Abstract

Background: Bladder cancer is rare in young women, and advanced presentations are exceptionally uncommon. We report a de-identified case of a previously healthy 31-year-old female who developed rapidly progressive stage IV bladder cancer within 12 months of completing a three-dose Moderna mRNA vaccination series (May 2021, June 2021, December 2021). Case Findings: Comprehensive multi-omic profiling was performed using PBIMA (Molecular Surveillance and Individualized Targeted Immunotherapy Peptide Editing) and REViSS (Spike-associated Transcription-al/Translational Instability Surveillance), incorporating analyses of plasma-derived circulating tumor DNA, whole-blood RNA, and urine exosome proteomics. Dysregulated gene expressions were identified across oncogenic driver genes (KRAS, ATM, MAPK1, NRAS, CHD4, PIK3CA, and SF3B1), auxiliary tumor-promoting signals (TOP1, PSIP1, and ERBB2), and broad evidence of genome instability with impaired DNA repair (ATM, MSH2). Within circulating tumor DNA, a host–vector chimeric read mapped to chr19:55,482,637–55,482,674 (GRCh38), in cytoband 19q13.42, positioned ~367 kb downstream of the canonical AAVS1 safe harbor and ~158 kb upstream of ZNF580 at the proximal edge of the zinc-finger (ZNF) gene cluster. This sequence aligned with perfect 20/20 bp identity to a segment (bases 5905–5924) within the Spike open reading frame (ORF) coding region (bases 3674–7480) of the Pfizer BNT162b2 DNA plasmid reference (GenBank accession OR134577.1), despite the patient only receiving Moderna vaccinations. This apparent paradox is best explained by shared Spike ORF sequences within the expression cassette across both vaccine platforms; because Moderna has not deposited its proprietary plasmid sequence in NCBI, BLAST defaults to Pfizer’s published reference as the nearest available match. The integration site was located outside the canonical AAVS1 “safe harbor” and within a gene-dense, recombination-prone regulatory region, raising concern for transcriptional disruption, fusion transcript formation, and oncogenic potential. The probability of a random 20-base sequence perfectly matching a predefined target is approximately 1 in a trillion, making this alignment statistically compelling and highly unlikely to be an incidental artifact. Conclusions: This sentinel case report provides the first documented evidence of genomic integration of mRNA vaccine-derived genetic material in a human subject, documenting a temporal association between COVID-19 mRNA vaccination and aggressive malignancy, reproducible multi-omic evidence of oncogenic signaling, and a non–safe harbor host–vector integration event. While causality cannot be established from a single case, the con-vergence of (i) close temporal proximity to vaccination, (ii) genomic integration of a vaccine plasmid–derived spike gene fragment, and (iii) consistent transcriptomic and proteomic instability across biospecimens represents a highly unusual and biologically plausible pattern. These findings highlight an urgent need for systematic genomic sur-veillance, orthogonal validation with long-read sequencing, and larger cohort studies to rigorously define the im-pact of synthetic mRNA vaccine platforms on genome integrity and cancer risk.

References

1. Bekele, R.; Samant, A.; Hanlon, T.; Mouw, K. Abstract 1709: MAPK pathway alterations are a targetable vulnerability in bladder cancer. Cancer Research 2023, 83, 1709-1709, doi:10.1158/1538-7445.am2023-1709.
2. Bekele, R.T.; Samant, A.S.; Nassar, A.H.; So, J.; Garcia, E.P.; Curran, C.R.; Hwang, J.H.; Mayhew, D.L.; Nag, A.; Thorner, A.R.; et al. RAF1 amplification drives a subset of bladder tumors and confers sensitivity to MAPK-directed therapeutics. Journal of Clinical Investigation 2021, 131, doi:10.1172/jci147849.
3. Cheng, G.; Zhou, Z.; Li, S.; Yang, S.; Wang, Y.; Ye, Z.; Ren, C. Predicting bladder cancer survival with high accuracy: insights from MAPK pathway-related genes. Scientific Reports 2024, 14, doi:10.1038/s41598-024-61302-0.
4. Columbres, R.C.; Chakraborty, A.; Apolo, A.; Banday, R. Abstract 5640: Unlocking the therapeutic potential of SF3B1 inhibitors for bladder cancer treatment. Cancer Research 2024, 84, 5640-5640, doi:10.1158/1538-7445.am2024-5640.
5. Dadhania, V.; Zhang, M.; Zhang, L.; Bondaruk, J.; Majewski, T.; Siefker-Radtke, A.; Guo, C.C.; Dinney, C.; Cogdell, D.E.; Zhang, S.; et al. Meta-Analysis of the Luminal and Basal Subtypes of Bladder Cancer and the Identification of Signature Immunohistochemical Markers for Clinical Use. EBioMedicine 2016, 12, 105-117, doi:10.1016/j.ebiom.2016.08.036.
6. Deng, Z.; Shen, D.; Yu, M.; Zhou, F.; Shan, D.; Fang, Y.; Jin, W.; Qian, K.; Li, S.; Wang, G.; et al. Pectolinarigenin inhibits bladder urothelial carcinoma cell proliferation by regulating DNA damage/autophagy pathways. Cell Death Discovery 2023, 9, doi:10.1038/s41420-023-01508-9.
7. Dueñas, M.; Martínez-Fernández, M.; García-Escudero, R.; Villacampa, F.; Marqués, M.; Saiz-Ladera, C.; Duarte, J.; Martínez, V.; Gómez, M.J.; Martín, M.L.; et al. PIK3CA gene alterations in bladder cancer are frequent and associate with reduced recurrence in non-muscle invasive tumors. Molecular Carcinogenesis 2013, 54, 566-576, doi:10.1002/mc.22125.
8. Hoffmann, M.J.; Schulz, W.A. Alterations of Chromatin Regulators in the Pathogenesis of Urinary Bladder Urothelial Carcinoma. Cancers 2021, 13, 6040, doi:10.3390/cancers13236040.
9. Hou, S.; Nieva, J.; Desai, N. KRAS G12C mutated NSCLC and bladder cancer xenografts treated with sotorasib and adagrasib in combination with mTOR inhibitors show improved antitumor activity of nab-sirolimus vs everolimus. European Journal of Cancer 2022, 174, S58-S59, doi:10.1016/S0959-8049(22)00957-1.
10. Kompier, L.C.; Lurkin, I.; van der Aa, M.N.M.; van Rhijn, B.W.G.; van der Kwast, T.H.; Zwarthoff, E.C. FGFR3, HRAS, KRAS, NRAS and PIK3CA Mutations in Bladder Cancer and Their Potential as Biomarkers for Surveillance and Therapy. PLoS ONE 2010, 5, e13821, doi:10.1371/journal.pone.0013821.
11. Lee, K.-H.; Ku, J.H. Targeting splicing factors as molecular non-muscle invasive bladder cancer predictors. Translational Andrology and Urology 2018, 7, S715-S717, doi:10.21037/tau.2018.10.09.
12. López-Knowles, E.; Hernández, S.; Malats, N.r.; Kogevinas, M.; Lloreta, J.; Carrato, A.; Tardón, A.; Serra, C.; Real, F.X. PIK3CA Mutations Are an Early Genetic Alteration Associated with FGFR3 Mutations in Superficial Papillary Bladder Tumors. Cancer Research 2006, 66, 7401-7404, doi:10.1158/0008-5472.can-06-1182.
13. Monnin, K.A.; Bronstein, I.B.; Gaffney, D.K.; Holden, J.A. Elevations of DNA topoisomerase I in transitional cell carcinoma of the urinary bladder: Correlation with DNA topoisomerase II-alpha and p53 expression. Human Pathology 1999, 30, 384-391, doi:10.1016/s0046-8177(99)90112-0.
14. Montero‐Hidalgo, A.J.; Pérez‐Gómez, J.M.; Martínez‐Fuentes, A.J.; Gómez‐Gómez, E.; Gahete, M.D.; Jiménez‐Vacas, J.M.; Luque, R.M. Alternative splicing in bladder cancer: potential strategies for cancer diagnosis, prognosis, and treatment. WIREs RNA 2022, 14, doi:10.1002/wrna.1760.
15. Ouerhani, S.; Bougatef, K.; Soltani, I.; Elgaaied, A.B.A.; Abbes, S.; Menif, S. The prevalence and prognostic significance of KRAS mutation in bladder cancer, chronic myeloid leukemia and colorectal cancer. Molecular Biology Reports 2013, 40, 4109-4114, doi:10.1007/s11033-013-2512-8.
16. Ouerhani, S.; Elgaaied, A.B.A. The mutational spectrum of HRAS, KRAS, NRAS and FGFR3 genes in bladder cancer. Cancer Biomarkers 2012, 10, 259-266, doi:10.3233/cbm-2012-0254.
17. Pan, Y.-H.; Zhang, J.-X.; Chen, X.; Liu, F.; Cao, J.-Z.; Chen, Y.; Chen, W.; Luo, J.-H. Predictive Value of the TP53/PIK3CA/ATM Mutation Classifier for Patients With Bladder Cancer Responding to Immune Checkpoint Inhibitor Therapy. Frontiers in Immunology 2021, 12, doi:10.3389/fimmu.2021.643282.
18. Shuman, L.; Pham, J.; Wildermuth, T.; Wu, X.-R.; Walter, V.; Warrick, J.I.; DeGraff, D.J. Urothelium-Specific Expression of Mutationally Activated Pik3ca Initiates Early Lesions of Noninvasive Bladder Cancer. The American Journal of Pathology 2023, 193, 2133-2143, doi:10.1016/j.ajpath.2023.07.001.
19. Stenehjem, D.; Tran, D.; Nkrumah, M.; Gupta, S. PD1/PDL1 inhibitors for the treatment of advanced urothelial bladder cancer. OncoTargets and Therapy 2018, Volume 11, 5973-5989, doi:10.2147/ott.s135157.
20. Wang, Z.; Shang, J.; Li, Z.; Li, H.; Zhang, C.; He, K.; Li, S.; Ju, W. PIK3CA Is Regulated by CUX1, Promotes Cell Growth and Metastasis in Bladder Cancer via Activating Epithelial-Mesenchymal Transition. Frontiers in Oncology 2020, 10, doi:10.3389/fonc.2020.536072.
21. Wu, T.-Z.; Lei, C.-Y.; Li, J.-M.; Guo, Y.-F. Sex discrepancy characterization revealed by somatic DNA alterations in muscle-invasive bladder carcinoma. Chinese Medical Journal 2019, 132, 2492-2494, doi:10.1097/cm9.0000000000000487.
22. Yi, R.; Lin, A.; Cao, M.; Xu, A.; Luo, P.; Zhang, J. ATM Mutations Benefit Bladder Cancer Patients Treated With Immune Checkpoint Inhibitors by Acting on the Tumor Immune Microenvironment. Frontiers in Genetics 2020, 11, doi:10.3389/fgene.2020.00933.
23. Zargar, P.; Koochakkhani, S.; Hassanzadeh, M.; Ashouri Taziani, Y.; Nasrollahi, H.; Eftekhar, E. Downregulation of topoisomerase 1 and 2 with acriflavine sensitizes bladder cancer cells to cisplatin-based chemotherapy. Molecular Biology Reports 2022, 49, 2755-2763, doi:10.1007/s11033-021-07087-1.
24. Zheng, Y.; Izumi, K.; Yao, J.L.; Miyamoto, H. Dihydrotestosterone upregulates the expression of epidermal growth factor receptor and ERBB2 in androgen receptor-positive bladder cancer cells. Endocrine-Related Cancer 2011, 18, 451-464, doi:10.1530/erc-11-0010.
25. Zhou, Y.; Börcsök, J.; Adib, E.; Kamran, S.C.; Neil, A.J.; Stawiski, K.; Freeman, D.; Stormoen, D.R.; Sztupinszki, Z.; Samant, A.; et al. ATM deficiency confers specific therapeutic vulnerabilities in bladder cancer. Science Advances 2023, 9, doi:10.1126/sciadv.adg2263.
26. Aldén, M.; Olofsson Falla, F.; Yang, D.; Barghouth, M.; Luan, C.; Rasmussen, M.; De Marinis, Y. Intracellular Reverse Transcription of Pfizer BioNTech COVID-19 mRNA Vaccine BNT162b2 In Vitro in Human Liver Cell Line. Current Issues in Molecular Biology 2022, 44, 1115-1126, doi:10.3390/cimb44030073.
27. Solomon, A.L.; Ratchford, E.V.; Armitage, K.B.; Kovacic, J.C. Vascular Disease Patient Information Page: Vascular considerations with COVID-19 vaccines. Vascular Medicine 2021, 27, 102-106, doi:10.1177/1358863X211066128.
28. Speicher, D.J.; Rose, J.; McKernan, K. Quantification of residual plasmid DNA and SV40 promoter-enhancer sequences in Pfizer/BioNTech and Moderna modRNA COVID-19 vaccines from Ontario, Canada. Autoimmunity 2025, 58, 2551517, doi:10.1080/08916934.2025.2551517.
29. von Ranke, N.; Zhang, W.; Anokin, P.; Hulscher, N.; McKernan, K.J.; McCullough, P.A.; Catanzaro, J.A. Synthetic mRNA Vaccines and Transcriptomic Dysregulation: Evidence from New-Onset Adverse Events and Cancers Post-Vaccination. ResearchGate, 2025.https://www.researchgate.net/publication/394007467_Synthetic_mRNA_Vaccines_and_Transcriptomic_Dysregulation_Evidence_from_New-Onset_Adverse_Events_and_Cancers_Post-Vaccination
30. Li, Y.E.; Wang, S.; Reiter, R.J.; Ren, J. Clinical cardiovascular emergencies and the cellular basis of COVID-19 vaccination: from dream to reality? International Journal of Infectious Diseases 2022, 124, 1-10, doi:10.1016/j.ijid.2022.08.026.
31. Mohseni Afshar, Z.; Tavakoli Pirzaman, A.; Liang, J.J.; Sharma, A.; Pirzadeh, M.; Babazadeh, A.; Hashemi, E.; Deravi, N.; Abdi, S.; Allahgholipour, A.; et al. Do we miss rare adverse events induced by COVID-19 vaccination? Frontiers in Medicine 2022, Volume 9 - 2022.
32. Bartolomucci, A.; Nobrega, M.; Ferrier, T.; Dickinson, K.; Kaorey, N.; Nadeau, A.; Castillo, A.; Burnier, J.V. Circulating tumor DNA to monitor treatment response in solid tumors and advance precision oncology. npj Precision Oncology 2025, 9, doi:10.1038/s41698-025-00876-y.
33. Hanahan, D.; Weinberg, Robert A. Hallmarks of Cancer: The Next Generation. Cell 2011, 144, 646-674, doi:10.1016/j.cell.2011.02.013.
34. Kim, H.; Park, K.U. Clinical Circulating Tumor DNA Testing for Precision Oncology. Cancer Research and Treatment 2023, 55, 351-366, doi:10.4143/crt.2022.1026.
35. Raghavan, S.; Kundumani-Sridharan, V.; Kumar, S.; White, C.W.; Das, K.C. Thioredoxin Prevents Loss of UCP2 in Hyperoxia via MKK4–p38 MAPK–PGC1α Signaling and Limits Oxygen Toxicity. American Journal of Respiratory Cell and Molecular Biology 2022, 66, 323-336, doi:10.1165/rcmb.2021-0219oc.
36. Torgovnick, A.; Schumacher, B. DNA repair mechanisms in cancer development and therapy. Frontiers in Genetics 2015, 6, doi:10.3389/fgene.2015.00157.
37. Cassandri, M.; Smirnov, A.; Novelli, F.; Pitolli, C.; Agostini, M.; Malewicz, M.; Melino, G.; Raschellà, G. Zinc-finger proteins in health and disease. Cell Death Discovery 2017, 3, doi:10.1038/cddiscovery.2017.71.
38. Kamaliyan, Z.; Clarke, T.L. Zinc finger proteins: guardians of genome stability. Frontiers in Cell and Developmental Biology 2024, 12, doi:10.3389/fcell.2024.1448789.
39. Li, X.; Han, M.; Zhang, H.; Liu, F.; Pan, Y.; Zhu, J.; Liao, Z.; Chen, X.; Zhang, B. Structures and biological functions of zinc finger proteins and their roles in hepatocellular carcinoma. Biomarker Research 2022, 10, doi:10.1186/s40364-021-00345-1.
40. Dutheil, N.; Yoon-Robarts, M.; Ward, P.; Henckaerts, E.; Skrabanek, L.; Berns, K.I.; Campagne, F.; Linden, R.M. Characterization of the Mouse Adeno-Associated Virus AAVS1 Ortholog. Journal of Virology 2004, 78, 8917-8921, doi:10.1128/jvi.78.16.8917-8921.2004.
41. Hamilton, H.; Gomos, J.; Berns, K.I.; Falck-Pedersen, E. Adeno-Associated Virus Site-Specific Integration and AAVS1 Disruption. Journal of Virology 2004, 78, 7874-7882, doi:10.1128/jvi.78.15.7874-7882.2004.
42. Cheng, Y.; Liang, P.; Geng, H.; Wang, Z.; Li, L.; Cheng, S.H.; Ying, J.; Su, X.; Ng, K.M.; Ng, M.H.L.; et al. A Novel 19q13 Nucleolar Zinc Finger Protein Suppresses Tumor Cell Growth through Inhibiting Ribosome Biogenesis and Inducing Apoptosis but Is Frequently Silenced in Multiple Carcinomas. Molecular Cancer Research 2012, 10, 925-936, doi:10.1158/1541-7786.mcr-11-0594.
43. Jen, J.; Wang, Y.-C. Zinc finger proteins in cancer progression. Journal of Biomedical Science 2016, 23, doi:10.1186/s12929-016-0269-9.
44. Petre, G.; Lorès, P.; Sartelet, H.; Truffot, A.; Poreau, B.; Brandeis, S.; Martinez, G.; Satre, V.; Harbuz, R.; Ray, P.F.; et al. Genomic duplication in the 19q13.42 imprinted region identified as a new genetic cause of intrauterine growth restriction. Clinical Genetics 2018, 94, 575-580, doi:10.1111/cge.13449.
45. Oceguera-Yanez, F.; Kim, S.-I.; Matsumoto, T.; Tan, G.W.; Xiang, L.; Hatani, T.; Kondo, T.; Ikeya, M.; Yoshida, Y.; Inoue, H.; et al. Engineering the AAVS1 locus for consistent and scalable transgene expression in human iPSCs and their differentiated derivatives. Methods 2016, 101, 43-55, doi:10.1016/j.ymeth.2015.12.012.
46. Chang HHY, Pannunzio NR, Adachi N, Lieber MR. Non-homologous DNA end joining and alternative pathways to double-strand break repair. Nat Rev Mol Cell Biol. 2017;18(8):495-506. doi:10.1038/nrm.2017.48
47. Huang R, Zhou PK. DNA damage repair: historical perspectives, mechanistic pathways and clinical translation for targeted cancer therapy. Signal Transduct Target Ther. 2021;6(1):254. Published 2021 Jul 9. doi:10.1038/s41392-021-00648-7
48. Schaafsma, E.; Fugle, C.M.; Wang, X.; Cheng, C.A.-O. Pan-cancer association of HLA gene expression with cancer prognosis and immunotherapy efficacy.
49. Szolek, A.; Schubert, B.; Mohr, C.; Sturm, M.; Feldhahn, M.; Kohlbacher, O. OptiType: precision HLA typing from next-generation sequencing data.
50. Bahar, M.E.; Kim, H.J.; Kim, D.R. Targeting the RAS/RAF/MAPK pathway for cancer therapy: from mechanism to clinical studies. Signal Transduction and Targeted Therapy 2023, 8, doi:10.1038/s41392-023-01705-z.
51. Kodaz, H. Frequency of RAS Mutations (KRAS, NRAS, HRAS) in Human Solid Cancer. Eurasian Journal of Medicine and Oncology 2017, doi:10.14744/ejmo.2017.22931.
52. Braicu, C.; Buse, M.; Busuioc, C.; Drula, R.; Gulei, D.; Raduly, L.; Rusu, A.; Irimie, A.; Atanasov, A.G.; Slaby, O.; et al. A Comprehensive Review on MAPK: A Promising Therapeutic Target in Cancer. Cancers 2019, 11, 1618, doi:10.3390/cancers11101618.
53. Clark-Garvey, S.; Kim, W.Y. RAF1 amplification: an exemplar of MAPK pathway activation in urothelial carcinoma. Journal of Clinical Investigation 2021, 131, doi:10.1172/jci154095.
54. Novillo, A.; Fernández-Santander, A.; Gaibar, M.; Galán, M.; Romero-Lorca, A.; El Abdellaoui-Soussi, F.; Gómez-del Arco, P. Role of Chromodomain-Helicase-DNA-Binding Protein 4 (CHD4) in Breast Cancer. Frontiers in Oncology 2021, 11, doi:10.3389/fonc.2021.633233.
55. Wang, J.; Zhong, F.; Li, J.; Yue, H.; Li, W.; Lu, X. The epigenetic factor CHD4 contributes to metastasis by regulating the EZH2/β-catenin axis and acts as a therapeutic target in ovarian cancer. Journal of Translational Medicine 2023, 21, doi:10.1186/s12967-022-03854-1.
56. Liu, Z.; Yoshimi, A.; Wang, J.; Cho, H.; Chun-Wei Lee, S.; Ki, M.; Bitner, L.; Chu, T.; Shah, H.; Liu, B.; et al. Mutations in the RNA Splicing Factor SF3B1 Promote Tumorigenesis through MYC Stabilization. Cancer Discovery 2020, 10, 806-821, doi:10.1158/2159-8290.cd-19-1330.
57. Samy, A.; Ozdemir, M.K.; Alhajj, R. Studying the connection between SF3B1 and four types of cancer by analyzing networks constructed based on published research. Scientific Reports 2023, 13, doi:10.1038/s41598-023-29777-5.
58. Goswami, K.; Venkatachalam, K.; Singh, S.P.; Rao, C.V.; Madka, V. Chromatin Remodulator CHD4: A Potential Target for Cancer Interception. Genes 2025, 16, 225, doi:10.3390/genes16020225.
59. Xia, L.; Huang, W.; Bellani, M.; Seidman, M.M.; Wu, K.; Fan, D.; Nie, Y.; Cai, Y.; Zhang, Y.W.; Yu, L.-R.; et al. CHD4 Has Oncogenic Functions in Initiating and Maintaining Epigenetic Suppression of Multiple Tumor Suppressor Genes. Cancer Cell 2017, 31, 653-668.e657, doi:10.1016/j.ccell.2017.04.005.
60. Madsen, R.R.; Vanhaesebroeck, B.; Semple, R.K. Cancer-Associated PIK3CA Mutations in Overgrowth Disorders. Trends in Molecular Medicine 2018, 24, 856-870, doi:10.1016/j.molmed.2018.08.003.
61. Ross, R.L.; McPherson, H.R.; Kettlewell, L.; Shnyder, S.D.; Hurst, C.D.; Alder, O.; Knowles, M.A. PIK3CA dependence and sensitivity to therapeutic targeting in urothelial carcinoma. BMC Cancer 2016, 16, doi:10.1186/s12885-016-2570-0.
62. Lee, J.-H. Targeting the ATM pathway in cancer: Opportunities, challenges and personalized therapeutic strategies. Cancer Treatment Reviews 2024, 129, 102808, doi:10.1016/j.ctrv.2024.102808.
63. Acuti Martellucci, C.; Capodici, A.; Soldato, G.; et al. COVID-19 vaccination, all-cause mortality, and hospitalization for cancer: 30-month cohort study in an Italian province. EXCLI Journal 2025, 24, 690–707. doi:10.17179/excli2025-8400.
64. Marik, P.; Hope, J. COVID-19 mRNA-Induced “Turbo Cancers.” Journal of Independent Medicine 2025, 1, 185–194. doi:10.71189/JIM/2025/V01N03A02.
65. Mills, A.A. The Chromodomain Helicase DNA-Binding Chromatin Remodelers: Family Traits that Protect from and Promote Cancer. Cold Spring Harbor Perspectives in Medicine 2017, 7, a026450, doi:10.1101/cshperspect.a026450.