Distinct genomic and immunologic tumor evolution in germline TP53-driven breast cancers

Abstract

Pathogenic germline TP53 alterations cause Li-Fraumeni Syndrome (LFS), and breast cancer is the most common cancer in LFS females. We performed first of its kind multimodal analysis of LFS breast cancer (LFS-BC) compared to sporadic premenopausal BC. Nearly all LFS-BC underwent biallelic loss of TP53 with no recurrent oncogenic variants except ERBB2 (HER2) amplification. Compared to sporadic BC, in situ and invasive LFS-BC exhibited a high burden of short amplified aneuploid segments (SAAS). Pro-apoptotic p53 target genes BAX and TP53I3 failed to be up-regulated in LFS-BC as was seen in sporadic BC compared to normal breast tissue. LFS-BC had lower CD8+ T-cell infiltration compared to sporadic BC yet higher levels of proliferating cytotoxic T-cells. Within LFS-BC, progression from in situ to invasive BC was marked by an increase in chromosomal instability with a decrease in proliferating cytotoxic T-cells. Our study uncovers critical events in mutant p53-driven tumorigenesis in breast tissue.

Keywords: Germline; TP53; inherited; p53 tumor suppressor; tumor development; tumor immunology.

Figure 1:. Genomic features of LFS-BC compared to early onset nonLFS-BC.

(a) Heat-map showing overall clinical and genomic features of LFS-BC and early-onset nonLFS-BC, including self-reported ethnicity, TP53 germline and somatic variant status and variant alteration type, breast cancer histology and hormone receptor class, TP53 biallelic loss status, tumor mutational burden, overall homologous recombination deficiency (HRD) score, genomic loss of heterozygosity score, large state transition score, non-telomeric allelic imbalance score, and aneuploidy score, oncogenic mutations, gene-level amplifications/deletions, and mutated gene oncogenic pathways. (b) State of the germline TP53 locus in LFS-BC, tumors from TCGA with germline TP53 PGVs and tumors from TCGA with germline TP53 LB/B-VUS. In tumors with biallelic loss, the mechanism of biallelic loss is shown, including copy neutral loss of heterozygosity (CN-LOH), copy neutral gain loss of heterozygosity (CNG-LOH), deletion loss of heterozygosity (Del-LOH), and a second somatic mutation in TP53 (2^nd hit). (c) Amplification of ERBB2 (HER2) in LFS-BC and normal breast tissues. Copy number state for ERBB2 was normalized to the copy number state of chr17p. Abbreviations: CN, copy neutral; DBD, DNA binding domain; DCIS, ductal carcinoma in situ; Del, deletion; ER, Estrogen Receptor; HRD, homologous recombination deficiency; HER2: receptor tyrosine-protein kinase erbB-2; IDC, invasive ductal carcinoma; LB/B: Likely Benign/Benign variants; LFS, Li Fraumeni Syndrome; LOF, loss of function; LOH, loss of heterozygosity; LP/P, Likely Pathogenic/Pathogenic variants; LST, large state transitions; SNV, single nucleotide variant; TAI, telomeric allelic imbalances; TMB, tumor mutational burden; VUS, variants of uncertain significance.

Figure 2:. Copy number alterations in LFS-BC compared to early onset nonLFS-BC.

(a) Genomic instability measures including microsatellite instability (MSI), tumor mutational burden (TMB), aneuploidy score (AS), chromosomal instability (CIN) score, homologous recombination deficiency (HRD) score, genomic loss of heterozygosity (gLOH), large state transitions (LST) and non-telomeric allelic imbalances (TAI) in normal LFS breast tissue versus LFS-BC. (b) Genomic instability measures (MSI, TMB, CIN, HRD, gLOH, LST, nTAI) stratified by hormone receptor status in LFS-BC versus nonLFS-BC. (c) Distribution of the number of amplified (total copy number ≥5) segments of allelic imbalance, aka short amplified aneuploid segments (SAAS) across LFS-BC and nonLFS-BC and box plot of number of amplified AI segments in LFS-BC versus nonLFS-BC. (d) Distribution of the lengths of amplified AI segments in LFS-BC versus nonLFS-BC and box plot of average length of amplified AI segments in LFS-BC versus nonLFS-BC. Abbreviations: AI, allelic imbalance; Avg, average; CNA, copy number alteration; CNt, copy number total. ER, Estrogen Receptor; HRD, homologous recombination deficiency; HER2: receptor tyrosine-protein kinase erbB-2; No, number; IDC, invasive ductal carcinoma; LFS, Li Fraumeni Syndrome; TNBC, triple negative breast cancer. *p<0.05; **p<0.01; p<0.001; ***p<0.0001, uncorrected.

Figure 3:. Gene expression profiling in LFS-BC compared to early onset nonLFS-BC.

(a) Volcano plot showing differentially expressed genes (DEGs) in LFS-BC compared to normal LFS breast tissue. (b) Volcano plot showing DEGs in nonLFS-BC compared to normal breast tissue. (c) Differential expression of canonical p53 targets in the indicated tumor types (LFS-BC, early onset nonLFS-BC, TCGA ER+Her2− nonLFS-BC stratified by somatic TP53 mutation status, TCGA HER2+BC nonLFS-BC stratified by somatic TP53 mutation status, and TCGA non-LFS TNBC stratified by somatic TP53 mutation status) compared to normal breast tissue. (d) Volcano plot showing DEGs in LFS-BC compared to early-onset nonLFS-BC. (e) Hallmark pathways significantly differentially regulated between LFS-BC and nonLFS-BC. (f) Log2 fold change (FC) of significantly regulated genes in these pathways in LFS-BC compared to nonLFS-BC. (g) Normalized enrichment scores for Hallmark pathways in differential gene expression analyses between LFS-BC, nonLFS-BC and TCGA ER+, Her2+ and TNBC, each stratified by somatic p53 mutation status and each group compared to breast tissue normal. Pathways shown were significantly enriched in at least one comparison, non-significantly enriched pathways are indicated by “ns”; red arrow indicates pathway significantly regulated in all comparisons except LFS-BC versus normal. (h) Log2 fold change (FC) of significantly regulated genes in MYC targets pathway in indicated tumor type compared to normal breast tissue. Abbreviations: BC, breast cancer; DEG, differentially expressed genes; ER, Estrogen receptor; FC, fold change; FDR, false discovery rate; HER2: receptor tyrosine-protein kinase erbB-2; LFS, Li Fraumeni Syndrome; p53Mut, positive for somatic TP53 mutation; p53WT, negative for somatic TP53 mutation; TNBC, triple negative breast cancer.

Figure 4:. Immunological profiling in LFS-BC compared to early onset nonLFS-BC.

(a) xCell analysis of RNASeq data showing stroma, immune and microenvironment scores from invasive cancers from LFS-BC and early-onset nonLFS.. (b) Single stain IHC in LFS-BC compared to normal LFS breast tissue, fold enrichments in tumor versus normal are shown. (c) Representative images of multiplex immunohistochemistry (IHC) results staining for tumor infiltrating lymphocyte (TIL) and T-cell activation (ACT) panels. (d) Quantification of multiplex IHC results for TIL panel in LFS-BC, nonLFS ER+BC, nonLFS-TNBC. CD4, CD8 and CD20 in tumor and stroma are shown. Abbreviations: ACT, T-cell activation; CK, cytokeratin; BC, breast cancer; ER, estrogen receptor; HER2: receptor tyrosine-protein kinase erbB-2; LFS, Li Fraumeni Syndrome; TIL, tumor infiltrating lymphocytes; TNBC, triple negative breast cancer.

Figure 5:. Genomic, transcriptomic and immunological features of LFS-DCIS versus LFS-IDC.

(a) Genomic instability measures including microsatellite instability (MSI), tumor mutational burden (TMB), aneuploidy score (AS), chromosomal instability (CIN) score, homologous recombination deficiency (HRD) score, genomic loss of heterozygosity (gLOH), large state transitions (LST) and non-telomeric allelic imbalances (TAI) in LFS-DCIS compared to LFS-IDC. (b) Differentially expressed genes (DEGs) in LFS-DCIS versus normal LFS breast tissue and LFS-IDC versus normal LFS tissue. Involvement of these DEGs in Hallmark biological processes. (c) Quantification of multiplex immunohistochemistry (IHC) results of tumor infiltrating lymphocyte (TIL) panel components in LFS DCIS versus LFS-IDC. CD4, CD8, and CD20 in tumor are shown. (d) Quantification of multiplex IHC ACT panel components in LFS-DCIS versus LFS-IDC. Granzyme B, Ki67, and CD3 in tumor are shown. (e) Quantification of single stain IHC for FoxP3+ and CD68+ cells in LFS-DCIS versus LFS-IDC. Abbreviations: ACT, T-cell activation; CK, cytokeratin; DCIS, ductal carcinoma in situ; ER, estrogen receptor; Grzmb, granzyme B; HER2: receptor tyrosine-protein kinase erbB-2; IDC, invasive ductal carcinoma; LFS, Li Fraumeni Syndrome; TIL, tumor infiltrating lymphocytes; TNBC, triple negative breast cancer.