Performance of a novel spectroscopy-based tool for adjuvant therapy decision-making in hormone receptor-positive breast cancer: a validation study.

PURPOSE: Digistain Index (DI), measured using an inexpensive mid-infrared spectrometer, reflects the level of aneuploidy in unstained tissue sections and correlates with tumor grade. We investigated whether incorporating DI with other clinicopathological variables could predict outcomes in patients with early breast cancer. METHODS: DI was calculated in 801 patients with hormone receptor-positive, HER2-negative primary breast cancer and ≤ 3 positive lymph nodes. All patients were treated with systemic endocrine therapy and no chemotherapy. Multivariable proportional hazards modeling was used to incorporate DI with clinicopathological variables to generate the Digistain Prognostic Score (DPS). DPS was assessed for prediction of 5- and 10-year outcomes (recurrence, recurrence-free survival [RFS] and overall survival [OS]) using receiver operating characteristics and Cox proportional hazards regression models. Kaplan-Meier analysis evaluated the ability of DPS to stratify risk. RESULTS: DPS was consistently highly accurate and had negative predictive values for all three outcomes, ranging from 0.96 to 0.99 at 5 years and 0.84 to 0.95 at 10 years. DPS demonstrated statistically significant prognostic ability with significant hazard ratios (95% CI) for low- versus high-risk classification for RFS, recurrence and OS (1.80 [CI 1.31-2.48], 1.83 [1.32-2.52] and 1.77 [1.28-2.43], respectively; all P < 0.001). CONCLUSION: DPS showed high accuracy and predictive performance, was able to stratify patients into low or high-risk, and considering its cost and rapidity, has the potential to offer clinical utility.

Meta-Analysis of Circulating Tumor Cell PD-L1 Expression and the Association with Clinical Outcomes in Non-Small Cell Lung Cancer.

BACKGROUND: Programmed death ligand-1 (PD-L1) expression on circulating tumor cells (CTCs) has been suggested to provide prognostic information in non-small cell lung cancer (NSCLC), but consensus relative to treatment outcomes is lacking. We conducted the first comprehensive meta-analysis exploring its potential as a prognostic and predictive marker, and assessed the concordance between PD-L1 + CTCs and paired tumor tissue in NSCLC patients. METHOD: A comprehensive search was applied to PubMed and EMBASE to identify 26 studies that evaluated PD-L1 + CTCs and their association with survival outcomes in 1236 NSCLC patients. RESULTS: The meta-analysis estimated a mean PD-L1 + CTCs detection rate of 61% (95% CI, 49-72). Subgroup analysis based on treatment showed that PD-L1 + CTCs was not significantly associated with better overall survival (OS) in NSCLC patients treated with immune checkpoint inhibitors (ICIs) (Hazard Ratio (HR) = 0.96, 95% CI, 0.35-2.65, P = 0.944), but was predictive of worse OS in those treated with other therapies (HR = 2.11, 95% CI, 1.32-3.36, P = 0.002). Similarly, PD-L1 + CTCs was not significantly associated with superior progressing free survival (PFS) in NSCLCs treated with ICIs (HR = 0.67, 95% CI, 0.41-1.09, P = 0.121), but was significantly associated with shorter PFS in patients treated with other therapies (HR = 1.91, 95% CI, 1.24-2.94, P = 0.001). The overall estimate for the concordance between PD-L1 expression on CTCs and tumor cells was 63% (95% CI, 44-80). CONCLUSION: The average detection rate of PD-L1 + CTCs was comparable to the rate of PD-L1 expression in NSCLC tumors. There was a trend towards better PFS in ICI-treated NSCLC patients with PD-L1 + CTCs. Larger longitudinal studies on the association of PD-L1 + CTCs with clinical outcomes in NSCLC patients treated with ICIs are warranted.

A Rapid, Shallow Whole Genome Sequencing Workflow Applicable to Limiting Amounts of Cell-Free DNA.

BACKGROUND: Somatic copy number alterations (sCNAs) acquired during the evolution of breast cancer provide valuable prognostic and therapeutic information. Here we present a workflow for screening sCNAs using picogram amounts of cell-free DNA (cfDNA) and single circulating tumor cells (CTCs). METHODS: We repurposed the Ion ReproSeq PGS™ preimplantation genetic testing kit to perform shallow whole genome sequencing on 178 cfDNA samples (300 pg) and individual CTCs from 10 MBC patients with metastatic breast cancer (MBC) recovered by CellSearch®/DEPArray™. Results were analyzed using a tailored ichorCNA workflow. RESULTS: sCNAs were detected in cfDNA of 41/105 (39%) patients with MBC and 3/23 (13%) primary breast cancers on follow-up (PBC FU), all of whom subsequently relapsed. In 8 of 10 MBCs, individual CTCs had a higher copy number count than matched cfDNA. The median tumor fraction detected by ichorCNA was 0.34 (range 0.17-0.58) for MBC and 0.36 (range 0.31-0.37) for PBC FU. Patients with detectable tumor fraction (≥ 0.1) and TFx and OncomineTM variants had significantly lower overall survival rates (P values P = 0.002 and P < 0.0001 for the log-rank test, respectively). CONCLUSIONS: The ReproSeq PGS assay is rapid, at approximately $120 per sample, providing both a sCNA profile and estimation of the tumor DNA fraction from limiting cfDNA template (300pg) and individual CTCs. The approach could be used to examine the copy number landscape over time to guide treatment decisions, support future trial designs, and be applied to low volume blood spot samples enabling remote monitoring.

Shallow WGS of individual CTCs identifies actionable targets for informing treatment decisions in metastatic breast cancer.

BACKGROUND: We report copy-number profiling by low-pass WGS (LP-WGS) in individual circulating tumour cells (CTCs) for guiding treatment in patients with metastatic breast cancer (MBC), comparing CTC results with mutations detected in circulating tumour DNA (ctDNA) in the same blood samples. METHODS: Across 10 patients with MBC who were progressing at the time of blood sampling and that had >20 CTCs detected by CellSearch®, 63 single cells (50 CTCs and 13 WBCs) and 16 cell pools (8 CTC pools and 8 WBC pools) were recovered from peripheral blood by CellSearch®/DEPArray™ and sequenced with Ampli1 LowPass technology (Menarini Silicon Biosystems). Copy-number aberrations were identified using the MSBiosuite software platform, and results were compared with mutations detected in matched plasma cfDNA analysed by targeted next-generation sequencing using the Oncomine™ Breast cfDNA Assay (Thermo Fisher). RESULTS: LP-WGS data demonstrated copy-number gains/losses in individual CTCs in regions including FGFR1, JAK2 and CDK6 in five patients, ERBB2 amplification in two HER2-negative patients and BRCA loss in two patients. Seven of eight matched plasmas also had mutations in ctDNA in PIK3CA, TP53, ESR1 and KRAS genes with mutant allele frequencies (MAF) ranging from 0.05 to 33.11%. Combining results from paired CTCs and ctDNA, clinically actionable targets were identified in all ten patients. CONCLUSION: This combined analysis of CTCs and ctDNA may offer a new approach for monitoring of disease progression and to direct therapy in patients with advanced MBC, at a time when they are coming towards the end of other treatment options.

CDK7 inhibitors as anticancer drugs.

Cyclin-dependent kinase 7 (CDK7), along with cyclin H and MAT1, forms the CDK-activating complex (CAK), which directs progression through the cell cycle via T-loop phosphorylation of cell cycle CDKs. CAK is also a component of the general transcription factor, TFIIH. CDK7-mediated phosphorylation of RNA polymerase II (Pol II) at active gene promoters permits transcription. Cell cycle dysregulation is an established hallmark of cancer, and aberrant control of transcriptional processes, through diverse mechanisms, is also common in many cancers. Furthermore, CDK7 levels are elevated in a number of cancer types and are associated with clinical outcomes, suggestive of greater dependence on CDK7 activity, compared with normal tissues. These findings identify CDK7 as a cancer therapeutic target, and several recent publications report selective CDK7 inhibitors (CDK7i) with activity against diverse cancer types. Preclinical studies have shown that CDK7i cause cell cycle arrest, apoptosis and repression of transcription, particularly of super-enhancer-associated genes in cancer, and have demonstrated their potential for overcoming resistance to cancer treatments. Moreover, combinations of CDK7i with other targeted cancer therapies, including BET inhibitors, BCL2 inhibitors and hormone therapies, have shown efficacy in model systems. Four CDK7i, ICEC0942 (CT7001), SY-1365, SY-5609 and LY3405105, have now progressed to Phase I/II clinical trials. Here we describe the work that has led to the development of selective CDK7i, the current status of the most advanced clinical candidates, and discuss their potential importance as cancer therapeutics, both as monotherapies and in combination settings. ClinicalTrials.gov Identifiers: NCT03363893; NCT03134638; NCT04247126; NCT03770494.

FOXA1 is a determinant of drug resistance in breast cancer cells.

PURPOSE: Breast cancer is one of the most commonly diagnosed cancers in women. Five subtypes of breast cancer differ in their genetic expression profiles and carry different prognostic values, with no treatments available for some types, such as triple-negative, due to the absence of genetic signatures that could otherwise be targeted by molecular therapies. Although endocrine treatments are largely successful for estrogen receptor (ER)-positive cancers, a significant proportion of patients with metastatic tumors fail to respond and acquire resistance to therapy. FOXA1 overexpression mediates endocrine therapy resistance in ER-positive breast cancer, although the regulation of chemotherapy response by FOXA1 has not been addressed previously. FOXA1, together with EP300 and RUNX1, regulates the expression of E-cadherin, and is expressed in luminal, but absent in triple-negative and basal-like breast cancers. We have previously determined that EP300 regulates drug resistance and tumor initiation capabilities in breast cancer cells. METHODS: Here we describe the generation of breast cancer cell models in which FOXA1 expression has been modulated either by expression of hairpins targeting FOXA1 mRNA or overexpression plasmids. RESULTS: Upon FOXA1 knockdown in luminal MCF-7 and T47D cells, we found an increase in doxorubicin and paclitaxel sensitivity as well as a decrease in anchorage independence. Conversely, upregulation of FOXA1 in basal-like MDA-MB-231 cells led to an increase in drug resistance and anchorage independence. CONCLUSION: Together, these data suggest that FOXA1 plays a role in making tumors more aggressive.

Comparison of two targeted ultra-deep sequencing technologies for analysis of plasma circulating tumour DNA in endocrine-therapy-resistant breast cancer patients.

PURPOSE: There is growing interest in the application of circulating tumour DNA (ctDNA) as a sensitive tool for monitoring tumour evolution and guiding targeted therapy in patients with cancer. However, robust comparisons of different platform technologies are still required. Here we compared the InVisionSeq™ ctDNA Assay with the Oncomine™ Breast cfDNA Assay to assess their concordance and feasibility for the detection of mutations in plasma at low (< 0.5%) variant allele fraction (VAF). METHODS: Ninety-six plasma samples from 50 patients with estrogen receptor (ER)-positive metastatic breast cancer (mBC) were profiled using the InVision Assay. Results were compared to the Oncomine assay in 30 samples from 26 patients, where there was sufficient material and variants were covered by both assays. Longitudinal samples were analysed for 8 patients with endocrine resistance. RESULTS: We detected alterations in 59/96 samples from 34/50 patients analysed with the InVision assay, most frequently affecting ESR1, PIK3CA and TP53. Complete or partial concordance was found in 28/30 samples analysed by both assays, and VAF values were highly correlated. Excellent concordance was found for most genes, and most discordant calls occurred at VAF < 1%. In longitudinal samples from progressing patients with endocrine resistance, we detected consistent alterations in sequential samples, most commonly in ESR1 and PIK3CA. CONCLUSION: This study shows that both ultra-deep next-generation sequencing (NGS) technologies can detect genomic alternations even at low VAFs in plasma samples of mBC patients. The strong agreement of the technologies indicates sufficient reproducibility for clinical use as prognosic and predictive biomarker.

Plasma cell-free DNA (cfDNA) as a predictive and prognostic marker in patients with metastatic breast cancer.

BACKGROUND: Breast cancer (BC) is the most common cancer in women, and despite the introduction of new screening programmes, therapies and monitoring technologies, there is still a need to develop more useful tests for monitoring treatment response and to inform clinical decision making. The purpose of this study was to compare circulating cell-free DNA (cfDNA) and circulating tumour cells (CTCs) with conventional breast cancer blood biomarkers (CA15-3 and alkaline phosphatase (AP)) as predictors of response to treatment and prognosis in patients with metastatic breast cancer (MBC). METHODS: One hundred ninety-four female patients with radiologically confirmed MBC were recruited to the study. Total cfDNA levels were determined by qPCR and compared with CELLSEARCH® CTC counts and CA15-3 and alkaline phosphatase (AP) values. Blood biomarker data were compared with conventional tumour markers, treatment(s) and response as assessed by RECIST and survival. Non-parametric statistical hypothesis tests were used to examine differences, correlation analysis and linear regression to determine correlation and to describe its effects, logistic regression and receiver operating characteristic curve (ROC curve) to estimate the strength of the relationship between biomarkers and clinical outcomes and value normalization against standard deviation to make biomarker values comparable. Kaplan-Meier estimator and Cox regression models were used to assess survival. Univariate and multivariate models were performed where appropriate. RESULTS: Multivariate analysis showed that both the amount of total cfDNA (p value = 0.024, HR = 1.199, CI = 1.024-1.405) and the number of CTCs (p value = 0.001, HR = 1.243, CI = 1.088-1.421) are predictors of overall survival (OS), whereas total cfDNA levels is the sole predictor for progression-free survival (PFS) (p value = 0.042, HR = 1.193, CI = 1.007-1.415) and disease response when comparing response to non-response to treatment (HR = 15.917, HR = 12.481 for univariate and multivariate analysis, respectively). Lastly, combined analysis of CTCs and cfDNA is more informative than the combination of two conventional biomarkers (CA15-3 and AP) for prediction of OS. CONCLUSION: Measurement of total cfDNA levels, which is a simpler and less expensive biomarker than CTC counts, is associated with PFS, OS and response in MBC, suggesting potential clinical application of a cheap and simple blood-based test.

Dissecting the predictive value of MAPK/AKT/estrogen-receptor phosphorylation axis in primary breast cancer to treatment response for tamoxifen over exemestane: a Translational Report of the Intergroup Exemestane Study (IES)-PathIES.

PURPOSE: The prognostic and predictive values of the MAPK/AKT/ERα phosphorylation axis (pT202/T204MAPK, pT308AKT, pS473AKT, pS118ERα and pS167ERα) in primary tumours were assessed to determine whether these markers can differentiate between patient responses for switching adjuvant endocrine therapy after 2-3 years from tamoxifen to exemestane and continued tamoxifen monotherapy in the Intergroup Exemestane Study (IES). METHODS: Of the 4724 patients in IES, 1506 were managed in a subset of centres (N = 89) participating in PathIES. These centres recruited 1282 (85%, 1282/1506) women into PathIES of whom 1036 had phospho-marker data. All phospho-markers were analysed by immunohistochemistry staining. Multivariable Cox proportional hazards models of the phospho-markers for disease-free survival (DFS) and overall survival (OS) were adjusted for clinicopathological factors. Treatment effects on the biomarker expression were determined by interaction tests. Benjamini-Hochberg adjustment for multiple testing with a false discovery rate of 10% was applied (pBH). RESULTS: Phospho-T202/T204MAPK, pS118ERα and pS167ERα were all found to be correlated (pBH = 0.0002). These markers were not associated with either DFS or OS when controlling for the established clinicopathological factors. Interaction terms between the phospho-markers and treatment strategies for either DFS or OS were not statistically significant (pBH > 0.05 for all). CONCLUSIONS: This PathIES study confirmed previously described associations between the phosphorylation site markers of AKT, MAPK and ERα activity in postmenopausal breast cancer patients. No prognostic correlations between the phosphorylation markers and clinical outcome were found, nor were they predictive for clinical outcomes among patients who switched therapy over those treated with tamoxifen alone.

p53 controls expression of the DNA deaminase APOBEC3B to limit its potential mutagenic activity in cancer cells.

Cancer genome sequencing has implicated the cytosine deaminase activity of apolipoprotein B mRNA editing enzyme catalytic polypeptide-like (APOBEC) genes as an important source of mutations in diverse cancers, with APOBEC3B (A3B) expression especially correlated with such cancer mutations. To better understand the processes directing A3B over-expression in cancer, and possible therapeutic avenues for targeting A3B, we have investigated the regulation of A3B gene expression. Here, we show that A3B expression is inversely related to p53 status in different cancer types and demonstrate that this is due to a direct and pivotal role for p53 in repressing A3B expression. This occurs through the induction of p21 (CDKN1A) and the recruitment of the repressive DREAM complex to the A3B gene promoter, such that loss of p53 through mutation, or human papilloma virus-mediated inhibition, prevents recruitment of the complex, thereby causing elevated A3B expression and cytosine deaminase activity in cancer cells. As p53 is frequently mutated in cancer, our findings provide a mechanism by which p53 loss can promote cancer mutagenesis.

Correction to: Tumour suppressor EP300, a modulator of paclitaxel resistance and stemness, is downregulated in metaplastic breast cancer.

In the original publication, Fig. 1 depicting the blot for EP300 in CAL51 cells (Fig. 1c) was unintentionally duplicated with that from MDA-MB-231 cells (Fig. 1d). The new figure given in this erratum depicts the correct EP300 blot in Fig. 1c.

Evaluation of applying IHC4 as a prognostic model in the translational study of Intergroup Exemestane Study (IES): PathIES.

BACKGROUND: Intergroup Exemestane Study (IES) was a randomised study that showed a survival benefit of switching adjuvant endocrine therapy after 2-3 years from tamoxifen to exemestane. This PathIES aimed to assess the role of immunohistochemical (IHC)4 score in determining the relative sensitivity to either tamoxifen or sequential treatment with tamoxifen and exemestane. PATIENTS AND METHODS: Primary tumour samples were available for 1274 patients (27% of IES population). Only patients for whom the IHC4 score could be calculated (based on oestrogen receptor, progesterone receptor, HER2 and Ki67) were included in this analysis (N = 430 patients). The clinical score (C) was based on age, grade, tumour size and nodal status. The association of clinicopathological parameters, IHC4(+C) scores and treatment effect with time to distant recurrence-free survival (TTDR) was assessed in univariable and multivariable Cox regression analyses. A modified clinical score (PathIEscore) (N = 350) was also estimated. RESULTS: Our results confirm the prognostic importance of the original IHC4, alone and in conjunction with clinical scores, but no significant difference with treatment effects was observed. The combined IHC4 + Clinical PathIES score was prognostic for TTDR (P < 0.001) with a hazard ratio (HR) of 5.54 (95% CI 1.29-23.70) for a change from 1st quartile (Q1) to Q1-Q3 and HR of 15.54 (95% CI 3.70-65.24) for a change from Q1 to Q4. CONCLUSION: In the PathIES population, the IHC4 score is useful in predicting long-term relapse in patients who remain disease-free after 2-3 years. This is a first trial to suggest the extending use of IHC4+C score for prognostic indication for patients who have switched endocrine therapies at 2-3 years and who remain disease-free after 2-3 years.

Correction to: IRIS study: a phase II study of the steroid sulfatase inhibitor Irosustat when added to an aromatase inhibitor in ER-positive breast cancer patients.

In the original publication of the article, a part of acknowledgement section was missed out. The omitted acknowledgement is given below: 'The study was coordinated by the Imperial Clinical Trials Unit-Cancer, Imperial College London and Sponsored by Imperial College London. The Imperial Clinical Trials Unit receives funding from the National Institute for Health (NIHR) Biomedical Research Centre based at Imperial College Healthcare NHS Trust and Imperial College London. The views expressed are those of the author(s) and not necessarily those of the NHS, the NIHR or the Department of Health. This study was supported by Imperial Experimental Cancer Medicine Centre and Cancer Research UK Imperial Centre'.

Exploration of tumour-infiltrating lymphocytes as a predictive biomarker for adjuvant endocrine therapy in early breast cancer.

PURPOSE: Tumour-infiltrating lymphocytes (TILs) have been shown to be prognostic for disease-free survival and predictive for the benefit of chemotherapy in patients with early breast cancer, but have not been studied for endocrine therapy. EXPERIMENTAL DESIGN: The number of CD8-positive TILs was assessed in a subcohort of 236 patients in the Intergroup Exemestane Study. AQ After 2-3 years of adjuvant tamoxifen, AQpatients were randomized between the schemes of continuation for 5 years on tamoxifen and switching to exemestane. The numbers of CD8-positive TILs were analysed for correlations with disease-free survival (DFS) and overall survival (OS). A similar analysis was performed on 2596 patients in the TEAM trial who were randomized between the sequential scheme and the exemestane monotherapy. RESULTS: In the first cohort, patients with low (below median) numbers of CD8-positive TILs had a univariate hazard ratio (HR) for DFS of 0.27 (95% CI 0.13-0.55) in favour of treatment with exemestane, whereas this benefit was not observed in patients with high numbers of CD8-positive TILs (HR 1.34, 95% CI 0.71-2.50, HR for interaction 5.02, p = 0.001). In the second cohort, patients with low numbers of CD8-positive TILs showed a benefit of exemestane treatment on recurrence-free survival (RFS HR 0.67, 95% CI 0.45-0.99), and not with above-median numbers of CD8-positive TILs (HR 0.86, 95% CI 0.59-1.26, HR for interaction 1.29, p = 0.36). CONCLUSIONS: This study is the first to propose the number of CD8-positive TILs as potential predictive markers for endocrine therapy, with the low presence of CD8-positive TILs associated to benefit for exemestane-inclusive therapy. However, treatment-by-marker interactions were only significant in one cohort, indicating the need for further validation.

Clinical translation of [18F]ICMT-11 for measuring chemotherapy-induced caspase 3/7 activation in breast and lung cancer.

BACKGROUND: Effective anticancer therapy is thought to involve induction of tumour cell death through apoptosis and/or necrosis. [18F]ICMT-11, an isatin sulfonamide caspase-3/7-specific radiotracer, has been developed for PET imaging and shown to have favourable dosimetry, safety, and biodistribution. We report the translation of [18F]ICMT-11 PET to measure chemotherapy-induced caspase-3/7 activation in breast and lung cancer patients receiving first-line therapy. RESULTS: Breast tumour SUVmax of [18F]ICMT-11 was low at baseline and unchanged following therapy. Measurement of M30/M60 cytokeratin-18 cleavage products showed that therapy was predominantly not apoptosis in nature. While increases in caspase-3 staining on breast histology were seen, post-treatment caspase-3 positivity values were only approximately 1%; this low level of caspase-3 could have limited sensitive detection by [18F]ICMT-11-PET. Fourteen out of 15 breast cancer patients responded to first-line chemotherapy (complete or partial response); one patient had stable disease. Four patients showed increases in regions of high tumour [18F]ICMT-11 intensity on voxel-wise analysis of tumour data (classed as PADS); response was not exclusive to patients with this phenotype. In patients with lung cancer, multi-parametric [18F]ICMT-11 PET and MRI (diffusion-weighted- and dynamic contrast enhanced-MRI) showed that PET changes were concordant with cell death in the absence of significant perfusion changes. CONCLUSION: This study highlights the potential use of [18F]ICMT-11 PET as a promising candidate for non-invasive imaging of caspase3/7 activation, and the difficulties encountered in assessing early-treatment responses. We summarize that tumour response could occur in the absence of predominant chemotherapy-induced caspase-3/7 activation measured non-invasively across entire tumour lesions in patients with breast and lung cancer.

IPET study: an FLT-PET window study to assess the activity of the steroid sulfatase inhibitor irosustat in early breast cancer.

BACKGROUND: Steroid sulfatase (STS) is involved in oestrogen biosynthesis and irosustat is a first generation, irreversible steroid sulfatase inhibitor. A pre-surgical window-of-opportunity study with irosustat was undertaken in estrogen receptor-positive (ER+) breast cancer to assess the effect of irosustat on tumour cell proliferation as measured by 3'-deoxy-3'-[18F] fluorothymidine uptake measured by PET scanning (FLT-PET) and Ki67. METHODS: Postmenopausal women with untreated ER+ early breast cancer were recruited, and imaged with FLT-PET at baseline and after at least 2 weeks treatment with irosustat, 40 mg once daily orally. The primary endpoint was changed in FLT uptake; secondary endpoints included safety and tolerability of irosustat, changes in tumoral Ki67 and steroidogenic enzymes expression and circulating steroid hormone levels. RESULTS: Thirteen women were recruited, and ten started irosustat for 2 weeks, followed by repeat FLT-PET scans in eight. Defining response as decreases of ≥20% in standardized uptake value (SUV) or ≥30% in Ki, 1 (12.5% (95% CI 2-47%, p = 0.001)) and 3 (43% (95% CI 16-75%, p = <0.001) patients, respectively, responded. 6 out of 7 patients had a Ki67 reduction (range = -19.3 to 76.4%), and median percentage difference in Ki67 was 52.3% (p = 0.028). In one patient with a low baseline STS expression, a 19.7% increase in Ki67 was recorded. STS decreases were seen in tumours with high basal STS expression, significant decreases were also noted in aromatase, and 17ß-hydroxysteroid dehydrogenase type 1 and 2. Irosustat was generally well tolerated with all adverse event CTCAE Grade ≤2. CONCLUSIONS: Irosustat resulted in a significant reduction in FLT uptake and Ki67, and is well tolerated. These data are the first demonstrating clinical activity of irosustat in early breast cancer. Baseline expression of STS may be a biomarker of sensitivity to irosustat.

Tumour suppressor EP300, a modulator of paclitaxel resistance and stemness, is downregulated in metaplastic breast cancer.

PURPOSE: We have previously described a novel pathway controlling drug resistance, epithelial-to-mesenchymal transition (EMT) and stemness in breast cancer cells. Upstream in the pathway, three miRs (miR-106b, miR-93 and miR-25) target EP300, a transcriptional activator of E-cadherin. Upregulation of these miRs leads to the downregulation of EP300 and E-cadherin with initiation of an EMT. However, miRs regulate the expression of many genes, and the contribution to EMT by miR targets other than EP300 cannot be ruled out. METHODS: We used lentiviruses expressing EP300-targeting shRNA to downregulate its expression in MCF-7 cells as well as an EP300-knocked-out colon carcinoma cell line. An EP300-expression plasmid was used to upregulate its expression in basal-like CAL51 and MDA-MB-231 breast cancer cells. Drug resistance was determined by short-term proliferation and long-term colony formation assays. Stemness was determined by tumour sphere formation in both soft agar and liquid cultures as well as by the expression of CD44/CD24/ALDH markers. Gene expression microarray analysis was performed in MCF-7 cells lacking EP300. EP300 expression was analysed by immunohistochemistry in 17 samples of metaplastic breast cancer. RESULTS: Cells lacking EP300 became more resistant to paclitaxel whereas EP300 overexpression increased their sensitivity to the drug. Expression of cancer stem cell markers, as well as tumour sphere formation, was also increased in EP300-depleted cells, and was diminished in EP300-overexpressing cells. The EP300-regulated gene signature highlighted genes associated with adhesion (CEACAM5), cytoskeletal remodelling (CAPN9), stemness (ABCG2), apoptosis (BCL2) and metastasis (TGFB2). Some genes in this signature were also validated in a previously generated EP300-depleted model of breast cancer using minimally transformed mammary epithelial cells. Importantly, two key genes in apoptosis and stemness, BCL2 and ABCG2, were also upregulated in EP300-knockout colon carcinoma cells and their paclitaxel-resistant derivatives. Immunohistochemical analysis demonstrated that EP300 expression was low in metaplastic breast cancer, a rare, but aggressive form of the disease with poor prognosis that is characterized by morphological and physiological features of EMT. CONCLUSIONS: EP300 plays a major role in the reprogramming events, leading to a more malignant phenotype with the acquisition of drug resistance and cell plasticity, a characteristic of metaplastic breast cancer.

IRIS study: a phase II study of the steroid sulfatase inhibitor Irosustat when added to an aromatase inhibitor in ER-positive breast cancer patients.

PURPOSE: Irosustat is a first-generation, orally active, irreversible steroid sulfatase inhibitor. We performed a multicentre, open label phase II trial of the addition of Irosustat to a first-line aromatase inhibitor (AI) in patients with advanced BC to evaluate the safety of the combination and to test the hypothesis that the addition of Irosustat to AI may further suppress estradiol levels and result in clinical benefit. EXPERIMENTAL DESIGN: Postmenopausal women with ER-positive locally advanced or metastatic breast cancer who had derived clinical benefit from a first-line AI and who subsequently progressed were enrolled. The first-line AI was continued and Irosustat (40 mg orally daily) added. The primary endpoint was clinical benefit rate (CBR). Secondary endpoints included safety, tolerability, and pharmacodynamic end points. RESULTS: Twenty-seven women were recruited, four discontinued treatment without response assessment. Based on local reporting, the CBR was 18.5% (95% CI 6.3-38.1%) on an intent to treat basis, increasing to 21.7% (95% CI 7.4-43.7%) by per-protocol analysis. In those patients that achieved clinical benefit (n = 5), the median (interquartile range) duration was 9.4 months (8.1-11.3) months. The median progression-free survival time was 2.7 months (95% CI 2.5-4.6) in both the ITT and per-protocol analyses. The most frequently reported grade 3/4 toxicities were dry skin (28%), nausea (13%), fatigue (13%), diarrhoea (8%), headache (7%), anorexia (7%) and lethargy (7%). CONCLUSIONS: The addition of Irosustat to aromatase inhibitor therapy resulted in clinical benefit with an acceptable safety profile. The study met its pre-defined success criterion by both local and central radiological assessments.

Next Generation Sequencing of Circulating Cell-Free DNA for Evaluating Mutations and Gene Amplification in Metastatic Breast Cancer.

BACKGROUND: Breast cancer tissues are heterogeneous and show diverse somatic mutations and somatic copy number alterations (CNAs). We used a novel targeted next generation sequencing (NGS) panel to examine cell-free DNA (cfDNA) to detect somatic mutations and gene amplification in women with metastatic breast cancer (MBC). METHODS: cfDNA from pretreated patients (n = 42) and 9 healthy controls were compared with matched lymphocyte DNA by NGS, using a custom 158 amplicon panel covering hot-spot mutations and CNAs in 16 genes, with further validation of results by droplet digital PCR. RESULTS: No mutations were identified in cfDNA of healthy controls, whereas exactly half the patients with metastatic breast cancer had at least one mutation or amplification in cfDNA (mean 2, range 1-6) across a total of 13 genes. Longitudinal follow up showed dynamic changes to mutations and gene amplification in cfDNA indicating clonal and subclonal response to treatment that was more dynamic than cancer antigen 15-3 (CA15-3). Interestingly, at the time of blood sampling disease progression was occurring in 7 patients with erb-b2 receptor tyrosine kinase 2 (ERBB2) gene amplification in their cfDNA and 3 of these patients were human epidermal growth factor receptor 2 (HER2) negative at diagnosis, suggesting clonal evolution to a more aggressive phenotype. Lastly, 6 patients harbored estrogen receptor 1 (ESR1) mutations in cfDNA, suggesting resistance to endocrine therapy. Overall 9 of 42 patients (21%) had alterations in cfDNA that could herald a change in treatment. CONCLUSIONS: Targeted NGS of cfDNA has potential for monitoring response to targeted therapies through both mutations and gene amplification, for analysis of dynamic tumor heterogeneity and stratification to targeted therapy.