Pathogenic Variants in CHEK2 Are Associated With an Adverse Prognosis in Symptomatic Early-Onset Breast Cancer.

PURPOSE: Checkpoint kinase 2 (CHEK2) is frequently included in multigene panels. We describe the associated outcomes among carriers of CHEK2 pathogenic variants in young patients with symptomatic breast cancer. PATIENTS AND METHODS: Participants (N = 2,344) in the Prospective Outcomes in Sporadic Versus Hereditary Breast Cancer study had a diagnosis of primary invasive breast cancer at age ≤ 40 years. Summary statistics were used to compare tumor characteristics among CHEK2+ carriers with those who were CHEK2-. Kaplan-Meier curves were used to demonstrate overall survival (OS) and distant disease-free survival. RESULTS: Overall, 53 of the 2,344 participants (2.3%) had a pathogenic CHEK2 variant. CHEK2+-associated tumors were significantly more likely to be grade 2, estrogen receptor and progesterone receptor-positive compared with CHEK2- tumors (grade 2, n = 28 of 52 [53.8%] v n = 803 of 2,229 [36.0%]; P = .029). CHEK2-associated tumors were significantly more likely to have nodal involvement (N1, n = 37 of 53 [69.8%] v 1,169 of 2,253 [51.9%]; P = .0098) and demonstrated a trend toward multifocality. A higher proportion of participants with CHEK2+ variants with invasive breast cancer were obese than were those with CHEK2- variant (28.3% v 18.8%; P = .039). Univariate and multivariable analyses revealed that OS and distant disease-free survival were significantly worse in CHEK2+ versus CHEK2- carriers (OS hazard ratio, 1.58; 95% CI, 1.01 to 2.48; P = .043). CONCLUSION: This work highlights the adverse prognosis associated with breast cancer in carriers of CHEK2 pathogenic variants. It also identifies a potential association among obesity, family history, and breast cancer risk in young CHEK2 gene carriers.

Breast cancer in patients with germline TP53 pathogenic variants have typical tumour characteristics: the Cohort study of TP53 carrier early onset breast cancer (COPE study).

Germline TP53 pathogenic variants are rare but associated with a high risk of cancer; they are often identified in the context of clinically diagnosed Li-Fraumeni syndrome predisposing to a range of young onset cancers including sarcomas and breast cancer. The study aim was to conduct a detailed morphological review and immuno-phenotyping of breast cancer arising in carriers of a germline TP53 pathogenic variant. We compared breast cancers from five defined groups: (1) TP53 carriers with breast cancer (n = 59), (2) early onset HER2-amplified breast cancer, no germline pathogenic variant in BRCA1/2 or TP53 (n = 55), (3) BRCA1 pathogenic variant carriers (n = 60); (4) BRCA2 pathogenic variant carriers (n = 61) and (5) young onset breast cancer with no known germline pathogenic variant (n = 98). Pathologists assessed a pre-agreed set of morphological characteristics using light microscopy. Immunohistochemistry (IHC) for HER2, ER, PR, p53, integrin alpha v beta 6 (αvß6) integrin, α-smooth muscle actin (α-SMA) and pSMAD2/3 was performed on tissue microarrays of invasive carcinoma. We confirmed a previously reported high prevalence of HER2-amplified, ductal no special type invasive breast carcinoma amongst known TP53 germline pathogenic variant carriers 20 of 36 (56%). Furthermore we observed a high frequency of densely sclerotic tumour stroma in cancers from TP53 carriers (29/36, 80.6%) when compared with non-carriers, 50.9% (28/55), 34.7% (50/144), 41.4% (65/157), 43.8% (95/217) in groups 2-5 respectively. The majority of germline TP53 gene carrier breast tumours had a high intensity of integrin αvß6, α-SMA and pSMAD2/3 expression in the majority of cancer cells. In conclusion, aggressive HER2 positive breast cancers with densely sclerotic stroma are common in germline TP53 carriers. High levels of αvß6 integrin, α-SMA and pSMAD2/3 expression suggest that the dense stromal phenotype may be driven by upregulated transforming growth factor beta signalling.

Loss of MMP-8 in ductal carcinoma in situ (DCIS)-associated myoepithelial cells contributes to tumour promotion through altered adhesive and proteolytic function.

BACKGROUND: Normal myoepithelial cells (MECs) play an important tumour-suppressor role in the breast but display an altered phenotype in ductal carcinoma in situ (DCIS), gaining tumour-promoter functions. Matrix metalloproteinase-8 (MMP-8) is expressed by normal MECs but is lost in DCIS. This study investigated the function of MMP-8 in MECs and the impact of its loss in DCIS. METHODS: Primary normal and DCIS-associated MECs, and normal (N-1089) and DCIS-modified myoepithelial (ß6-1089) cell lines, were used to assess MMP-8 expression and function. ß6-1089 lacking MMP-8 were transfected with MMP-8 WT and catalytically inactive MMP-8 EA, and MMP-8 in N-1089 MEC was knocked down with siRNA. The effect on adhesion and migration to extracellular matrix (ECM), localisation of α6ß4 integrin to hemidesmosomes (HD), TGF-ß signalling and gelatinase activity was measured. The effect of altering MEC MMP-8 expression on tumour cell invasion was investigated in 2D and 3D organotypic models. RESULTS: Assessment of primary cells and MEC lines confirmed expression of MMP-8 in normal MEC and its loss in DCIS-MEC. Over-expression of MMP-8 WT but not MMP-8 EA in ß6-1089 cells increased adhesion to ECM proteins and reduced migration. Conversely, knock-down of MMP-8 in N-1089 reduced adhesion and increased migration. Expression of MMP-8 WT in ß6-1089 led to greater localisation of α6ß4 to HD and reduced retraction fibre formation, this being reversed by MMP-8 knock-down in N-1089. Over-expression of MMP-8 WT reduced TGF-ß signalling and gelatinolytic activity. MMP-8 knock-down enhanced TGF-ß signalling and gelatinolytic activity, which was reversed by blocking MMP-9 by knock-down or an inhibitor. MMP-8 WT but not MMP-8 EA over-expression in ß6-1089 reduced breast cancer cell invasion in 2D and 3D invasion assays, while MMP-8 knock-down in N-1089 enhanced cancer cell invasion. Staining of breast cancer cases for MMP-8 revealed a statistically significant loss of MMP-8 expression in DCIS with invasion versus pure DCIS (p = 0.001). CONCLUSIONS: These data indicate MMP-8 is a vital component of the myoepithelial tumour-suppressor function. It restores MEC interaction with the matrix, opposes TGF-ß signalling and MMP-9 proteolysis, which contributes to inhibition of tumour cell invasion. Assessment of MMP-8 expression may help to determine risk of DCIS progression.

The Initiator Methionine tRNA Drives Secretion of Type II Collagen from Stromal Fibroblasts to Promote Tumor Growth and Angiogenesis.

Expression of the initiator methionine tRNA (tRNAi(Met)) is deregulated in cancer. Despite this fact, it is not currently known how tRNAi(Met) expression levels influence tumor progression. We have found that tRNAi(Met) expression is increased in carcinoma-associated fibroblasts, implicating deregulated expression of tRNAi(Met) in the tumor stroma as a possible contributor to tumor progression. To investigate how elevated stromal tRNAi(Met) contributes to tumor progression, we generated a mouse expressing additional copies of the tRNAi(Met) gene (2+tRNAi(Met) mouse). Growth and vascularization of subcutaneous tumor allografts was enhanced in 2+tRNAi(Met) mice compared with wild-type littermate controls. Extracellular matrix (ECM) deposited by fibroblasts from 2+tRNAi(Met) mice supported enhanced endothelial cell and fibroblast migration. SILAC mass spectrometry indicated that elevated expression of tRNAi(Met) significantly increased synthesis and secretion of certain types of collagen, in particular type II collagen. Suppression of type II collagen opposed the ability of tRNAi(Met)-overexpressing fibroblasts to deposit pro-migratory ECM. We used the prolyl hydroxylase inhibitor ethyl-3,4-dihydroxybenzoate (DHB) to determine whether collagen synthesis contributes to the tRNAi(Met)-driven pro-tumorigenic stroma in vivo. DHB had no effect on the growth of syngeneic allografts in wild-type mice but opposed the ability of 2+tRNAi(Met) mice to support increased angiogenesis and tumor growth. Finally, collagen II expression predicts poor prognosis in high-grade serous ovarian carcinoma. Taken together, these data indicate that increased tRNAi(Met) levels contribute to tumor progression by enhancing the ability of stromal fibroblasts to synthesize and secrete a type II collagen-rich ECM that supports endothelial cell migration and angiogenesis.