Methodological Heterogeneity in the Assessment of X Chromosome Inactivation in Cancer: A Systematic Review

X chromosome inactivation (XCI) is an essential epigenetic mechanism that balances the dosage of X-linked genes in female cells. Disruption of XCI has been increasingly reported in cancer and may represent an important manifestation of sex chromosome dysregulation. This systematic review evaluated how XCI is assessed in cancer and how methodological differences influence the interpretation of XCI status. A systematic search of PubMed and Embase identified studies assessing XCI in the context of human cancer. Eligible studies employed methylation-based assays, loss-of-heterozygosity (LOH) analyses, transcription-based approaches, cytogenetic methods, or genomic and epigenomic strategies. Studies examining XIST expression alone without assessing XCI status were excluded. Sixty-one studies met the inclusion criteria. Considerable heterogeneity was observed in both the methodologies used and the definitions of XCI disruption. Epigenetic and genomic approaches were represented in 23 studies, followed by LOH-based analyses (n=19), methylation-based methods (n=14), transcription-based approaches (n=5) and cytogenetic methods (n=4). These categories were not mutually exclusive. A small number of genome-wide and integrative studies classified or quantified defective XCI, with reported frequencies varying across cancer types and analytical definitions. Across the literature, methods measuring distinct biological features were frequently interpreted as equivalent assessments of XCI status. Methodological heterogeneity limits the comparability of findings across cancer studies. Many commonly used approaches do not directly assess functional XCI, complicating interpretation of the prevalence and significance of XCI disruption. Standardised approaches integrating complementary structural, epigenetic and transcriptional measurements are needed to distinguish clonality, X-chromosome alterations and functional XCI dysregulation.