Retinoid dynamics in chicken eye during pre- and postnatal development.

Changes in the steady state level of retinols, retinaldehydes and retinyl esters in the trans and 11-cis forms and trans retinoic acid were measured in whole chicken eye during development from day 6 in ovo to day 3 post-hatch. These retinoids, quantified by different HPLC systems, were detected in this time sequence: trans-retinol and trans-retinyl esters in the first week in ovo, 11-cis-retinol in the second week. The highest level of 11-cis-retinaldehyde and 11-cis-retinyl esters was reached at the end of development in ovo; however, their levels increased further after hatching. The retinoic acid level decreased at the end of the first week, rising again at the end of the second week. The enzyme activities involved in the metabolism of these retinoids-acyl-CoA: retinol acyltransferase, trans-retinol dehydrogenase, 11-cis-retinol dehydrogenase, trans-retinyl ester hydrolase and trans: 11-cis-retinol isomerase were also estimated and they were detectable already in the first week of development in ovo. At day 6 of the biosynthesis of retinoic acid by the retinaldehyde dehydrogenase activity from retina cytosol was also shown.

Cardiac peroxisomal enzymes and starvation.

In mice subjected to 3-day periods of food deprivation an increase in plasma free fatty acids occurred together with a rise in the cardiac content of fatty acyl CoA-oxidase (+ 15.2%) and catalase (+ 136.2%) activities. Stimulation of hydrogen peroxide production by the heart was found after 30 hours of fasting and this phenomenon was almost completely eliminated by 6 hours of refeeding. These data suggest that high myocardial loads of free fatty acids involve the peroxisomal enzymes in the beta-oxidation process. The resulting increase in hydrogen peroxide production could be partly responsible for the myocardial injury caused by starvation.

Analysis of a soluble lipid-protein complex carrying endogenous 11-cis retinaldehyde from bovine retinal pigment epithelium.

A soluble lipid-protein complex in bovine retinal pigment epithelium is shown to carry endogenous 11-cis retinaldehyde, in the extent of 15% of the total 11-cis retinaldehyde found in this tissue. The complex, analyzed with respect to its chemical composition, exhibits a lipid composition close resembling the lipid composition of the rod outer segment membrane; the SDS-PAGE evidences the presence of a number of protein bands, two of which of 34 and 27 kDa appear glycoproteins. Finally, the lipid-protein complex exhibits a discrete level of a Cathepsin D-like protease activity. From the above, the possibility is discussed that the soluble lipid-protein complex could represent some phagolysosomal inclusion occurring in the pigment epithelial cells upon rod outer segment phagocytosis.

Retinyl ester hydrolases in retinal pigment epithelium.

In bovine retinal pigment epithelium membranes we have found three hydrolases which were active against trans-retinyl palmitate. This was possible by assaying different subcellular fractions as a function of pH in the range 3-9. Detection of these activities has been favored by the use in the enzyme assay of Triton X-100, which has an activating effect up to a concentration of 0.03% at a detergent-protein ratio of about 1.5-3.0. Apparent kinetic parameters for the retinyl ester hydrolases have been determined after a study of the optimization of assay conditions. Vmax values for hydrolases acting at pH 4.5, 6.0, and 7.0 were, respectively, 156, 55, and 70 nmol/h/mg. To identify the subcellular site for these hydrolytic activities, assays of marker enzymes from various organelles in each subcellular preparation were carried out, demonstrating the lysosomal origin of the pH 4.5 retinyl ester hydrolase and the microsomal origin of the pH 6.0 retinyl ester hydrolase and suggesting that the pH 7.0 retinyl ester hydrolase originates from the Golgi complex.