Uterine Serous Carcinoma Tumors Appear More Aggressive in Black Populations

PAX8, a marker for aggressive disease in numerous cancer types, was more highly expressed in Black patients with uterine serous carcinoma.

Uterine serous carcinoma (USC) tumors were found to be expressed more aggressively with greater immunosuppressive features in Black patients than White patients, according to findings from a study published in Proceedings of the National Academy of Sciences.¹ Furthermore, it was found that PAX8, a marker for aggressive disease in numerous cancer types, was more highly expressed in Black patients with USC than White patients.

Data revealed that 5 of 6 mutations at TP53 hotspot Arg273 were found in Black patients. Furthermore, nuclei from Black patients were found to have a distinct enrichment pathway from White patients and a higher degree of immunosuppression due to a lower density of CD8-positive cytotoxic T cells. Patients with high PAX8-expressing tumors for all subtypes had worse overall survival compared with patients with low ones (HR = 0.549; P = 0.00787)

“Our environment, socioeconomic status and societal stressors can all impact us psychologically,” senior author Julie Kim, PhD, Susy Y. Hung Research Professor of Obstetrics and Gynecology at Northwestern Feinberg School of Medicine, said in a news release on the study findings.² “If the environmental insults are chronic, they can have an impact on health. We are seeing differences in these tumors in terms of the genes that they express, in terms of the immune system and the immune response.”

In addition to TP53 Arg273 mutations, the presence of mismatch repair and POLE mutations was assessed. Ultimately, no POLE mutations and some mismatch repair mutations in genes such as MLH1 were found, the latter of which was added to cohort analysis.

An average of 4617 mRNA molecules per nucleus were found in an average of 2684 genes in 102,431 nuclei. Six major cell subtypes were revealed through transcriptomic tumor analysis: tumor epithelium (PAX8), ciliated epithelium (DNAH7), stromal fibroblast (LSAMP), myeloid (CD163), lymphoid (CD247), and endothelium (FLT1); tumor epithelium compromised more than 60% of cell types recovered from 10 of 13 patients.

Furthermore, of 4 tumor epithelium clusters—tumor epithelium 1 (TE1), tumor epithelium 2 (TE2), tumor epithelium 3 (TE3), and stem cell tumor epithelium (SCTE)—White patients were found to have the greatest presence of TE1 nuclei (67%), and Black patients were found to have the greatest presence of TE2 nuclei (88%). TE1 nuclei were notably highly enriched for p53, while TE2 nuclei were not, indicating differences in cluster pathways.

Single-nuclei RNA-sequencing was performed on tumors from 4 and 9 self-identified White and Black patients, respectively, and all tumors were pathologist-evaluated and diagnosed as having serous histology. All tumors were found to be missense TP53-positive through exome sequencing.

Resected tumors specimens were frozen, snRNA sequence libraries were constructed, and the libraries were pooled and sequenced. Genomic DNA from these specimens were extracted and paired-end 150 bp sequencing was performed. ARK2 cells were obtained and grown in DMEM supplemented with penicillin, streptomycin, 1X NEAA, and 10% FBS; THP-1 cells were cultured similarly, except with 0.05 mM ß-mercaptoethanol in place of 1X NEAA.

Immunohistochemistry was conducted through cutting and deparaffinizing 4 µm sections, with antigen retrieval performed using a 1 M citrate buffer, and 1:100 of dilution PAX8 antibody into antibody diluent. Multiplex immunohistochemistry was performed on tumor and matching benign tissue, defined as matched tissue adjacent to the endometrium. To achieve siRNA knockdown of PAX8, ARK2 cells were cultured and washed, a reagent was used to transfect multiple siRNAs against PAX8 into ARK2 cells, and 24 hours later, cells were washed and cultured again.

The RNA was harvested, sent for library preparation and sequencing, and differential gene and gene ontology enrichment analyses were conducted. Media were collected from ARK2 and ARK2 PAX8 cells, filtered through a 20 µm strainer, and then centrifuged to eliminate ARK2 cells; media were frozen until needed. Then, THP-1 cells were grown until 80% to 90% confluent and differentiated for 24 hours; they were then washed and treated with a 1:2 ratio of RPMI media and ARK2 PAX8 KD-CM.

The THP-1 cell RNA was harvested 72 hours after it was cultured in CM. THP-1 cell media were collected following 72 hours of treatment and the release of 80 growth factors and cytokines was determined through use of human cytokine array C5.

References

1. Foley GK, Adli M, Kim JJ. Single-nuclei sequencing of uterine serous carcinoma reveals racial differences in immune signaling. Proc Natl Acad Sci. 2024;121(34): e2402998121 doi:10.1073/pnas.2402998121
2. Paul M. Cancerous uterine tumors more aggressive in Black patients than white patients. News release. Northwestern Now. August 14, 2024. Accessed August 20, 2024. https://tinyurl.com/2zn4weeb