Abstract B21: Protein quantitation assays for Akt, PI3K p110α, and PTEN to assess PI3K pathway activity in tumor tissue
Notice bibliographique
Résumé
Abstract Introduction: Many clinical trials for inhibitors targeting the PI3K pathway now include genetic screening of tumors to try to select probable treatment responders. Genetic testing identifies some relevant mutations, but this approach does not address many mechanisms of PI3K pathway regulation. Epigenetic changes and post-translational modifications may increase pathway activity without any detectable DNA mutation. Where mutations are detected, mutated proteins may not be expressed or may be quickly degraded. Direct measurement of oncogenic proteins may therefore offer more useful molecular profiles for characterizing cancer-driving alterations and matching patients with appropriate treatments. Purpose: The purpose of this study was to develop and implement protein quantitation assays to accurately assess PI3K pathway activity in tumors, as a means of matching patients with targeted cancer treatments. Methods: Protein extracted from tumor tissue lysate is subjected to proteolytic digestion to generate proteotypic peptides used for protein quantitation. Synthetic stable isotope-labeled peptides are added to the digest as an internal standard, after which the target peptides are immuno-enriched with antibodies coupled to magnetic beads. MS analysis is then performed by either eluting peptides for measurement with liquid chromatography multiple reaction monitoring tandem MS (LC-MRM-MS/MS) or by spotting beads onto a plate for matrix-assisted laser desorption MS (MALDI-MS). External calibration curves are used for accurate quantitation. Results: We developed and implemented MS-based methods to determine the concentration of PI3K pathway proteins in tumor samples. The developed methods enable precise and reproducible quantitation of Akt1, Akt2, PI3K P110α, and PTEN. We were able to distinguish and quantify specific isoforms, including phospho-Akt1 (Ser473), phospho-Akt2 (Ser474), and mutant-Akt (E17K). Endogenous protein levels were quantified in cell lines, fresh-frozen tumor samples, and formalin-fixed, paraffin embedded tumor tissue. 10 μg of protein extract per analyte was sufficient for quantitation. Subsets of assays could be multiplexed through simultaneous or sequential enrichment without compromising assay performance. Novel Aspect: The multiplexing of immuno-MALDI assays through sequential enrichment and the immuno-MRM-MS assays for PTEN and mutant Akt represent new developments. Recent adoption of monoclonal antibodies for Akt1 will facilitate this assay's translation to a clinical setting. Conclusions: This panel of quantitative proteomics assays enables comprehensive PI3K pathway assessment in tumor tissues. Previously collected genetically screened tumor samples were obtained with patient consent (as approved by the Jewish General Hospital Research Ethics Committee). Samples will be analyzed to assess PI3K pathway activity and determine its correlation to clinical features and response to PI3K-targeted treatments. Note: This abstract was not presented at the conference. Citation Format: Constance A. Sobsey, Robert Popp, Sahar Ibrahim, Bjoern C Froehlich, Adriana Aguilar-Mahecha, Mark Basik, Gerald Batist, Christoph Borchers. Protein quantitation assays for Akt, PI3K p110α, and PTEN to assess PI3K pathway activity in tumor tissue [abstract]. In: Proceedings of the AACR Special Conference on Targeting PI3K/mTOR Signaling; 2018 Nov 30-Dec 8; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Res 2020;18(10_Suppl):Abstract nr B21.
Récupéré en direct depuis OpenAlex et désinversé. Les résumés ne sont pas conservés dans cette base de données : les index inversés représentent 8,6 Go des 9,3 Go de texte de la base, et le serveur dispose de 13 Go libres.
Comment cette classification a été obtenuedéplier
Prédiction distillée sur la base complète
Imitation des enseignantsNi prévalence calibrée, ni vérité terrain. Validation humaine à venir. Apprise à partir de 10 348 étiquettes directes de Codex et de 10 348 étiquettes directes de Gemma. Le mode candidate est l'union des têtes enseignantes seuillées; le consensus est leur intersection. Ces sorties portent le statut machine_predicted_unvalidated et ne sont ni des étiquettes humaines ni des étiquettes directes de modèles de pointe.
Scores Codex et Gemma par catégorie
| Catégorie | Codex | Gemma |
|---|---|---|
| Métarecherche | 0,001 | 0,001 |
| Méta-épidémiologie (sens strict) | 0,000 | 0,000 |
| Méta-épidémiologie (sens large) | 0,000 | 0,000 |
| Bibliométrie | 0,000 | 0,000 |
| Études des sciences et des technologies | 0,000 | 0,000 |
| Communication savante | 0,000 | 0,000 |
| Science ouverte | 0,000 | 0,000 |
| Intégrité de la recherche | 0,000 | 0,000 |
| Charge utile insuffisante (le modèle a refusé de juger) | 0,000 | 0,000 |
Scores machine (provisoires)
Les deux têtes enseignantes du modèle étudiant, lues sur ce travail. Un score ordonne la base pour la relecture; il n'affirme jamais une catégorie, et le statut de validation accompagne chaque rangée tel quel.
Scores de référence d'un modèle non mature (critères de maturité non atteints, 7 itérations). Un score ordonne; il n'affirme jamais une catégorie.
score_only:v0-immature-baseline · tel quel depuis la passe de notation : score_only signifie que le nombre peut ordonner les travaux, et qu'aucune étiquette de catégorie n'en découleClassification
machine, non validéePrédiction automatique; un appel candidat d’une seule tête enseignante, pas un consensus.
Le détail, modèle par modèle et score par score, se trouve en fin de page sous « Comment cette classification a été obtenue ».