Introduction Breast cancer (BC) is the most common cancer affecting women in the United States. Ductal carcinoma in situ (DCIS) is the earliest identifiable pre-invasive BC lesion. Estimates show that 14 to 50% of DCIS cases progress to invasive BC. Methods Our objective was to identify nuclear matrix proteins (NMP) with specifically altered expression in DCIS and later stages of BC compared to non-diseased breast reduction mammoplasty and a contralateral breast explant culture using mass spectrometry and RNA sequencing to accurately identify aggressive DCIS. Results Sixty NMPs were significantly differentially expressed between the DCIS and non-diseased breast epithelium in an isogenic contralateral pair of patient-derived extended explants. Ten of the sixty showed significant mRNA expression level differences that matched the protein expression. These 10 proteins were similarly expressed in non-diseased breast reduction cells. Three NMPs (RPL7A, RPL11, RPL31) were significantly upregulated in DCIS and all other BC stages compared to the matching contralateral breast culture and an unrelated non-diseased breast reduction culture. RNA sequencing analyses showed that these three genes were increasingly upregulated with BC progression. Finally, we identified three NMPs (AHNAK, CDC37 and DNAJB1) that were significantly downregulated in DCIS and all other BC stages compared to the isogenically matched contralateral culture and the non-diseased breast reduction culture using both proteomics and RNA sequencing techniques. Discussion These genes should form the basis of, or contribute to, a molecular diagnostic panel that could identify DCIS lesions likely to be indolent and therefore not requiring aggressive treatment.

Background and Objectives: In 2021 breast cancer (BC) was the most common cancer affecting women and the second deadliest cancer in the U.S. Ductal Carcinoma In Situ (DCIS) is the earliest identifiable pre-invasive breast cancer lesion. Approximately 20-25% of diagnosed breast cancer cases are ductal carcinoma in situ (DCIS). Estimates show that 14 to 50% of DCIS cases progress to invasive breast cancer. We used a novel tissue-engineering system established in the Latimer laboratory to generate model systems representative of different molecular and histological subtypes of all stages of BC, including DCIS. Using a mass spectrometry (MS)-based relative quantitative methodology, we analyzed one novel DCIS cell line, the matching isogenic contralateral cell line and the non-tumor adjacent cell line, as well as two non-diseased breast reduction mammoplasty cell lines and the established stage IV BC cell lines MCF7 and MDA MB231. RNA sequencing analysis included additional cell lines from stages I, II and III. In order to limit the number of proteins for proteomic assessment, nuclear matrix proteins (NMPs) were targeted for analysis. NMPs have previously been linked with cancers of the prostate, colon, and breast. Maldi-TOF/TOF-MS analysis identified 270 NMPs with high confidence. Our objective was to identify NMPs with specifically altered expression in DCIS and later stages of BC compared to non-diseased breast and contralateral breast. Such markers may help to accurately identify invasive DCIS and improve early detection of BC. Results: We identified two nuclear matrix proteins that are significantly downregulated in DCIS and all other stages of BC in our collection. This downregulation was also apparent through mRNA expression analysis. These proteins are upregulated in non-diseased breast reduction tissue and contra-lateral DCIS derived from the same patient. We also identified four NMPs that were significantly upregulated in DCIS and all other BC stages compared to non-diseased breast reduction tissue and contralateral DCIS. Gene expression analyses showed that these four genes were upregulated with BC progression. Finally, we identified nine NMPS with significant differences in expression between DCIS and all stages of BC versus non-diseased breast reduction tissue and contralateral DCIS where gene expression did not follow the same trend as proteomic analysis. Conclusion: Our findings suggest that the changes that occur in the nuclear matrix of DCIS vs. normal breast epithelium could provide a basis for the development of prognostic biomarkers for DCIS and allow for targeted prevention of progression to invasive disease. This work was supported by grant W81XWH-0701-0660 from the DOD CDMRP BC program. Citation Format: Ali Almutairy, Abdullah Alhamed, Stephen G. Grant, Miranda Sarachine Falso, Billy W. Day, Jean J. Latimer. Identification of cancer-specific alterations in expression of nuclear matrix proteins through proteomic and RNAsequencing analyses of ductal carcinoma in situ [abstract]. In: Proceedings of the AACR Special Conference on the Evolutionary Dynamics in Carcinogenesis and Response to Therapy; 2022 Mar 14-17. Philadelphia (PA): AACR; Cancer Res 2022;82(10 Suppl):Abstract nr A033.

Aims: Gulf War illness (GWI) is thought to be associated with exposures experienced by soldiers deployed in the 1991 Gulf War. A major question is how these exposures continue to influence the health of these individuals three decades later. One potentially permanent effect of such exposures is the induction of genetic mutations. We investigated whether veterans with GWI exhibited persistently elevated levels of somatic mutation. Materials and methods: We applied the blood-based glycophorin A (GPA) somatic mutation assay to a cohort of veterans diagnosed with GWI and a set of both concurrent and historic, age-matched controls. This assay quantifies red blood cells with a phenotype consistent with loss of one allele at the genetic determinant for the MN blood group, the GPA gene. Key findings: As a population, those affected with GWI exhibited an uninduced mutation frequency at the GPA locus that was effectively twice that observed in controls, a result that was statistically significant. This result was influenced by an increase in the incidence of individuals with aberrantly high mutation frequencies, seemingly higher than would be expected by dose extrapolation and consistent with the induction of localized genomic instability in the hematopoietic bone marrow stem cells. When these "outliers" were removed from consideration, the remaining affected population retained a significantly higher mean allele loss mutation frequency, suggesting that both dose-dependent bone marrow genotoxicity and induction of genomic instability are contributing to the elevation in mutation frequency in these affected veterans. Significance: This study provides evidence that manifestation of GWI is associated with increased cumulative exposure to agents capable of inducing persistent mutations in bone marrow stem cells. Whether these mutations are involved in the clinical aspects of the condition or are simply biomarkers of overall exposure has yet to be determined. The increased incidence of genomic instability suggests that this persistent mutation can have important delayed effects on cellular integrity.

Introduction Veterans of the 1991 Gulf War were potentially exposed to a mixture of stress, chemicals and radiation that may have contributed to the persistent symptoms of Gulf War Illness (GWI). The genotoxic effects of some of these exposures are mediated by the DNA nucleotide excision repair (NER) pathway. We hypothesized that individuals with relatively low DNA repair capacity would suffer greater damage from cumulative genotoxic exposures, some of which would persist, causing ongoing problems. Materials and Methods Blood samples were obtained from symptomatic Gulf War veterans and age-matched controls. The unscheduled DNA synthesis assay, a functional measurement of NER capacity, was performed on cultured lymphocytes, and lymphocyte mRNA was extracted and analyzed by sequencing. Results Despite our hypothesis that GWI would be associated with DNA repair deficiency, NER capacity in lymphocytes from affected GWI veterans actually exhibited a significantly elevated level of DNA repair (p = 0.016). Both total gene expression and NER gene expression successfully differentiated individuals with GWI from unaffected controls. The observed functional increase in DNA repair capacity was accompanied by an overexpression of genes in the NER pathway, as determined by RNA sequencing analysis. Conclusion We suggest that the observed elevations in DNA repair capacity and NER gene expression are indicative of a “hormetic,” i.e., induced or adaptive protective response to battlefield exposures. Normally such effects are short-term, but in these individuals this response has resulted in a long-term metabolic shift that may also be responsible for the persistent symptoms of GWI.