Calcification-associated molecular traits and therapeutic strategies in hormone receptor-positive HER2-negative breast cancer

Clinical characteristics and pathological patterns of different calcification groups. (A) The box plot shows tumor sizes among groups. The sample sizes for the calcification-negative, probably benign, and high suspicion groups were 103, 90, and 100, respectively. (B) The stacked bar chart compares the lymph node stages across groups. P values were calculated with Fisher’s exact test. The sample sizes for the calcification-negative, probably benign, and high suspicion groups were 103, 90, and 100, respectively. (C) The stacked bar chart shows the lympho-vascular invasion status across groups. P values were calculated with Fisher’s exact test. The sample sizes for the calcification-negative, probably benign, and high suspicion groups were 103, 90, and 100, respectively. (D) The box plot illustrates the ages among groups. The sample sizes for the calcification-negative, probably benign, and high suspicion groups were 103, 90, and 100, respectively. (E) The box plot shows the immunohistochemistry scores for ER, PR, and Ki-67. The sample sizes for the calcification-negative, probably benign, and high suspicion groups were 103, 90, and 100, respectively. (F) Relapse-free survival (RFS) among groups with endocrine therapy. P values were calculated from a two-stage test (probably benign vs. the others). (G) A stacked bar chart illustrates the calcification status for different recurrence outcomes. P values were calculated with Fisher’s exact test.

Genomic features of tumors in different calcification groups. (A) The box plot illustrates tumor mutational burden (TMB) scores among calcification groups. The sample sizes for the calcification-negative, probably benign, and high suspicion groups were 87, 77, and 87, respectively. (B) The box plot shows homologous recombination deficiency (HRD) scores across groups. The sample sizes for the calcification-negative, probably benign, and high suspicion groups were 93, 83, and 88, respectively. (C) The box plot shows the comparison of chromosomal instability (CIN) scores. The sample sizes for the calcification-negative, probably benign, and high suspicion groups were 93, 83, and 88, respectively. (D) The proportion of mutation signatures in each sample, on the basis of the Catalog of Somatic Mutations in Cancer (COSMIC) database. (E) Somatic mutations of the top mutated genes. Genes are ordered by total mutation frequency. P values were calculated with Fisher’s exact test (^#P value < 0.1). (F) Somatic CNV among groups, and comparison between tumors with high suspicious calcifications and tumors in the other groups. The P values were calculated with Fisher’s exact test and adjusted for multiple comparisons with the Bonferroni method; the horizontal line in the bottom panel represents the adjusted P value of 0.05. (G) The CNV frequencies of cell cycle genes across groups. P values were determined with Fisher’s exact test.

Exploration of transcriptomic features and therapeutic strategies among calcification groups. (A) The stacked bar chart shows the composition of PAM50 subtypes across groups. P values were calculated with Fisher’s exact test. The sample sizes for the calcification-negative, probably benign, and high suspicion groups were 103, 89, and 98, respectively. (B) The stacked bar chart compares the composition of SNF subtypes across groups. P values were calculated with Fisher’s exact test. The sample sizes for the calcification-negative, probably benign, and high suspicion groups were 59, 51, and 59, respectively. (C) The heatmap shows the comparison of biomarker scores. P values were determined with Mann-Whitney Wilcoxon tests (one group vs. the others) (*P value < 0.05; **P value < 0.01; ***P value < 0.001). (D) The heatmap reveals the estimated abundance of 24 microenvironment cell types. (E) The box plot shows the single-sample gene set enrichment analysis (ssGSEA) scores for ER-associated pathways. The sample sizes for the calcification-negative, probably benign, and high suspicion groups were 102, 89, and 96, respectively. (F) The box plots illustrate the sensitivity scores for endocrine therapy among groups. The sample sizes for the calcification-negative, probably benign, and high suspicion groups were 102, 89, and 96, respectively. (G) The box plot shows the single-sample gene set enrichment analysis (ssGSEA) scores for cell cycle-associated pathways. The sample sizes for the calcification-negative, probably benign, and high suspicion groups were 102, 89, and 96, respectively. (H) The box plots compare the sensitivity scores for CDK4/6 inhibitors among groups. The sample sizes for the calcification-negative, probably benign, and high suspicion groups were 102, 89, and 96, respectively.