Helena M. Earl

The somatic mutation profiles of 2,433 breast cancers refines their genomic and transcriptomic landscapes

The genomic landscape of breast cancer is complex, and inter- and intra-tumour heterogeneity are important challenges in treating the disease. In this study, we sequence 173 genes in 2,433 primary breast tumours that have copy number aberration (CNA), gene expression and long-term clinical follow-up data. We identify 40 mutation-driver (Mut-driver) genes, and determine associations between mutations, driver CNA profiles, clinical-pathological parameters and survival. We assess the clonal states of Mut-driver mutations, and estimate levels of intra-tumour heterogeneity using mutant-allele fractions. Associations between PIK3CA mutations and reduced survival are identified in three subgroups of ER-positive cancer (defined by amplification of 17q23, 11q13–14 or 8q24). High levels of intra-tumour heterogeneity are in general associated with a worse outcome, but highly aggressive tumours with 11q13–14 amplification have low levels of intra-tumour heterogeneity. These results emphasize the importance of genome-based stratification of breast cancer, and have important implications for designing therapeutic strategies.

Association between CD8+ T-cell infiltration and breast cancer survival in 12 439 patients

BACKGROUND T-cell infiltration in estrogen receptor (ER)-negative breast tumours has been associated with longer survival. To investigate this association and the potential of tumour T-cell infiltration as a prognostic and predictive marker, we have conducted the largest study of T cells in breast cancer to date. PATIENTS AND METHODS Four studies totalling 12 439 patients were used for this work. Cytotoxic (CD8+) and regulatory (forkhead box protein 3, FOXP3+) T cells were quantified using immunohistochemistry (IHC). IHC for CD8 was conducted using available material from all four studies (8978 samples) and for FOXP3 from three studies (5239 samples)-multiple imputation was used to resolve missing data from the remaining patients. Cox regression was used to test for associations with breast cancer-specific survival. RESULTS In ER-negative tumours [triple-negative breast cancer and human epidermal growth factor receptor 2 (human epidermal growth factor receptor 2 (HER2) positive)], presence of CD8+ T cells within the tumour was associated with a 28% [95% confidence interval (CI) 16% to 38%] reduction in the hazard of breast cancer-specific mortality, and CD8+ T cells within the stroma with a 21% (95% CI 7% to 33%) reduction in hazard. In ER-positive HER2-positive tumours, CD8+ T cells within the tumour were associated with a 27% (95% CI 4% to 44%) reduction in hazard. In ER-negative disease, there was evidence for greater benefit from anthracyclines in the National Epirubicin Adjuvant Trial in patients with CD8+ tumours [hazard ratio (HR) = 0.54; 95% CI 0.37-0.79] versus CD8-negative tumours (HR = 0.87; 95% CI 0.55-1.38). The difference in effect between these subgroups was significant when limited to cases with complete data (P heterogeneity = 0.04) and approached significance in imputed data (P heterogeneity = 0.1). CONCLUSIONS The presence of CD8+ T cells in breast cancer is associated with a significant reduction in the relative risk of death from disease in both the ER-negative [supplementary Figure S1, available at Annals of Oncology online] and the ER-positive HER2-positive subtypes. Tumour lymphocytic infiltration may improve risk stratification in breast cancer patients classified into these subtypes. NEAT ClinicalTrials.gov: NCT00003577.

Randomized Phase II Placebo-Controlled Trial of Maintenance Therapy Using the Oral Triple Angiokinase Inhibitor BIBF 1120 After Chemotherapy for Relapsed Ovarian Cancer

PURPOSE Inhibiting angiogenesis is one of the most promising avenues for new therapies for ovarian cancer. We investigated the efficacy and safety of a novel agent, BIBF 1120, a triple angiokinase inhibitor, after chemotherapy for relapsed disease. PATIENTS AND METHODS We conducted a randomized, double-blind, controlled phase II trial in 83 patients who had just completed chemotherapy for relapsed ovarian cancer, with evidence of response, but at high risk of further early recurrence. The patients were randomly assigned to receive maintenance therapy using BIBF 1120 250 mg or placebo, twice per day, continuously for 36 weeks. End points were progression-free survival (PFS), toxicity, and overall survival. RESULTS Thirty-six-week PFS rates were 16.3% and 5.0% in the BIBF 1120 and placebo groups, respectively (hazard ratio, 0.65; 95% CI, 0.42 to 1.02; P = .06). Four patients continued on BIBF 1120, including two patients for another year or more. The proportion of patients with any grade 3 or 4 adverse events was similar between the groups (34.9% for BIBF 1120 v 27.5% for placebo; P = .49; mostly grade 3). However, more patients on BIBF 1120 experienced diarrhea, nausea, or vomiting (mainly grade 1 or 2 and no grade 4). There was a higher rate of grade 3 or 4 hepatotoxicity in patients on BIBF 1120 (51.2%) compared with patients on placebo (7.5%; P < .001), but this was rarely of clinical significance, and patients continued with the trial treatment. A single-level dose reduction to 150 mg was made in 15 patients, all on active drug. CONCLUSION BIBF 1120 is well tolerated and associated with a potential improvement in PFS. The observed treatment effect is sufficient to justify further study within a large phase III trial.

Phase III Randomized Trial of Doxorubicin and Docetaxel Versus Doxorubicin and Cyclophosphamide As Primary Medical Therapy in Women With Breast Cancer: An Anglo-Celtic Cooperative Oncology Group Study

PURPOSE To compare the clinical and pathologic response rates of doxorubicin and cyclophosphamide (AC) with doxorubicin and docetaxel (AD) as primary chemotherapy in women with primary or locally advanced breast cancer. PATIENTS AND METHODS Eligible patients with histologically proven breast cancer with primary tumors >/= 3 cm, inflammatory or locally advanced disease, and no evidence of metastases were randomly assigned to receive a maximum of six cycles of either doxorubicin (60 mg/m(2)) plus cyclophosphamide (600 mg/m(2)) administered intravenously (IV) every 3 weeks or doxorubicin (60 mg/m(2)) plus docetaxel (75 mg/m(2)) IV every 3 weeks, followed by surgery on completion of chemotherapy. Results A total of 363 patients were randomly assigned to AC (n = 180) or AD (n = 183). A complete clinical response was observed in 17% and 20% of patients treated with AC and AD, respectively (P = .42). Overall (complete and partial) clinical response rates for AC and AD were 61% and 70%, respectively (P = .06). There was no significant difference in either the pathologic complete response rates in the breast with AC (24%) and AD (21%; P = .61) or in the number of patients with positive axillary nodes at surgery with AC (61%) and AD (66%; P = .28). At a median follow-up of 32 months, there is no significant difference between the two groups for the number of relapses. CONCLUSION In contrast to the positive results reported for sequential docetaxel after AC as primary chemotherapy of breast cancer, our data do not suggest a benefit for simultaneous AD over AC.

Sequential docetaxel as adjuvant chemotherapy for early breast cancer (TACT): an open-label, phase III, randomised controlled trial

Summary Background Incorporation of a taxane as adjuvant treatment for early breast cancer offers potential for further improvement of anthracycline-based treatment. The UK TACT study (CRUK01/001) investigated whether sequential docetaxel after anthracycline chemotherapy would improve patient outcome compared with standard chemotherapy of similar duration. Methods In this multicentre, open-label, phase III, randomised controlled trial, 4162 women (aged >18 years) with node-positive or high-risk node-negative operable early breast cancer were randomly assigned by computer-generated permuted block randomisation to receive FEC (fluorouracil 600 mg/m2, epirubicin 60 mg/m2, cyclophosphamide 600 mg/m2 at 3-weekly intervals) for four cycles followed by docetaxel (100 mg/m2 at 3-weekly intervals) for four cycles (n=2073) or control (n=2089). For the control regimen, centres chose either FEC for eight cycles (n=1265) or epirubicin (100 mg/m2 at 3-weekly intervals) for four cycles followed by CMF (cyclophosphamide 600 mg/m2, methotrexate 40 mg/m2, and fluorouracil 600 mg/m2 at 4-weekly intervals) for four cycles (n=824). The primary endpoint was disease-free survival. Analysis was by intention to treat (ITT). This study is registered as an International Standard Randomised Controlled Trial, number ISRCTN79718493. Findings All randomised patients were included in the ITT population. With a median follow-up of 62 months, disease-free survival events were seen in 517 of 2073 patients in the experimental group compared with 539 of 2089 controls (hazard ratio [HR] 0·95, 95% CI 0·85–1·08; p=0·44). 75·6% (95% CI 73·7–77·5) of patients in the experimental group and 74·3% (72·3–76·2) of controls were alive and disease-free at 5 years. The proportion of patients who reported any acute grade 3 or 4 adverse event was significantly greater in the experimental group than in the control group (p<0·0001); the most frequent events were neutropenia (937 events vs 797 events), leucopenia (507 vs 362), and lethargy (456 vs 272). Interpretation This study did not show any overall gain from the addition of docetaxel to standard anthracycline chemotherapy. Exploration of predictive biomarker-defined subgroups might have the potential to better target the use of taxane-based therapy. Funding Cancer Research UK (CRUK 01/001), Sanofi-Aventis, Pfizer, and Roche.

The Extracellular Matrix Protein TGFBI Induces Microtubule Stabilization and Sensitizes Ovarian Cancers to Paclitaxel

Summary The extracellular matrix (ECM) can induce chemotherapy resistance via AKT-mediated inhibition of apoptosis. Here, we show that loss of the ECM protein TGFBI (transforming growth factor beta induced) is sufficient to induce specific resistance to paclitaxel and mitotic spindle abnormalities in ovarian cancer cells. Paclitaxel-resistant cells treated with recombinant TGFBI protein show integrin-dependent restoration of paclitaxel sensitivity via FAK- and Rho-dependent stabilization of microtubules. Immunohistochemical staining for TGFBI in paclitaxel-treated ovarian cancers from a prospective clinical trial showed that morphological changes of paclitaxel-induced cytotoxicity were restricted to areas of strong expression of TGFBI. These data show that ECM can mediate taxane sensitivity by modulating microtubule stability.

Spatial and Temporal Heterogeneity in High-Grade Serous Ovarian Cancer: A Phylogenetic Analysis

Background The major clinical challenge in the treatment of high-grade serous ovarian cancer (HGSOC) is the development of progressive resistance to platinum-based chemotherapy. The objective of this study was to determine whether intra-tumour genetic heterogeneity resulting from clonal evolution and the emergence of subclonal tumour populations in HGSOC was associated with the development of resistant disease. Methods and Findings Evolutionary inference and phylogenetic quantification of heterogeneity was performed using the MEDICC algorithm on high-resolution whole genome copy number profiles and selected genome-wide sequencing of 135 spatially and temporally separated samples from 14 patients with HGSOC who received platinum-based chemotherapy. Samples were obtained from the clinical CTCR-OV03/04 studies, and patients were enrolled between 20 July 2007 and 22 October 2009. Median follow-up of the cohort was 31 mo (interquartile range 22–46 mo), censored after 26 October 2013. Outcome measures were overall survival (OS) and progression-free survival (PFS). There were marked differences in the degree of clonal expansion (CE) between patients (median 0.74, interquartile range 0.66–1.15), and dichotimization by median CE showed worse survival in CE-high cases (PFS 12.7 versus 10.1 mo, p = 0.009; OS 42.6 versus 23.5 mo, p = 0.003). Bootstrap analysis with resampling showed that the 95% confidence intervals for the hazard ratios for PFS and OS in the CE-high group were greater than 1.0. These data support a relationship between heterogeneity and survival but do not precisely determine its effect size. Relapsed tissue was available for two patients in the CE-high group, and phylogenetic analysis showed that the prevalent clonal population at clinical recurrence arose from early divergence events. A subclonal population marked by a NF1 deletion showed a progressive increase in tumour allele fraction during chemotherapy. Conclusions This study demonstrates that quantitative measures of intra-tumour heterogeneity may have predictive value for survival after chemotherapy treatment in HGSOC. Subclonal tumour populations are present in pre-treatment biopsies in HGSOC and can undergo expansion during chemotherapy, causing clinical relapse.

Predictive markers of anthracycline benefit: a prospectively planned analysis of the UK National Epirubicin Adjuvant Trial (NEAT/BR9601)

BACKGROUND The NEAT/BR9601 trial showed benefit for addition of anthracyclines to cyclophosphamide, methotrexate, and fluorouracil (CMF) as adjuvant treatment for early breast cancer. We investigated prospectively predictive biomarkers of anthracycline benefit including HER2 and TOP2A. METHODS 1941 tumours from 2391 women recruited to NEAT/BR9601 were analysed on tissue microarrays for HER2 and TOP2A amplification and deletion, HER1-3 and Ki67 expression, and duplication of chromosome 17 centromere enumeration probe (Ch17CEP). Log-rank analyses identified factors affecting relapse-free and overall survival, and regression models tested independent prognostic effect of markers, with adjustment for known prognostic factors (age, nodal status, oestrogen-receptor status, grade, and tumour size). The predictive value of markers was tested by treatment interactions for relapse-free and overall survival. FINDINGS 1762 patients were analysed. 21% of tumours (n=367) were HER2 amplified, 10% were TOP2A amplified (n=169), 11% showed TOP2A deleted (n=191), 23% showed Ch17CEP duplication (n=406), and 61% had high (>13.0%) Ki67 (n=1136). In univariate analyses, only HER2 amplification and TOP2A deletion were significant prognostic factors for relapse-free (hazard ratio [HR] 1.59, 95% CI 1.32-1.92, p<0.0001; and 1.52, 1.20-1.92, p=0.0006, respectively) and overall survival (1.79, 1.47-2.19, p<0.0001; and 1.62, 1.26-2.08, p=0.0002 respectively). We detected no significant interaction with anthracycline benefit for Ki67, HER2, HER1-3, or TOP2A. By contrast, in multivariate analyses, Ch17CEP duplication was associated with significant improvements in both relapse-free (HR 0.92, 95% CI 0.72-1.18 for tumours with normal Ch17CEP vs 0.52, 0.34-0.81 for tumours with abnormal Ch17CEP; p for interaction=0.004) and overall survival (0.94, 0.72-1.24 vs 0.57, 0.36-0.92; p for interaction=0.02) with anthracycline use. INTERPRETATION In women with early breast cancer receiving adjuvant chemotherapy, the most powerful predictor of benefit from anthracyclines is Ch17CEP duplication. In view of the location of HER2/TOP2A on chromosome 17, Ch17CEP duplication might explain the inconsistencies in previous studies of factors predicting benefit from anthracyclines. FUNDING Cancer Research UK and the Scottish Breast Cancer Clinical Trials Group.

Can breast MRI help in the management of women with breast cancer treated by neoadjuvant chemotherapy

Contrast-enhanced (CE) MRI was used to monitor breast cancer response to neoadjuvant chemotherapy. Patients underwent CE MRI before and after therapy, together with conventional assessment methods (CAM). CE MRI was carried out at 1.5 T in the coronal plain with 3D sequences before and after bolus injection. An expert panel determined chemotherapy response using both CE MRI and CAM. Histopathological response in the surgical specimen was then used to determine the sensitivity and specificity of CE MRI and CAM. In total, 67 patients with 69 breast cancers were studied (mean age of 46 years). Tumour characteristics showed a high-risk tumour population: median size 49 mm: histopathological grade 3 (55%): oestrogen receptor (ER) negative (48%). Histopathological response was as follows: – complete pathological response (pCR) 17%; partial response (pPR) 68%; no response (NR) 15%. Sensitivity of CAM for pCR or pPR was 98% (CI 91–100%) and specificity was 50% (CI 19–81%). CE MRI sensitivity was 100% (CI 94–100%), and specificity was 80% (CI 44–97%). The absolute agreement between assessment methods and histopathology was marginally higher for CE MRI than CAM (81 vs 68%; P=0.09). In 71%, CE MRI increased diagnostic knowledge, although in 20% it was judged confusing or incorrect. The 2nd MRI study significantly increased diagnostic confidence, and in 19% could have changed the treatment plan. CE MRI persistently underestimated minimal residual disease. In conclusion, CE MRI of breast cancer proved more reliable for predicting histopathological response to neoadjuvant chemotherapy than conventional assessment methods.

PD-L1 protein expression in breast cancer is rare, enriched in basal-like tumours and associated with infiltrating lymphocytes

BACKGROUND Expression of programmed death ligand 1 (PD-L1) in solid tumours has been shown to predict whether patients are likely to respond to anti-PD-L1 therapies. To estimate the therapeutic potential of PD-L1 inhibition in breast cancer, we evaluated the prevalence and significance of PD-L1 protein expression in a large collection of breast tumours. PATIENTS AND METHODS Correlations between CD274 (PD-L1) copy number, transcript and protein levels were evaluated in tumours from 418 patients recruited to the METABRIC genomic study. Immunohistochemistry was used to detect PD-L1 protein in breast tumours in tissue microarrays from 5763 patients recruited to the SEARCH population-based study (N = 4079) and the NEAT randomised, controlled trial (N = 1684). RESULTS PD-L1 protein data was available for 3916 of the possible 5763 tumours from the SEARCH and NEAT studies. PD-L1 expression by immune cells was observed in 6% (235/3916) of tumours and expression by tumour cells was observed in just 1.7% (66/3916). PD-L1 was most frequently expressed in basal-like tumours. This was observed both where tumours were subtyped by combined copy number and expression profiling [39% (17/44) of IntClust 10 i.e. basal-like tumours were PD-L1 immune cell positive; P < 0.001] and where a surrogate IHC-based classifier was used [19% (56/302) of basal-like tumours were PD-L1 immune cell positive; P < 0.001]. Moreover, CD274 (PD-L1) amplification was observed in five tumours of which four were IntClust 10. Expression of PD-L1 by either tumour cells or infiltrating immune cells was positively correlated with infiltration by both cytotoxic and regulatory T cells (P < 0.001). There was a nominally significant association between PD-L1 and improved disease-specific survival (hazard ratio 0.53, 95% confidence interval 0.26-1.07; P = 0.08) in ER-negative disease. CONCLUSIONS Expression of PD-L1 is rare in breast cancer, markedly enriched in basal-like tumours and is correlated with infiltrating lymphocytes. PD-L1 inhibition may benefit the 19% of patients with basal-like tumours in which the protein is expressed. NEAT CLINICALTRIALSGOV NCT00003577.