{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2026,7,30]],"date-time":"2026-07-30T14:13:36Z","timestamp":1785420816590,"version":"3.56.0"},"reference-count":35,"publisher":"MDPI AG","issue":"12","license":[{"start":{"date-parts":[[2026,6,11]],"date-time":"2026-06-11T00:00:00Z","timestamp":1781136000000},"content-version":"vor","delay-in-days":0,"URL":"https:\/\/creativecommons.org\/licenses\/by\/4.0\/"}],"funder":[{"name":"University of South Alabama, Pathology department","award":["#112000-730910-4100"],"award-info":[{"award-number":["#112000-730910-4100"]}]},{"award":["#112000-730910-4100"],"award-info":[{"award-number":["#112000-730910-4100"]}],"id":[{"id":"https:\/\/ror.org\/01s7b5y08","id-type":"ROR","asserted-by":"publisher"}]},{"name":"NIH NCI","award":["5RO1 CA294584"],"award-info":[{"award-number":["5RO1 CA294584"]}]}],"content-domain":{"domain":[],"crossmark-restriction":false},"short-container-title":["Diagnostics"],"abstract":"<jats:p>Background\/Objectives: Colorectal cancer (CRC) is relatively uncommon in individuals under 40 years of age; however, its rising incidence is a growing concern. This study aimed to investigate clinicopathologic features, genetic alterations, and protein expression patterns in early-onset colorectal cancer (EOCRC) to better understand the underlying mechanisms and prognostic factors. Methods: We retrospectively analyzed 18 patients diagnosed with EOCRC (&lt;40 years) at our institution between 2018 and 2023. Next-generation sequencing (NGS) and immunohistochemistry (IHC) were used to assess genomic alterations and protein expression profiles. Clinicopathologic data were correlated with molecular findings and outcomes. Results: The cohort included ten females and eight males (mean age, 32.7 years; range, 17\u201338 years). Tumors most frequently arose in the rectum (56%) and were predominantly high stage (T3\u2013T4, 67%) and moderately differentiated (78%). Lymphovascular invasion occurred in 50% of cases, and lymph node metastasis in 39%. Most tumors were microsatellite stable (MSS, 89%) and mismatch repair\u2013proficient; two cases (11%) were MSI-high with germline MMR mutations. Among 17 patients who underwent NGS, the most frequent mutations involved KRAS (35%), APC (24%), TP53 (18%), and SMAD4 (18%). Notably, SMAD4 protein downregulation by IHC was observed in 67% of cases, including 60% of SMAD4 wild-type tumors. Loss of SMAD4 expression was significantly associated with lymph node metastasis (p = 0.037) and poor survival (p = 0.045). Conclusions: SMAD4 alteration\u2014on both the genetic and protein levels\u2014is common in EOCRC and is significantly correlated with aggressive clinicopathologic features and worse prognosis.<\/jats:p>","DOI":"10.3390\/diagnostics16121804","type":"journal-article","created":{"date-parts":[[2026,6,11]],"date-time":"2026-06-11T12:14:58Z","timestamp":1781180098000},"page":"1804","update-policy":"https:\/\/doi.org\/10.3390\/mdpi_crossmark_policy","source":"Crossref","is-referenced-by-count":0,"title":["SMAD4 Protein Alterations in Early-Onset Colorectal Cancer: Implications as a Potential Marker for Aggressive Disease and Prognosis\u2014A Clinicopathological and Molecular Analysis of 18 Cases in Patients &lt; 40 Years of Age"],"prefix":"10.3390","volume":"16","author":[{"given":"Lingling","family":"Xian","sequence":"first","affiliation":[{"name":"Department of Pathology, University of South Alabama, Mobile, AL 36617, USA"},{"name":"Department of Pathology, Yale University School of Medicine, New Haven, CT 06520, USA"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Jim","family":"Lu","sequence":"additional","affiliation":[{"name":"GoPath Diagnostics, Buffalo Grove, IL 60089, USA"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Lan","family":"Zhou","sequence":"additional","affiliation":[{"name":"Weill Cornell Medical College, Cornell University, Huston, TX 14850, USA"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0003-0987-0443","authenticated-orcid":false,"given":"Wei","family":"Xin","sequence":"additional","affiliation":[{"name":"Department of Pathology, University of South Alabama, Mobile, AL 36617, USA"}],"role":[{"vocabulary":"crossref","role":"author"}]}],"member":"1968","published-online":{"date-parts":[[2026,6,11]]},"reference":[{"key":"ref_1","first-page":"233","article-title":"Colorectal cancer statistics","volume":"73","author":"Siegel","year":"2023","journal-title":"CA Cancer J. Clin."},{"key":"ref_2","doi-asserted-by":"crossref","first-page":"1547","DOI":"10.1056\/NEJMra2200869","article-title":"Increasing incidence of early-onset colorectal cancer","volume":"386","author":"Sinicrope","year":"2022","journal-title":"N. Engl. J. Med."},{"key":"ref_3","first-page":"747","article-title":"Treatment patterns and survival differ between early-onset and late-onset colorectal cancer patients: The patient outcomes to advance learning network","volume":"30","author":"Powers","year":"2019","journal-title":"Cancer Causes Control"},{"key":"ref_4","doi-asserted-by":"crossref","first-page":"728","DOI":"10.1016\/j.cgh.2016.10.038","article-title":"Advanced-stage colorectal cancer in persons younger than 50 years not associated with longer duration of symptoms or time to diagnosis","volume":"15","author":"Chen","year":"2017","journal-title":"Clin. Gastroenterol. Hepatol."},{"key":"ref_5","doi-asserted-by":"crossref","first-page":"230","DOI":"10.1038\/s41571-020-00445-1","article-title":"Rising incidence of early-onset colorectal cancer: A call to action","volume":"18","author":"Akimoto","year":"2021","journal-title":"Nat. Rev. Clin. Oncol."},{"key":"ref_6","doi-asserted-by":"crossref","unstructured":"Xu, T., Zhang, Y., Zhang, J., Qi, C., Liu, D., Wang, Z., Li, Y., Ji, C., Li, J., and Lin, X. (2020). Germline profiling and molecular characterization of early-onset metastatic colorectal cancer. Front. Oncol., 10.","DOI":"10.3389\/fonc.2020.568911"},{"key":"ref_7","doi-asserted-by":"crossref","first-page":"2165","DOI":"10.1136\/gutjnl-2019-320019","article-title":"Organoid cultures of early-onset colorectal cancers reveal distinct and rare genetic profiles","volume":"69","author":"Yan","year":"2020","journal-title":"Gut"},{"key":"ref_8","doi-asserted-by":"crossref","first-page":"100974","DOI":"10.1016\/j.xcrm.2023.100974","article-title":"Integrated multi-omics approach to distinct molecular characterization and classification of early-onset colorectal cancer","volume":"4","author":"Du","year":"2023","journal-title":"Cell Rep. Med."},{"key":"ref_9","doi-asserted-by":"crossref","first-page":"27","DOI":"10.1186\/s13053-023-00267-z","article-title":"SMAD4 variants and genotype\u2013phenotype correlations in juvenile polyposis syndrome","volume":"21","author":"Cao","year":"2023","journal-title":"Hered. Cancer Clin. Pract."},{"key":"ref_10","doi-asserted-by":"crossref","first-page":"2044","DOI":"10.1053\/j.gastro.2010.01.054","article-title":"Hereditary and familial colon cancer","volume":"138","author":"Jasperson","year":"2010","journal-title":"Gastroenterology"},{"key":"ref_11","doi-asserted-by":"crossref","first-page":"965","DOI":"10.1136\/gut.2006.116913","article-title":"Risk of colorectal cancer in juvenile polyposis","volume":"56","author":"Brosens","year":"2007","journal-title":"Gut"},{"key":"ref_12","doi-asserted-by":"crossref","first-page":"3037","DOI":"10.1158\/1078-0432.CCR-15-0939","article-title":"Reduced expression of SMAD4 is associated with poor survival in colon cancer","volume":"22","author":"Yan","year":"2016","journal-title":"Clin. Cancer Res."},{"key":"ref_13","doi-asserted-by":"crossref","first-page":"873","DOI":"10.1111\/his.13805","article-title":"SMAD4 alteration associates with invasive-front pathological markers and poor prognosis in colorectal cancer","volume":"74","author":"Oyanagi","year":"2019","journal-title":"Histopathology"},{"key":"ref_14","doi-asserted-by":"crossref","first-page":"319","DOI":"10.1634\/theoncologist.2018-0117","article-title":"Impact of patient age on molecular alterations of left-sided colorectal tumors","volume":"24","author":"Puccini","year":"2019","journal-title":"Oncologist"},{"key":"ref_15","first-page":"1095","article-title":"Loss of SMAD4 expression predicts liver metastasis in human colorectal cancer","volume":"17","author":"Losi","year":"2007","journal-title":"Oncol. Rep."},{"key":"ref_16","doi-asserted-by":"crossref","first-page":"385","DOI":"10.1053\/j.gastro.2004.04.022","article-title":"Risk factors for an adverse outcome in early invasive colorectal carcinoma","volume":"127","author":"Ueno","year":"2004","journal-title":"Gastroenterology"},{"key":"ref_17","doi-asserted-by":"crossref","first-page":"438","DOI":"10.1007\/DCR.0b013e318197e37f","article-title":"Indications for subsequent surgery after endoscopic resection of submucosally invasive colorectal carcinomas: A prospective cohort study","volume":"52","author":"Choi","year":"2009","journal-title":"Dis. Colon Rectum"},{"key":"ref_18","doi-asserted-by":"crossref","first-page":"211","DOI":"10.1016\/j.jss.2011.11.008","article-title":"Germline mutations in SMAD4 disrupt bone morphogenetic protein signaling","volume":"174","author":"Carr","year":"2012","journal-title":"J. Surg. Res."},{"key":"ref_19","first-page":"113","article-title":"Vessels\u2019 morphology in SMAD4- and BMPR1A-related juvenile polyposis","volume":"138","author":"Condroyer","year":"2005","journal-title":"Am. J. Med. Genet. A"},{"key":"ref_20","doi-asserted-by":"crossref","first-page":"823","DOI":"10.1158\/1541-7786.MCR-20-0721","article-title":"Colorectal cancer\u2013associated SMAD4 R361 hotspot mutations boost Wnt\/\u03b2-catenin signaling through enhanced SMAD4\u2013LEF1 binding","volume":"19","author":"Lanauze","year":"2021","journal-title":"Mol. Cancer Res."},{"key":"ref_21","doi-asserted-by":"crossref","first-page":"725","DOI":"10.1158\/0008-5472.CAN-12-2706","article-title":"SMAD2, SMAD3, and SMAD4 mutations in colorectal cancer","volume":"73","author":"Fleming","year":"2013","journal-title":"Cancer Res."},{"key":"ref_22","doi-asserted-by":"crossref","unstructured":"Mehrvarz Sarshekeh, A., Advani, S., Overman, M.J., Manyam, G., Kee, B.K., Fogelman, D.R., Dasari, A., Raghav, K., Vilar, E., and Manuel, S. (2017). Association of SMAD4 mutation with patient demographics, tumor characteristics, and clinical outcomes in colorectal cancer. PLoS ONE, 12.","DOI":"10.1371\/journal.pone.0173345"},{"key":"ref_23","doi-asserted-by":"crossref","first-page":"454","DOI":"10.1186\/s13059-014-0454-7","article-title":"Comparative sequencing analysis reveals high genomic concordance between matched primary and metastatic colorectal cancer lesions","volume":"15","author":"Brannon","year":"2014","journal-title":"Genome Biol."},{"key":"ref_24","doi-asserted-by":"crossref","first-page":"1493","DOI":"10.1016\/S0969-2126(00)88340-9","article-title":"Crystal structure of a transcriptionally active SMAD4 fragment","volume":"7","author":"Qin","year":"1999","journal-title":"Structure"},{"key":"ref_25","doi-asserted-by":"crossref","first-page":"764","DOI":"10.1056\/NEJMcp1714533","article-title":"Lynch syndrome\u2013associated colorectal cancer","volume":"379","author":"Sinicrope","year":"2018","journal-title":"N. Engl. J. Med."},{"key":"ref_26","doi-asserted-by":"crossref","first-page":"3544","DOI":"10.1200\/JCO.2015.61.4503","article-title":"High prevalence of hereditary cancer syndromes in adolescents and young adults with colorectal cancer","volume":"33","author":"Mork","year":"2015","journal-title":"J. Clin. Oncol."},{"key":"ref_27","doi-asserted-by":"crossref","first-page":"464","DOI":"10.1001\/jamaoncol.2016.5194","article-title":"Prevalence and spectrum of germline cancer susceptibility gene mutations among patients with early-onset colorectal cancer","volume":"3","author":"Pearlman","year":"2017","journal-title":"JAMA Oncol."},{"key":"ref_28","doi-asserted-by":"crossref","first-page":"897","DOI":"10.1053\/j.gastro.2017.11.004","article-title":"Germline genetic features of young individuals with colorectal cancer","volume":"154","author":"Stoffel","year":"2018","journal-title":"Gastroenterology"},{"key":"ref_29","doi-asserted-by":"crossref","first-page":"3208","DOI":"10.1158\/1055-9965.EPI-08-0512","article-title":"Molecular characterization of MSI-H colorectal cancer by MLH1 promoter methylation, immunohistochemistry, and mismatch repair germline mutation screening","volume":"17","author":"Poynter","year":"2008","journal-title":"Cancer Epidemiol. Biomark. Prev."},{"key":"ref_30","doi-asserted-by":"crossref","unstructured":"Liao, X., Hao, Y., Zhang, X., Ward, S., Houldsworth, J., Polydorides, A.D., and Harpaz, N. (2019). Clinicopathological characterization of SMAD4-mutated intestinal adenocarcinomas: A case\u2013control study. PLoS ONE, 14.","DOI":"10.1371\/journal.pone.0212142"},{"key":"ref_31","doi-asserted-by":"crossref","first-page":"3098","DOI":"10.1038\/sj.onc.1202642","article-title":"Higher frequency of SMAD4 gene mutation in human colorectal cancer with distant metastasis","volume":"18","author":"Miyaki","year":"1999","journal-title":"Oncogene"},{"key":"ref_32","doi-asserted-by":"crossref","first-page":"2606","DOI":"10.1158\/1078-0432.CCR-04-1458","article-title":"SMAD4 as a prognostic marker in colorectal cancer","volume":"11","author":"Alazzouzi","year":"2005","journal-title":"Clin. Cancer Res."},{"key":"ref_33","doi-asserted-by":"crossref","first-page":"684","DOI":"10.1016\/j.ejso.2018.02.247","article-title":"SMAD4 gene mutation predicts poor prognosis in patients undergoing resection for colorectal liver metastases","volume":"44","author":"Mizuno","year":"2018","journal-title":"Eur. J. Surg. Oncol."},{"key":"ref_34","doi-asserted-by":"crossref","first-page":"2644","DOI":"10.1158\/1078-0432.CCR-14-2391","article-title":"Hotspot mutation panel testing reveals clonal evolution in a study of 265 paired primary and metastatic tumors","volume":"21","author":"Goswami","year":"2015","journal-title":"Clin. Cancer Res."},{"key":"ref_35","doi-asserted-by":"crossref","first-page":"1683","DOI":"10.1093\/jnci\/djab124","article-title":"A comprehensive comparison of early-onset and average-onset colorectal cancers","volume":"113","author":"Cercek","year":"2021","journal-title":"J. Natl. Cancer Inst."}],"container-title":["Diagnostics"],"original-title":[],"language":"en","link":[{"URL":"https:\/\/www.mdpi.com\/2075-4418\/16\/12\/1804\/pdf","content-type":"unspecified","content-version":"vor","intended-application":"similarity-checking"}],"deposited":{"date-parts":[[2026,6,11]],"date-time":"2026-06-11T12:37:56Z","timestamp":1781181476000},"score":1,"resource":{"primary":{"URL":"https:\/\/www.mdpi.com\/2075-4418\/16\/12\/1804"}},"subtitle":[],"short-title":[],"issued":{"date-parts":[[2026,6,11]]},"references-count":35,"journal-issue":{"issue":"12","published-online":{"date-parts":[[2026,6]]}},"alternative-id":["diagnostics16121804"],"URL":"https:\/\/doi.org\/10.3390\/diagnostics16121804","relation":{},"ISSN":["2075-4418"],"issn-type":[{"value":"2075-4418","type":"electronic"}],"subject":[],"published":{"date-parts":[[2026,6,11]]}}}