Acquired ESR1 mutations are a major mechanism of resistance to aromatase inhibitors (AI). We developed ultra-high sensitivity multiplexed digital PCR assays for ESR1 mutations in circulating tumor DNA (ctDNA) and used these to investigate the clinical relevance and origin of ESR1 mutations in a cohort of 171 women with advanced breast cancer. ESR1 mutation status in ctDNA showed high concordance with contemporaneous tumor biopsies, and could be assessed in samples shipped at room temperature in preservative tubes without loss of accuracy. ESR1 mutations were found exclusively in patients with estrogen receptor positive breast cancer previously exposed to AI. Patients with ESR1 mutations had a substantially shorter progression-free survival on subsequent AI-based therapy (HR 3.1, 95%CI 1.9-23.1, log rank p=0.0041). ESR1 mutation prevalence differed markedly between patients that were first exposed to AI during the adjuvant and metastatic settings (5.8% (3/52) vs 36.4% (16/44) respectively, p=0.0002). In an independent cohort, ESR1 mutations were identified in 0% (0/32, 95%CI 0-10.9%) tumor biopsies taken after progression on adjuvant AI. In a patient with serial samples taken during metastatic treatment, ESR1 mutation was selected during metastatic AI therapy, to become the dominant clone in the cancer. ESR1 mutations can be robustly identified with ctDNA analysis and predict for resistance to subsequent AI therapy. ESR1 mutations are rarely acquired during adjuvant AI therapy, but are commonly selected by therapy for metastatic disease, providing evidence that the mechanisms of resistance to targeted therapy may be substantially different between the treatment of micro-metastatic and overt metastatic cancer.

ROR provides more prognostic information in endocrine-treated patients with ER-positive, node-negative disease than RS, with better differentiation of intermediate- and higher-risk groups.

Inositol polyphosphate 4-phosphatase-II (INPP4B) is a regulator of the phosphoinositide 3-kinase (PI3K) signaling pathway and is implicated as a tumor suppressor in epithelial carcinomas. INPP4B loss of heterozygosity (LOH) is detected in some human breast cancers; however, the expression of INPP4B protein in breast cancer subtypes and the normal breast is unknown. We report here that INPP4B is expressed in nonproliferative estrogen receptor (ER)-positive cells in the normal breast, and in ER-positive, but not negative, breast cancer cell lines. INPP4B knockdown in ER-positive breast cancer cells increased Akt activation, cell proliferation, and xenograft tumor growth. Conversely, reconstitution of INPP4B expression in ER-negative, INPP4B-null human breast cancer cells reduced Akt activation and anchorage-independent growth. INPP4B protein expression was frequently lost in primary human breast carcinomas, associated with high clinical grade and tumor size and loss of hormone receptors and was lost most commonly in aggressive basal-like breast carcinomas. INPP4B protein loss was also frequently observed in phosphatase and tensin homolog (PTEN)-null tumors. These studies provide evidence that INPP4B functions as a tumor suppressor by negatively regulating normal and malignant mammary epithelial cell proliferation through regulation of the PI3K/Akt signaling pathway, and that loss of INPP4B protein is a marker of aggressive basal-like breast carcinomas. phosphatidylinositol 3,4-bisphosphate

SUMMARY BACKGROUND Greater than 50% of recurrences in estrogen receptor-positive (ER+) breast cancer occur after 5 years of adjuvant endocrine therapy. Biomarkers capable of improving the risk-benefit of extended adjuvant endocrine therapy for these late recurrences would be clinically valuable. We compared the prognostic ability of the Breast Cancer Index (BCI), Oncotype DX Recurrence Score (RS) and IHC4 for both early and late recurrence among patients with ER+, node negative (N0) disease within the ATAC clinical trial. METHODS BCI was performed from 1102 primary tumor samples from ER+ patients and two versions (BCI-C (primary) and BCI-L (secondary), based on cubic and linear combinations of the variables) were evaluated. RS and IHC4 values were previously derived. Prognostic discrimination for early (<5y) and late recurrence (5–10y) was assessed. To evaluate the ability of the biomarkers to predict recurrence beyond standard clinicopathological parameters, the likelihood-ratio chi-square (LR-Δχ2) was calculated from Cox proportional hazards models. The primary endpoint was distant recurrence (DR). FINDINGS In the primary analysis of 665 ER+ N0 patients, categorical BCI-C demonstrated significant differences in risk of DR over 10 years (P<0·0001). In the secondary analysis, BCI-L proved to be a much stronger predictor, and BCI-L, IHC4 and RS had significant prognostic performance for early DR (BCI-L, p<0·0002), while only BCI-L was significant for late DR (LR-Δχ2: 7·97, p=0·0048). For risk of early DR at 5 years, BCI-L classified 59% (390/665), 25% (166/665) and 16% (109/665) of patients with 1.3% (0.5% – 3.1%), 5.6% (2.9% – 10.5%) and 18.1% (12.0% – 27.0%) for low, intermediate and high risk, respectively. For risk of late DR at 10 years, BCI-L classified 61% (366/596), 25% (146/596) and 14% (84/596) of patients with 3.5% (2.0% – 6.1%), 13.4% (8.5% – 20.8%) and 13.3% (7.4% – 23.4%) for low, intermediate and high, respectively. INTERPRETATION While all three biomarkers predicted for early DR, BCI-L was the only significant prognostic for risk of late DR. The three BCI-L groups identified two risk populations for both early and late DR with 84% (556/665) of patients having low risk for early DR, and a smaller population (39%, 230/596) having high risk for late DR who may benefit from extended endocrine or other therapy. FUNDING Avon Foundation, National Institutes of Health, Breast Cancer Foundation, DOD Breast Cancer Research Program, Susan G. Komen for the Cure, Breakthrough Breast Cancer through the Mary-Jean Mitchell Green Foundation, Astrazeneca, NIHR Biomedical Research Centre at the Royal Marsden.

The findings of this large study strongly support the notion that FGFR3 mutations characterize a subgroup of bladder cancers with good prognosis; patients with mutant TaG1 tumors have a higher risk of recurrence; and the F386L variant is selectively associated with low-grade tumors.

Urothelial bladder cancer (UBC) is heterogeneous at the clinical, pathological, and genetic levels. Tumor invasiveness (T) and grade (G) are the main factors associated with outcome and determine patient management (1). A discovery exome sequencing screen (n=17), followed by a prevalence screen (n=60), identified new genes mutated in this tumor coding for proteins involved in chromatin modification (MLL2, ASXL2, BPTF), cell division (STAG2, SMC1A, SMC1B), and DNA repair (ATM, ERCC2, FANCA). STAG2, a subunit of cohesin, was significantly and commonly mutated/lost in UBC, mainly in tumors of low stage/grade, and its loss was associated with improved outcome. Loss of expression was often observed in chromosomally-stable tumors and STAG2 knockdown in bladder cancer cells did not increase aneuploidy. STAG2 reintroduction in non-expressing cells led to reduced colony formation. Our findings indicate that STAG2 is a novel UBC tumor suppressor acting through mechanisms that are different from its role to prevent aneuploidy.

BackgroundAdjuvant endocrine therapy beyond 5 years reduces recurrence in patients with estrogen receptor–positive breast cancer. We have previously shown that immunohistochemical markers (IHC4) and two gene expression profile tests (recurrence score [RS] and PAM50 risk of recurrence [ROR]) are associated with time to distant recurrence, and we have now assessed the value of each of these scores and routine clinical variables for predicting outcome, specifically in years 5 to 10.MethodsWe used univariate and multivariable proportional hazards models to determine the prognostic value of all variables and scores (IHC4, RS, ROR) for distant recurrence, separately in years 0 to 5 and specifically for years 5 to 10 for all patients. All statistical tests were two-sided.ResultsNodal status and tumor size were at least as strong in years 5 to 10 as in years 0 to 5 (nodal status, years 5–10: χ2 = 21.72 vs years 0–5: χ2 = 11.08, both P < .001; tumor size, years 5–10: χ2 = 10.52 vs years 0–5: χ2 = 10.82, both P = .001). Ki67 and the overall IHC4 score were the only statistically significant biomarkers related to distant recurrence univariablely in the 5 to 10 year period (χ2 = 8.67, χ2 = 13.22, respectively). The ROR score was the strongest molecular prognostic factor in the late follow-up period (χ2 = 16.29; P < .001), whereas IHC4 (χ2 = 7.41) and RS (χ2 = 5.55) were only weakly prognostic in this period. Similar results were seen for all subgroups and for all recurrences.ConclusionsNone of the IHC4 markers provided statistically significant prognostic information in years 5 to 10, except for nodal status and tumor size. ROR gave the strongest prognostic information in years 5 to 10. These results may help select patients who could benefit most from hormonal therapy beyond 5 years of treatment.

Breast cancer is a common malignancy with current biological therapies tailored to steroid hormone (ER, PR) and HER2 receptor status. Understanding the biological basis of resistance to current targeted therapies and the identification of new potential therapeutic targets is an ongoing challenge. The PI3K pathway is altered in a high proportion of breast cancers and may contribute to therapeutic resistance. We undertook an integrative study of mutational, copy number and expression analyses of key regulators of the PI3K pathway in a cohort of 292 invasive breast cancer patients with known treatment outcomes. The alterations identified in this cohort included PIK3CA mutations (12/168, i.e. 7%), PIK3CA copy number gain (28/209, i.e. 14%), PTEN loss (73/258, i.e. 28%) and AKT activation (62/258, i.e. 24%). Overall at least 1 parameter was altered in 72% (139/193) of primary breast cancers. PI3K pathway activation was significantly associated with ER negative (p 5 0.0008) and PR negative (p 5 0.006) status, high tumor grade (p 5 0.032) and a ''basal-like'' phenotype (p 5 0.01), where 92% (25/27) of tumors had an altered pathway. In univariate analysis, PI3K pathway aberrations were associated with death from breast cancer; however, this relationship was not maintained in multivariate analysis. No association was identified between an activated pathway and outcome in tamoxifen-or chemotherapy-treated patients. We concluded that >70% of breast cancers have an alteration in at least 1 component of the PI3K pathway and this might be exploited to therapeutic advantage especially in ''basal-like'' cancers.The phosphatidylinositol 3 kinase (PI3K) pathway regulates many cellular functions, mainly associated with cell proliferation, survival and migration.1 Activation of PI3K can occur in response to a variety of extracellular signals through growth factor receptor-or integrin-mediated pathways. Upon receptor activation, the p85 regulatory subunit of Class 1a PI3K is recruited to phosphotyrosine residues within the receptor. Once recruited to the membrane, the p110a catalytic subunit, encoded by PIK3CA, phosphorylates phosphatidylinositol biphosphate to generate phosphatidylinositol triphosphate (PIP3), which recruits phosphoinositide-dependent kinase 1 (PDK1) and V-Akt murine thymoma viral oncogene homolog 1 (AKT) to the cell membrane. PDK1 and PDK2 (likely the TORC2 complex) then phosphorylate AKT leading to the regulation of an array of downstream pathways including forkhead rhabdomyosarcoma, GSK3b and BAD with consequent effects on cell proliferation, cell metabolism and apoptosis, respectively. PIP3 is a substrate for phosphatase and tensin homologue deleted on chromosome 10 (PTEN), a well-described tumor suppressor protein, which dephosphorylates PIP3, inactivating the PI3K pathway.Alterations in the PI3K pathway have been documented in many human cancers.2 In breast cancer, PIK3CA activating mutations, in exons 9 and 20, have been identified in $24% (range, 12-40%) of patient specimens. [3][4][5][6][7][8][9][10][11][1...