Epigenetic activation of SOX11 is associated with recurrence and progression of ductal carcinoma in situ to invasive breast cancer.

BACKGROUND: Risk of recurrence and progression of ductal carcinoma in situ (DCIS) to invasive cancer remains uncertain, emphasizing the need for developing predictive biomarkers of aggressive DCIS. METHODS: Human cell lines and mouse models of disease progression were analyzed for candidate risk predictive biomarkers identified and validated in two independent DCIS cohorts. RESULTS: RNA profiling of normal mammary and DCIS tissues (n = 48) revealed that elevated SOX11 expression correlates with MKI67, EZH2, and DCIS recurrence score. The 21T human cell line model of DCIS progression to invasive cancer and two mouse models developing mammary intraepithelial neoplasia confirmed the findings. AKT activation correlated with chromatin accessibility and EZH2 enrichment upregulating SOX11 expression. AKT and HER2 inhibitors decreased SOX11 expression along with diminished mammosphere formation. SOX11 was upregulated in HER2+ and basal-like subtypes (P < 0.001). Longitudinal DCIS cohort (n = 194) revealed shorter recurrence-free survival in SOX11+ than SOX11- patients (P = 0.0056 in all DCIS; P < 0.0001 in HER2+ subtype) associated with increased risk of ipsilateral breast event/IBE (HR = 1.9, 95%CI = 1.2-2.9; P = 0.003). DISCUSSION: Epigenetic activation of SOX11 drives recurrence of DCIS and progression to invasive cancer, suggesting SOX11 as a predictive biomarker of IBE.

Archival single-cell genomics reveals persistent subclones during DCIS progression.

Ductal carcinoma in situ (DCIS) is a common precursor of invasive breast cancer. Our understanding of its genomic progression to recurrent disease remains poor, partly due to challenges associated with the genomic profiling of formalin-fixed paraffin-embedded (FFPE) materials. Here, we developed Arc-well, a high-throughput single-cell DNA-sequencing method that is compatible with FFPE materials. We validated our method by profiling 40,330 single cells from cell lines, a frozen tissue, and 27 FFPE samples from breast, lung, and prostate tumors stored for 3-31 years. Analysis of 10 patients with matched DCIS and cancers that recurred 2-16 years later show that many primary DCIS had already undergone whole-genome doubling and clonal diversification and that they shared genomic lineages with persistent subclones in the recurrences. Evolutionary analysis suggests that most DCIS cases in our cohort underwent an evolutionary bottleneck, and further identified chromosome aberrations in the persistent subclones that were associated with recurrence.

Genomic analysis defines clonal relationships of ductal carcinoma in situ and recurrent invasive breast cancer.

Ductal carcinoma in situ (DCIS) is the most common form of preinvasive breast cancer and, despite treatment, a small fraction (5-10%) of DCIS patients develop subsequent invasive disease. A fundamental biologic question is whether the invasive disease arises from tumor cells in the initial DCIS or represents new unrelated disease. To address this question, we performed genomic analyses on the initial DCIS lesion and paired invasive recurrent tumors in 95 patients together with single-cell DNA sequencing in a subset of cases. Our data show that in 75% of cases the invasive recurrence was clonally related to the initial DCIS, suggesting that tumor cells were not eliminated during the initial treatment. Surprisingly, however, 18% were clonally unrelated to the DCIS, representing new independent lineages and 7% of cases were ambiguous. This knowledge is essential for accurate risk evaluation of DCIS, treatment de-escalation strategies and the identification of predictive biomarkers.

Frequency of Pathogenic Germline Variants in CDH1, BRCA2, CHEK2, PALB2, BRCA1, and TP53 in Sporadic Lobular Breast Cancer.

BACKGROUND: Invasive lobular breast cancer (ILC) accounts for approximately 15% of invasive breast carcinomas and is commonly associated with lobular carcinoma in situ (LCIS). Both have been shown to have higher familial risks than the more common ductal cancers. However, there are little data on the prevalence of the known high and moderate penetrance breast cancer predisposition genes in ILC. The aim of this study was to assess the frequency of germline variants in CDH1, BRCA2, BRCA1, CHEK2, PALB2, and TP53 in sporadic ILC and LCIS diagnosed in women ages ≤60 years. METHODS: Access Array technology (Fluidigm) was used to amplify all exons of CDH1, BRCA2, BRCA1, TP53, CHEK2, and PALB2 using a custom-made targeted sequencing panel in 1,434 cases of ILC and 368 cases of pure LCIS together with 1,611 controls. RESULTS: Case-control analysis revealed an excess of pathogenic variants in BRCA2, CHEK2, PALB2, and CDH1 in women with ILC. CHEK2 was the only gene that showed an association with pure LCIS [OR = 9.90; 95% confidence interval (CI), 3.42-28.66, P = 1.4 × 10-5] with a larger effect size seen in LCIS compared with ILC (OR = 4.31; 95% CI, 1.61-11.58, P = 1.7 × 10-3). CONCLUSIONS: Eleven percent of patients with ILC ages ≤40 years carried germline variants in known breast cancer susceptibility genes. IMPACT: Women with ILC ages ≤40 years should be offered genetic screening using a panel of genes that includes BRCA2, CHEK2, PALB2, and CDH1.

Frequency of pathogenic germline variants in BRCA1, BRCA2, PALB2, CHEK2 and TP53 in ductal carcinoma in situ diagnosed in women under the age of 50 years.

INTRODUCTION: Ductal carcinoma in situ (DCIS) is a non-obligate precursor of invasive ductal breast cancer, and approximately 20% of screen-detected tumours are pure DCIS. Most risk factors for breast cancer have similar associations with DCIS and IDC; however, there is limited data on the prevalence of the known high and moderate penetrance breast cancer predisposition genes in DCIS and which women with DCIS should be referred for genetic screening. The aim of this study was to assess the frequency of germline variants in BRCA2, BRCA1, CHEK2, PALB2 and TP53 in DCIS in women aged less than 50 years of age. METHODS: After DNA extraction from the peripheral blood, Access Array technology (Fluidigm) was used to amplify all exons of these five known breast cancer predisposition genes using a custom made targeted sequencing panel in 655 cases of pure DCIS presenting in women under the age of 50 years together with 1611 controls. RESULTS: Case-control analysis revealed an excess of pathogenic variants in BRCA2 (OR = 27.96, 95%CI 6.56-119.26, P = 2.0 × 10-10) and CHEK2 (OR = 8.04, 95%CI 2.93-22.05, P = 9.0 × 10-6), with weaker associations with PALB2 (P = 0.003), BRCA1 (P = 0.007) and TP53 (P = 0.02). For oestrogen receptor (ER)-positive DCIS the frequency of pathogenic variants was 9% under the age of 50 (14% with a family history of breast cancer) and 29% under the age of 40 (42% with a family history of breast cancer). For ER-negative DCIS, the frequency was 9% (16% with a family history of breast cancer) and 8% (11% with a family history of breast cancer) under the ages of 50 and 40, respectively. CONCLUSIONS: This study has shown that breast tumourigenesis in women with pathogenic variants in BRCA2, CHEK2, PALB2, BRCA1 and TP53 can involve a DCIS precursor stage and that the focus of genetic testing in DCIS should be on women under the age of 40 with ER-positive DCIS.

Carboplatin in BRCA1/2-mutated and triple-negative breast cancer BRCAness subgroups: the TNT Trial.

Germline mutations in BRCA1/2 predispose individuals to breast cancer (termed germline-mutated BRCA1/2 breast cancer, gBRCA-BC) by impairing homologous recombination (HR) and causing genomic instability. HR also repairs DNA lesions caused by platinum agents and PARP inhibitors. Triple-negative breast cancers (TNBCs) harbor subpopulations with BRCA1/2 mutations, hypothesized to be especially platinum-sensitive. Cancers in putative 'BRCAness' subgroups-tumors with BRCA1 methylation; low levels of BRCA1 mRNA (BRCA1 mRNA-low); or mutational signatures for HR deficiency and those with basal phenotypes-may also be sensitive to platinum. We assessed the efficacy of carboplatin and another mechanistically distinct therapy, docetaxel, in a phase 3 trial in subjects with unselected advanced TNBC. A prespecified protocol enabled biomarker-treatment interaction analyses in gBRCA-BC and BRCAness subgroups. The primary endpoint was objective response rate (ORR). In the unselected population (376 subjects; 188 carboplatin, 188 docetaxel), carboplatin was not more active than docetaxel (ORR, 31.4% versus 34.0%, respectively; P = 0.66). In contrast, in subjects with gBRCA-BC, carboplatin had double the ORR of docetaxel (68% versus 33%, respectively; biomarker, treatment interaction P = 0.01). Such benefit was not observed for subjects with BRCA1 methylation, BRCA1 mRNA-low tumors or a high score in a Myriad HRD assay. Significant interaction between treatment and the basal-like subtype was driven by high docetaxel response in the nonbasal subgroup. We conclude that patients with advanced TNBC benefit from characterization of BRCA1/2 mutations, but not BRCA1 methylation or Myriad HRD analyses, to inform choices on platinum-based chemotherapy. Additionally, gene expression analysis of basal-like cancers may also influence treatment selection.

MED12, TERT promoter and RBM15 mutations in primary and recurrent phyllodes tumours.

BACKGROUND: MED12 and TERT promoter mutations have been shown to be the most common somatic mutations in phyllodes tumours (PTs). The aims of this study were to determine the frequency of these mutations in recurrent PTs, assess whether TERT promoter mutations could be helpful in distinguishing fibroadenomas (FAs) from PTs and identify novel mutations that may be driving malignant progression. METHODS: MED12 and the TERT promoter were Sanger sequenced in 75 primary PTs, 21 recurrences, 19 single FAs and 2 cases of multiple FAs with benign PTs. Whole-exome sequencing was performed on one borderline PT. RESULTS: Recurrent PTs and multiple FAs showed temporal discordance in MED12 but not TERT. Recurrent samples did acquire TERT mutations, with recurrent benign PTs more likely to have mutations in both genes. TERT mutations were not helpful in differentiating between benign PTs and FAs in cases of multiple FAs/PTs. Exome sequencing revealed a nonsense mutation in RBM15 and Sanger sequencing revealed another three RBM15 mutations in malignant/borderline PTs. CONCLUSIONS: This study has shown that MED12 mutations can be heterogeneous in both synchronous and recurrent PTs unlike TERT mutations. We have also shown that RBM15 mutations may be important in the pathogenesis of borderline/malignant PTs.

SOX11 promotes invasive growth and ductal carcinoma in situ progression.

Here, we show that SOX11, an embryonic mammary marker that is normally silent in postnatal breast cells, is expressed in many oestrogen receptor-negative preinvasive ductal carcinoma in situ (DCIS) lesions. Mature mammary epithelial cells engineered to express SOX11 showed alterations in progenitor cell populations, including an expanded basal-like population with increased aldehyde dehydrogenase (ALDH) activity, and increased mammosphere-forming capacity. DCIS.com cells engineered to express SOX11 showed increased ALDH activity, which is a feature of cancer stem cells. The CD44+/CD24-/ALDH+ cell population was increased in DCIS.com cells that expressed SOX11. Upregulating SOX11 expression in DCIS.com cells led to increased invasive growth both in vitro and when they were injected intraductally in a mouse model of DCIS that recapitulates human disease. Invasive lesions formed sooner and tumour growth was augmented in vivo, suggesting that SOX11 contributes to the progression of DCIS to invasive breast cancer. We identified potential downstream effectors of SOX11 during both microinvasive and invasive tumour growth stages, including several with established links to regulation of progenitor cell function and prenatal developmental growth. Our findings suggest that SOX11 is a potential biomarker for DCIS lesions containing cells harbouring distinct biological features that are likely to progress to invasive breast cancer. © 2017 The Authors. The Journal of Pathology published by John Wiley & Sons Ltd on behalf of Pathological Society of Great Britain and Ireland.

PIK3CA mutations are common in lobular carcinoma in situ, but are not a biomarker of progression.

BACKGROUND: Lobular carcinoma in situ (LCIS) is a non-invasive breast lesion that is typically found incidentally on biopsy and is often associated with invasive lobular carcinoma (ILC). LCIS is considered by some to be a risk factor for future breast cancer rather than a true precursor lesion. The aim of this study was to identify genetic changes that could be used as biomarkers of progression of LCIS to invasive disease using cases of pure LCIS and comparing their genetic profiles to LCIS which presented contemporaneously with associated ILC, on the hypothesis that the latter represents LCIS that has already progressed. METHODS: Somatic copy number aberrations (SCNAs) were assessed by SNP array in three subgroups: pure LCIS, LCIS associated with ILC and the paired ILC. In addition exome sequencing was performed on seven fresh frozen samples of LCIS associated with ILC, to identify recurrent somatic mutations. RESULTS: The copy number profiles of pure LCIS and LCIS associated with ILC were almost identical. However, four SCNAs were more frequent in ILC than LCIS associated with ILC, including gain/amplification of CCND1. CCND1 protein over-expression assessed by immunohistochemical analysis in a second set of samples from 32 patients with pure LCIS and long-term follow up, was associated with invasive recurrence (P = 0.02, Fisher's exact test). Exome sequencing revealed that PIK3CA mutations were as frequent as CDH1 mutations in LCIS, but were not a useful biomarker of LCIS progression as they were as frequent in pure LCIS as in LCIS associated with ILC. We also observed heterogeneity of PIK3CA mutations and evidence of sub-clonal populations in LCIS irrespective of whether they were associated with ILC. CONCLUSIONS: Our data shows that pure LCIS and LCIS co-existing with ILC have very similar SCNA profiles, supporting the hypothesis that LCIS is a true precursor lesion. We have provided evidence that over-expression of CCND1 may identify a subgroup of patients with pure LCIS who are more likely to develop invasive disease, in contrast to PIK3CA mutations, which occur too early in lobular tumorigenesis to be informative.

Genetic predisposition to ductal carcinoma in situ of the breast.

BACKGROUND: Ductal carcinoma in situ (DCIS) is a non-invasive form of breast cancer. It is often associated with invasive ductal carcinoma (IDC), and is considered to be a non-obligate precursor of IDC. It is not clear to what extent these two forms of cancer share low-risk susceptibility loci, or whether there are differences in the strength of association for shared loci. METHODS: To identify genetic polymorphisms that predispose to DCIS, we pooled data from 38 studies comprising 5,067 cases of DCIS, 24,584 cases of IDC and 37,467 controls, all genotyped using the iCOGS chip. RESULTS: Most (67 %) of the 76 known breast cancer predisposition loci showed an association with DCIS in the same direction as previously reported for invasive breast cancer. Case-only analysis showed no evidence for differences between associations for IDC and DCIS after considering multiple testing. Analysis by estrogen receptor (ER) status confirmed that loci associated with ER positive IDC were also associated with ER positive DCIS. Analysis of DCIS by grade suggested that two independent SNPs at 11q13.3 near CCND1 were specific to low/intermediate grade DCIS (rs75915166, rs554219). These associations with grade remained after adjusting for ER status and were also found in IDC. We found no novel DCIS-specific loci at a genome wide significance level of P < 5.0x10(-8). CONCLUSION: In conclusion, this study provides the strongest evidence to date of a shared genetic susceptibility for IDC and DCIS. Studies with larger numbers of DCIS are needed to determine if IDC or DCIS specific loci exist.

Genomic Complexity Profiling Reveals That HORMAD1 Overexpression Contributes to Homologous Recombination Deficiency in Triple-Negative Breast Cancers.

UNLABELLED: Triple-negative breast cancers (TNBC) are characterized by a wide spectrum of genomic alterations, some of which might be caused by defects in DNA repair processes such as homologous recombination (HR). Despite this understanding, associating particular patterns of genomic instability with response to therapy has been challenging. Here, we show that allelic-imbalanced copy-number aberrations (AiCNA) are more prevalent in TNBCs that respond to platinum-based chemotherapy, thus providing a candidate predictive biomarker for this disease. Furthermore, we show that a high level of AiCNA is linked with elevated expression of a meiosis-associated gene, HORMAD1. Elevated HORMAD1 expression suppresses RAD51-dependent HR and drives the use of alternative forms of DNA repair, the generation of AiCNAs, as well as sensitizing cancer cells to HR-targeting therapies. Our data therefore provide a mechanistic association between HORMAD1 expression, a specific pattern of genomic instability, and an association with response to platinum-based chemotherapy in TNBC. SIGNIFICANCE: Previous studies have shown correlation between mutational "scars" and sensitivity to platinums extending beyond associations with BRCA1/2 mutation, but do not elucidate the mechanism. Here, a novel allele-specific copy-number characterization of genome instability identifies and functionally validates the inappropriate expression of the meiotic gene HORMAD1 as a driver of HR deficiency in TNBC, acting to induce allelic imbalance and moderate platinum and PARP inhibitor sensitivity with implications for the use of such "scars" and expression of meiotic genes as predictive biomarkers.

The proteasomal de-ubiquitinating enzyme POH1 promotes the double-strand DNA break response.

The regulation of Ubiquitin (Ub) conjugates generated by the complex network of proteins that promote the mammalian DNA double-strand break (DSB) response is not fully understood. We show here that the Ub protease POH1/rpn11/PSMD14 resident in the 19S proteasome regulatory particle is required for processing poly-Ub formed in the DSB response. Proteasome activity is required to restrict tudor domain-dependent 53BP1 accumulation at sites of DNA damage. This occurs both through antagonism of RNF8/RNF168-mediated lysine 63-linked poly-Ub and through the promotion of JMJD2A retention on chromatin. Consistent with this role POH1 acts in opposition to RNF8/RNF168 to modulate end-joining DNA repair. Additionally, POH1 acts independently of 53BP1 in homologous recombination repair to promote RAD51 loading. Accordingly, POH1-deficient cells are sensitive to DNA damaging agents. These data demonstrate that proteasomal POH1 is a key de-ubiquitinating enzyme that regulates ubiquitin conjugates generated in response to damage and that several aspects of the DSB response are regulated by the proteasome.