{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2026,2,25]],"date-time":"2026-02-25T07:45:36Z","timestamp":1772005536667,"version":"3.50.1"},"reference-count":32,"publisher":"MDPI AG","issue":"1","license":[{"start":{"date-parts":[[2026,1,2]],"date-time":"2026-01-02T00:00:00Z","timestamp":1767312000000},"content-version":"vor","delay-in-days":0,"URL":"https:\/\/creativecommons.org\/licenses\/by\/4.0\/"}],"funder":[{"name":"National Cancer Institute","award":["R01 CA276301"],"award-info":[{"award-number":["R01 CA276301"]}]},{"name":"Pelatonia","award":["IRP CC13702"],"award-info":[{"award-number":["IRP CC13702"]}]},{"name":"The Ohio State University Department of Pathology and Comprehensive Cancer Center"}],"content-domain":{"domain":[],"crossmark-restriction":false},"short-container-title":["Genes"],"abstract":"<jats:p>Background: Genetic mutations have proven to be the epicenters of cancer and disease progression. Traditional WXS sequencing and BLOSUM scoring can be used to infer the evolutionary conservation of amino acid substitutions, though these approaches are not informed by probable base pair sequence changes. Within gene mutation analysis, most tools focus on amino acid conservation or codon switching independently, limiting their ability to contextualize observed mutations against stochastic mutational processes. In the clinical setting, variants of unspecified significance remain difficult to interpret, as clinicians are often unable to determine whether observed mutations arise from oncogenic selection or from stochastic mutational degradation. Methods: We analyzed mutation sequences from the TCGA BRCA cohort for TP53 and PIK3CA and developed a model that integrates BLOSUM scoring with statistical modeling of base pair changes to evaluate deviation from codon-aware neutral expectations. Observed mutational distributions were compared against a stochastic neutral model to assess statistical significance. Results: Within the TCGA BRCA cohort, TP53 mutations were significantly more evolutionarily radical than expected under the codon-aware neutral model, while PIK3CA mutations were significantly more evolutionarily conservative, as determined using chi-square testing. These opposing patterns are consistent with the distinct functional roles of TP53 and PIK3CA in oncogenesis, where TP53 is inhibited through disruptive loss-of-function mutations, whereas PIK3CA is recurrently mutated in a manner that preserves protein structure and promotes constitutive pathway activation. This contrast reflects selective pressure toward disabling tumor suppressor function while maintaining persistent oncogenic signaling. Conclusions: Codon-aware neutral modeling provides a statistical framework for distinguishing mutations that deviate from stochastic expectations and may aid in the interpretation of variants of unspecified significance. By contextualizing mutational severity relative to neutral processes, this approach offers insight into tumor evolution and may support prognostic assessment without relying on predefined gene-level neutrality.<\/jats:p>","DOI":"10.3390\/genes17010055","type":"journal-article","created":{"date-parts":[[2026,1,2]],"date-time":"2026-01-02T12:23:44Z","timestamp":1767356624000},"page":"55","update-policy":"https:\/\/doi.org\/10.3390\/mdpi_crossmark_policy","source":"Crossref","is-referenced-by-count":1,"title":["GenBlosum: On Determining Whether Cancer Mutations Are Functional or Random"],"prefix":"10.3390","volume":"17","author":[{"ORCID":"https:\/\/orcid.org\/0009-0002-0731-9383","authenticated-orcid":false,"given":"Alejandro","family":"Leyva","sequence":"first","affiliation":[{"name":"Department of Biomedical Engineering, The Ohio State University, 2255 Kenny Rd., Colombus, OH 43210, USA"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-1278-7512","authenticated-orcid":false,"given":"Muhammad Khalid","family":"Khan Niazi","sequence":"additional","affiliation":[{"name":"Department of Pathology, The Ohio State University, 2255 Kenny Rd., Colombus, OH 43210, USA"}],"role":[{"role":"author","vocabulary":"crossref"}]}],"member":"1968","published-online":{"date-parts":[[2026,1,2]]},"reference":[{"key":"ref_1","doi-asserted-by":"crossref","first-page":"333","DOI":"10.1038\/nature12634","article-title":"Mutational Landscape Across 12 Major Cancer Types","volume":"502","author":"Kandoth","year":"2013","journal-title":"Nature"},{"key":"ref_2","doi-asserted-by":"crossref","first-page":"8368","DOI":"10.1093\/nar\/gkt584","article-title":"Structure of P53 Binding to the BAX Response Element Reveals DNA Unwinding and Compression to Accommodate Base-Pair Insertion","volume":"41","author":"Chen","year":"2013","journal-title":"Nucleic Acids Res."},{"key":"ref_3","doi-asserted-by":"crossref","first-page":"741","DOI":"10.1016\/j.molcel.2006.05.015","article-title":"Structural Basis of DNA Recognition by p53 Tetramers","volume":"22","author":"Kitayner","year":"2006","journal-title":"Mol. Cell."},{"key":"ref_4","doi-asserted-by":"crossref","first-page":"3210","DOI":"10.1080\/15384101.2016.1241917","article-title":"P53 Oligomerization Status Modulates Cell Fate Decisions between Growth, Arrest and Apoptosis","volume":"15","author":"Fischer","year":"2016","journal-title":"Cell Cycle"},{"key":"ref_5","doi-asserted-by":"crossref","first-page":"1099","DOI":"10.1158\/2159-8290.CD-24-1638","article-title":"Mutant P53 Gain of Function: Why Many See It, Why Some Do Not","volume":"15","author":"Lozano","year":"2025","journal-title":"Cancer Discov."},{"key":"ref_6","doi-asserted-by":"crossref","first-page":"11","DOI":"10.1146\/annurev-med-062913-051343","article-title":"The PI3K\/AKT Pathway as a Target for Cancer Treatment","volume":"67","author":"Mayer","year":"2016","journal-title":"Annu. Rev. Med."},{"key":"ref_7","doi-asserted-by":"crossref","first-page":"455","DOI":"10.1038\/sj.bjc.6602970","article-title":"Mutation of the PIK3CA Oncogene in Human Cancers","volume":"94","author":"Karakas","year":"2006","journal-title":"Br. J. Cancer"},{"key":"ref_8","doi-asserted-by":"crossref","first-page":"554","DOI":"10.1126\/science.1096502","article-title":"High Frequency of Mutations of the PIK3CA Gene in Human Cancers","volume":"304","author":"Samuels","year":"2004","journal-title":"Science"},{"key":"ref_9","doi-asserted-by":"crossref","first-page":"856","DOI":"10.1016\/j.molmed.2018.08.003","article-title":"Cancer-Associated PIK3CA Mutations in Overgrowth Disorders","volume":"24","author":"Madsen","year":"2018","journal-title":"Trends Mol. Med."},{"key":"ref_10","doi-asserted-by":"crossref","first-page":"169","DOI":"10.1016\/j.dnarep.2014.03.014","article-title":"DNA Double-Strand Break Repair Pathway Choice and Cancer","volume":"19","author":"Aparicio","year":"2014","journal-title":"DNA Repair"},{"key":"ref_11","doi-asserted-by":"crossref","first-page":"1029","DOI":"10.1016\/j.cell.2017.09.042","article-title":"Universal Patterns of Selection in Cancer and Somatic Tissues","volume":"171","author":"Martincorena","year":"2017","journal-title":"Cell"},{"key":"ref_12","doi-asserted-by":"crossref","first-page":"1642","DOI":"10.1158\/2159-8290.CD-22-0145","article-title":"Somatic Mutation: What Shapes the Mutational Landscape of Normal Epithelia?","volume":"12","author":"Fowler","year":"2022","journal-title":"Cancer Discov."},{"key":"ref_13","doi-asserted-by":"crossref","first-page":"35","DOI":"10.1186\/s13073-019-0648-4","article-title":"Somatic Mutation and Clonal Expansion in Human Tissues","volume":"11","author":"Martincorena","year":"2019","journal-title":"Genome Med."},{"key":"ref_14","doi-asserted-by":"crossref","first-page":"415","DOI":"10.1038\/nature12477","article-title":"Signatures of Mutational Processes in Human Cancer","volume":"500","author":"Alexandrov","year":"2013","journal-title":"Nature"},{"key":"ref_15","doi-asserted-by":"crossref","first-page":"1902","DOI":"10.1093\/molbev\/msu142","article-title":"The Influence of Selection for Protein Stability on dN\/dS","volume":"31","author":"Angelis","year":"2014","journal-title":"Mol. Biol. Evol."},{"key":"ref_16","doi-asserted-by":"crossref","first-page":"10915","DOI":"10.1073\/pnas.89.22.10915","article-title":"Amino Acid Substitution Matrices from Protein Blocks","volume":"89","author":"Henikoff","year":"1992","journal-title":"Proc. Natl. Acad. Sci. USA"},{"key":"ref_17","doi-asserted-by":"crossref","first-page":"pl1","DOI":"10.1126\/scisignal.2004088","article-title":"Integrative Analysis of Complex Cancer Genomics and Clinical Profiles Using the cBioPortal","volume":"6","author":"Gao","year":"2013","journal-title":"Sci. Signal."},{"key":"ref_18","doi-asserted-by":"crossref","first-page":"248","DOI":"10.1038\/nmeth0410-248","article-title":"A method and server for predicting damaging missense mutations","volume":"7","author":"Adzhubei","year":"2010","journal-title":"Nat. Methods"},{"key":"ref_19","doi-asserted-by":"crossref","first-page":"533","DOI":"10.1089\/gtmb.2010.0036","article-title":"Using SIFT and PolyPhen to Predict Loss-of-Function and Gain-of-Function Mutations","volume":"14","author":"Flanagan","year":"2010","journal-title":"Genet. Test. Mol. Biomarkers"},{"key":"ref_20","doi-asserted-by":"crossref","first-page":"661","DOI":"10.1002\/humu.21490","article-title":"Prediction of Missense Mutation Functionality Depends on Both the Algorithm and Sequence Alignment Employed","volume":"32","author":"Hicks","year":"2011","journal-title":"Hum. Mutat."},{"key":"ref_21","doi-asserted-by":"crossref","first-page":"3812","DOI":"10.1093\/nar\/gkg509","article-title":"SIFT: Predicting Amino Acid Changes that Affect Protein Function","volume":"31","author":"Ng","year":"2003","journal-title":"Nucleic Acids Res."},{"key":"ref_22","doi-asserted-by":"crossref","first-page":"704","DOI":"10.1158\/2159-8290.CD-15-0344","article-title":"APOBEC Enzymes: Mutagenic Fuel for Cancer Evolution and Heterogeneity","volume":"5","author":"Swanton","year":"2015","journal-title":"Cancer Discov."},{"key":"ref_23","doi-asserted-by":"crossref","first-page":"994","DOI":"10.7326\/M21-4109","article-title":"The Challenge of Genetic Variants of Uncertain Clinical Significance","volume":"175","author":"Burke","year":"2022","journal-title":"Ann. Intern. Med."},{"key":"ref_24","doi-asserted-by":"crossref","first-page":"405","DOI":"10.1038\/gim.2015.30","article-title":"Standards and Guidelines for the Interpretation of Sequence Variants","volume":"17","author":"Richards","year":"2015","journal-title":"Genet. Med."},{"key":"ref_25","first-page":"725","article-title":"A Codon-Based Model of Nucleotide Substitution for Protein-Coding DNA Sequences","volume":"11","author":"Goldman","year":"1994","journal-title":"Mol. Biol. Evol."},{"key":"ref_26","doi-asserted-by":"crossref","first-page":"3258","DOI":"10.1128\/MCB.00471-15","article-title":"P110\u03b1 Hot Spot Mutations E545K and H1047R Exert Metabolic Reprogramming Independently of P110\u03b1 Kinase Activity","volume":"35","author":"Chaudhari","year":"2015","journal-title":"Mol. Cell. Biol."},{"key":"ref_27","doi-asserted-by":"crossref","first-page":"5569","DOI":"10.1073\/pnas.0701005104","article-title":"Rare Cancer-Specific Mutations in PIK3CA Show Gain-of-Function Phenotypes","volume":"104","author":"Gymnopoulos","year":"2007","journal-title":"Proc. Natl. Acad. Sci. USA"},{"key":"ref_28","doi-asserted-by":"crossref","first-page":"2652","DOI":"10.1073\/pnas.0712169105","article-title":"Helical Domain and Kinase Domain Mutations in P110\u03b1 of Phosphatidylinositol 3-Kinase Induce Gain of Function by Different Mechanisms","volume":"105","author":"Zhao","year":"2008","journal-title":"Proc. Natl. Acad. Sci. USA"},{"key":"ref_29","doi-asserted-by":"crossref","first-page":"21","DOI":"10.1007\/82_2010_68","article-title":"Oncogenic Mutations of PIK3CA in Human Cancers","volume":"Volume 347","author":"Samuels","year":"2010","journal-title":"Phosphoinositide 3-kinase in Health and Disease"},{"key":"ref_30","doi-asserted-by":"crossref","first-page":"1","DOI":"10.1155\/2020\/3759179","article-title":"A Systematic Review of the Prevalence and Diagnostic Workup of PIK3CA Mutations in HR+\/HER2\u2212 Metastatic Breast Cancer","volume":"2020","author":"Anderson","year":"2020","journal-title":"Int. J. Breast Cancer"},{"key":"ref_31","doi-asserted-by":"crossref","first-page":"17082","DOI":"10.1038\/s41598-020-74086-w","article-title":"PIK3CA Mutation Enrichment and Quantitation from Blood and Tissue","volume":"10","author":"Keraite","year":"2020","journal-title":"Sci. Rep."},{"key":"ref_32","doi-asserted-by":"crossref","first-page":"483","DOI":"10.1007\/s10549-022-06637-w","article-title":"PIK3CA-Mutations in Breast Cancer","volume":"196","author":"Reinhardt","year":"2022","journal-title":"Breast Cancer Res. Treat."}],"container-title":["Genes"],"original-title":[],"language":"en","link":[{"URL":"https:\/\/www.mdpi.com\/2073-4425\/17\/1\/55\/pdf","content-type":"unspecified","content-version":"vor","intended-application":"similarity-checking"}],"deposited":{"date-parts":[[2026,1,4]],"date-time":"2026-01-04T05:16:55Z","timestamp":1767503815000},"score":1,"resource":{"primary":{"URL":"https:\/\/www.mdpi.com\/2073-4425\/17\/1\/55"}},"subtitle":[],"short-title":[],"issued":{"date-parts":[[2026,1,2]]},"references-count":32,"journal-issue":{"issue":"1","published-online":{"date-parts":[[2026,1]]}},"alternative-id":["genes17010055"],"URL":"https:\/\/doi.org\/10.3390\/genes17010055","relation":{},"ISSN":["2073-4425"],"issn-type":[{"value":"2073-4425","type":"electronic"}],"subject":[],"published":{"date-parts":[[2026,1,2]]}}}