Abrogated Response to Cellular Stress Identifies DCIS Associated with Subsequent Tumor Events and Defines Basal-like Breast Tumors

SummaryApproximately 15%–30% of women diagnosed with ductal carcinoma in situ (DCIS) develop a subsequent tumor event within 10 years after surgical lumpectomy. To date, little is known about the molecular pathways that confer this differential risk for developing subsequent disease. In this study, we demonstrate that expression of biomarkers indicative of an abrogated response to cellular stress predicts DCIS with worse outcome and is a defining characteristic of basal-like invasive tumors. Mechanistic studies identify the Rb pathway as a key regulator of this response. Conversely, biomarkers indicative of an intact response to cellular stress are strongly associated with a disease-free prognosis. Assessment of these biomarkers in DCIS begins to allow prediction of tumor formation years before it actually occurs

Development and validation of a targeted gene sequencing panel for application to disparate cancers

Next generation sequencing has revolutionised genomic studies of cancer, having facilitated the development of precision oncology treatments based on a tumour’s molecular profile. We aimed to develop a targeted gene sequencing panel for application to disparate cancer types with particular focus on tumours of the head and neck, plus test for utility in liquid biopsy. The final panel designed through Roche/Nimblegen combined 451 cancer-associated genes (2.01 Mb target region). 136 patient DNA samples were collected for performance and application testing. Panel sensitivity and precision were measured using well-characterised DNA controls (n = 47), and specificity by Sanger sequencing of the Aryl Hydrocarbon Receptor Interacting Protein (AIP) gene in 89 patients. Assessment of liquid biopsy application employed a pool of synthetic circulating tumour DNA (ctDNA). Library preparation and sequencing were conducted on Illumina-based platforms prior to analysis with our accredited (ISO15189) bioinformatics pipeline. We achieved a mean coverage of 395x, with sensitivity and specificity of >99% and precision of >97%. Liquid biopsy revealed detection to 1.25% variant allele frequency. Application to head and neck tumours/cancers resulted in detection of mutations aligned to published databases. In conclusion, we have developed an analytically-validated panel for application to cancers of disparate types with utility in liquid biopsy

The FANCM:p.Arg658* truncating variant is associated with risk of triple-negative breast cancer

Abstract: Breast cancer is a common disease partially caused by genetic risk factors. Germline pathogenic variants in DNA repair genes BRCA1, BRCA2, PALB2, ATM, and CHEK2 are associated with breast cancer risk. FANCM, which encodes for a DNA translocase, has been proposed as a breast cancer predisposition gene, with greater effects for the ER-negative and triple-negative breast cancer (TNBC) subtypes. We tested the three recurrent protein-truncating variants FANCM:p.Arg658*, p.Gln1701*, and p.Arg1931* for association with breast cancer risk in 67,112 cases, 53,766 controls, and 26,662 carriers of pathogenic variants of BRCA1 or BRCA2. These three variants were also studied functionally by measuring survival and chromosome fragility in FANCM−/− patient-derived immortalized fibroblasts treated with diepoxybutane or olaparib. We observed that FANCM:p.Arg658* was associated with increased risk of ER-negative disease and TNBC (OR = 2.44, P = 0.034 and OR = 3.79; P = 0.009, respectively). In a country-restricted analysis, we confirmed the associations detected for FANCM:p.Arg658* and found that also FANCM:p.Arg1931* was associated with ER-negative breast cancer risk (OR = 1.96; P = 0.006). The functional results indicated that all three variants were deleterious affecting cell survival and chromosome stability with FANCM:p.Arg658* causing more severe phenotypes. In conclusion, we confirmed that the two rare FANCM deleterious variants p.Arg658* and p.Arg1931* are risk factors for ER-negative and TNBC subtypes. Overall our data suggest that the effect of truncating variants on breast cancer risk may depend on their position in the gene. Cell sensitivity to olaparib exposure, identifies a possible therapeutic option to treat FANCM-associated tumors

Premalignant breast neoplasia: a paradigm of interlesional and intralesional molecular heterogeneity and its biological and clinical ramifications.

As is well established in invasive breast disease, it is becoming increasingly clear that molecular heterogeneity, both between and within lesions, is a prevalent, distinct phenotype of premalignant lesions of the breast. Key pathways of tumorigenesis modulate critical features of premalignant lesions such as proliferation, differentiation, stress response, and even the generation of diversity. Current studies show that evaluation of these lesions may provide clinically useful information on future tumor formation as well as biological insights into the origin and functional significance of this distinct phenotype

Abstract 333: The role of the retinoblastoma pathway (Rb) in high grade serous ovarian varcinoma (HGSC)

Abstract The Rb pathway functions as a cell cycle checkpoint and deregulation of its components, commonly found in malignancies, causes progression from G1 to S phase, promoting cellular proliferation. Because of Rb's central role in checkpoint regulation, abrogation of the pathway can occur through multiple non-redundant mechanisms including Rb loss, hypermethylation or mutation, and CCND1 or CCNE amplification. Emerging evidence shows that tumour types can often be distinguished by particular alterations in one member of the pathway suggesting that different mechanisms of Rb abrogation may regulate tumour behaviour. We hypothesize that Rb pathway deregulation is frequent in HGSC, the most common and most aggressive histotype of ovarian cancer, and that the mechanism of Rb pathway deregulation identifies clinically distinct subgroups of HGSC. Micro-dissected epithelium from HGSC and normal FTE samples were analyzed for differential gene expression using the Affymetrix U133 Plus 2.0 gene-chips, and expression values for p53, p21, p27, p16, CCND1, CCNE and Rb genes determined. Protein expression was assessed by immunohistochemistry (IHC) on tissue microarrays composed of ovarian/tubal carcinomas inclusive of the major histotypes. Digitized stained slides were quantified using automated image analysis and correlated with clinico-pathologic variables including outcome. Rb loss of heterozygosity (LOH) was tested by an Rb diagnostics protocol involving D13S153 and RB1.2 polymorphic marker analyses using PCR amplification, followed by comparisons of the tumour and its corresponding normal sample by MicroGene Clipper sequencers. Gene expression analysis showed statistically significant over-expression of p53, CCNE E2F1/3 and p16 and down-regulation of p21 and CCND1 in HGSC compared to normal fallopian tube epithelium (p<0.001). Protein expression determined by IHC analysis of HGSC revealed a similar pattern of expression when compared to normal fallopian tube, the site of origin of this carcinoma. There were important differences in the expression of these proteins between HGSC subgroups, where up-regulation was observed for p16, CCNE, CCND1 and BIRC5 in 58.3%, 57%, 33.3%, and 58.3% respectively. Rb however, showed no statistically significant differences at the RNA level, but 40% of all HGSC profiled had a significant decrease in protein expression. Interestingly, LOH analysis revealed 76% of HGSC had Rb inactivation at the gene level. Furthermore, we observed statistically significant correlations (p=0.029) between p16 over-expression and Rb protein loss, using Fisher's Exact test. HGSC is characterized by both genetic and protein abrogation in the Rb pathway. Additionally, we observed differences in the mechanism of this G1/S checkpoint inactivation amongst HGSC patient samples which may represent important biological/clinical differences amongst sub-groups of serous cancer patients. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 333. doi:10.1158/1538-7445.AM2011-33

p16INK4a modulates p53 in primary human mammary epithelial cells.

p16(INK4a) (p16) and p53 are tumor suppressor genes that are inactivated during carcinogenesis in many tumors. Here we show that p16 gene activity inversely modulates p53 status and function in primary human mammary epithelial cells. Reduced levels of p16 protein stabilize p53 protein through inhibition of proteolytic degradation, and this increase in p53 protein levels enhances the cellular response to radiation, represses proliferation, and transcriptionally activates downstream targets. Stabilization of p53 is mediated through the retinoblastoma/E2F/p14(ARF)/murine double minute-2 pathway. However, we have observed that p16 does not modulate p53 in fibroblasts, indicating a possible cell type-specific regulation of this pathway

Histologically normal human mammary epithelia with silenced p16(INK4a) overexpress COX-2, promoting a premalignant program.

Breast tissue from healthy women contains variant mammary epithelial cells (vHMEC) exhibiting p16INK4a promoter hypermethylation both in vivo and in vitro. When continuously cultured, vHMEC acquire telomeric dysfunction and produce the types of chromosomal abnormalities seen in premalignant lesions of cancer. We find that late passage vHMEC express elevated prostaglandin cyclo-oxygenase 2 (COX-2), which contributes to increased prostaglandin synthesis, angiogenic activity, and invasive ability. These data demonstrate the existence of human mammary epithelial cells with the potential to acquire multiple genomic alterations and phenotypes associated with malignant cells. Moreover, COX-2 overexpression coincides with focal areas of p16INK4a hypermethylation in vivo, creating ideal candidates as precursors to breast cancer. These putative precursors can be selectively eliminated upon exposure to COX-2 inhibitors in vitro

Histologically normal human mammary epithelia with silenced p16(INK4a) overexpress COX-2, promoting a premalignant program.

Breast tissue from healthy women contains variant mammary epithelial cells (vHMEC) exhibiting p16INK4a promoter hypermethylation both in vivo and in vitro. When continuously cultured, vHMEC acquire telomeric dysfunction and produce the types of chromosomal abnormalities seen in premalignant lesions of cancer. We find that late passage vHMEC express elevated prostaglandin cyclo-oxygenase 2 (COX-2), which contributes to increased prostaglandin synthesis, angiogenic activity, and invasive ability. These data demonstrate the existence of human mammary epithelial cells with the potential to acquire multiple genomic alterations and phenotypes associated with malignant cells. Moreover, COX-2 overexpression coincides with focal areas of p16INK4a hypermethylation in vivo, creating ideal candidates as precursors to breast cancer. These putative precursors can be selectively eliminated upon exposure to COX-2 inhibitors in vitro