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Background Prospective data on the risk of recurrence among women with hormone receptor-positive early breast cancer who temporarily discontinue endocrine therapy to attempt pregnancy are lacking. Methods We conducted a single-group trial in which we evaluated the temporary interruption of adjuvant endocrine therapy to attempt pregnancy in young women with previous breast cancer. Eligible women were 42 years of age or younger; had had stage I, II, or III disease; had received adjuvant endocrine therapy for 18 to 30 months; and desired pregnancy. The primary end point was the number of breast cancer events (defined as local, regional, or distant recurrence of invasive breast cancer or new contralateral invasive breast cancer) during follow-up. The primary analysis was planned to be performed after 1600 patient-years of follow-up. The prespecified safety threshold was the occurrence of 46 breast cancer events during this period. Breast cancer outcomes in this treatment-interruption group were compared with those in an external control cohort consisting of women who would have met the entry criteria for the current trial. Results Among 516 women, the median age was 37 years, the median time from breast cancer diagnosis to enrollment was 29 months, and 93.4% had stage I or II disease. Among 497 women who were followed for pregnancy status, 368 (74.0%) had at least one pregnancy and 317 (63.8%) had at least one live birth. In total, 365 babies were born. At 1638 patient-years of follow-up (median follow-up, 41 months), 44 patients had a breast cancer event, a result that did not exceed the safety threshold. The 3-year incidence of breast cancer events was 8.9% (95% confidence interval [CI], 6.3 to 11.6) in the treatment-interruption group and 9.2% (95% CI, 7.6 to 10.8) in the control cohort. Conclusions Among select women with previous hormone receptor-positive early breast cancer, temporary interruption of endocrine therapy to attempt pregnancy did not confer a greater short-term risk of breast cancer events, including distant recurrence, than that in the external control cohort. Further follow-up is critical to inform longer-term safety. (Funded by ETOP IBCSG Partners Foundation and others; POSITIVE ClinicalTrials.gov number, NCT02308085.).

Background: The landmark Suppression of Ovarian Function Trial (SOFT) in premenopausal breast cancer patients revealed that the addition of ovarian function suppression (OFS) to adjuvant endocrine therapy with either tamoxifen (T+OFS) or exemestane (E+OFS) reduces the risk of recurrence compared with adjuvant tamoxifen alone. The benefit from the addition of OFS was most clinically meaningful for patients at higher clinico-pathologic risk of recurrence. There are no biomarkers to aid decision-making about intensification of endocrine therapy with OFS and its resultant toxicities. The Breast Cancer Index (BCI) is a gene expression–based signature that stratifies patients based on the risk of overall (0-10 years) and late (post-5 years) distant recurrence (DR) and predicts the likelihood of benefit from extended endocrine therapy in early stage HR+ breast cancer. The purpose of this study is to assess BCI’s prognostic and predictive ability in premenopausal women randomly assigned to 5-years treatment with E+OFS or T+OFS vs T alone in the SOFT trial. Methods: All available FFPE tumor samples from the SOFT trial (n=1718 of 3047) were included in the study. BCI testing was performed blinded to clinical characteristics, treatment and outcome. Median follow-up was 13 years. Primary endpoint was breast cancer-free interval (BCFI). Secondary endpoints were distant recurrence-free interval (DRFI) and disease-free survival (DFS). Kaplan-Meier analysis and Cox proportional hazards regression models, stratified by prior chemotherapy and lymph node status, were used to evaluate the predictive performance of BCI (H/I) status (High vs Low), and secondarily H/I as a continuous score. Hypothesis testing for interaction was performed by stratified log-rank tests. Results: Tumor samples from 1687 (98%) patients (30.4% < 40 years, 64.1% T1, 50.1% G2, 65.8% N0, 85.5%% HER2-, 53.3% received prior chemotherapy) were successful in BCI testing and included in the final analysis. Patient characteristics in the translational cohort are representative of the parent SOFT trial. 42.4% of patients’ tumors had H/I-High status. Prognostic and predictive analyses are ongoing and will be presented at the meeting. Citation Format: Ruth O’Regan, Yi Zhang, Gini F. Fleming, Prudence Francis, Roswitha Kammler, Giuseppe Viale, István Láng, Meritxell Bellet Ezquerra, Hervé R. Bonnefoi, Sherene Loi, Marco Colleoni, Catherine A. Schnabel, Kai Treuner, Meredith Regan. Evaluation of the Breast Cancer Index in premenopausal women with early-stage HR+ breast cancer in the SOFT trial [abstract]. In: Proceedings of the 2022 San Antonio Breast Cancer Symposium; 2022 Dec 6-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2023;83(5 Suppl):Abstract nr GS1-06.

Background: The risk of late distant recurrence (≥ 5 years) in post-menopausal patients with ER+ BC remains significant even after standard adjuvant endocrine therapy (ET). Obesity, which has reached high proportions in Western Countries, is one of the modifiable factors that can contribute to BC recurrences. While body mass index (BMI) is an easily available surrogate measure of adiposity, it might not reflect accurately patient adiposity, especially in post-menopausal patients. We therefore hypothesize that mammary adipocyte measurements might better capture the adiposity of the patient. In this study, we retrospectively evaluated the prognostic role of both BMI and adipocyte measurements in the SOLE trial. Patients & Methods: The SOLE trial (NCT00553410) consisted of 4,884 postmenopausal women with hormone receptor positive, lymph node-positive BC treated with 5 years of letrozole after having completed 4-6 years of adjuvant ET. BMI was available for 3,606 patients with ER+/HER2- BC and categorized according to the WHO classification (underweight: < 18.5, normal weight 18.5 – 24.9, overweight 25 – 29.9, obesity ≥ 30 kg/m2). Considering the small sample size, patients with underweight (n=35) were grouped together with patients with normal weight for the subsequent analyses. We evaluated the association between clinico-pathological characteristics and BMI using Fisher exact test and Kruskal-Wallis test. Given the previously reported association of BMI with distant recurrences (PMID: 21115856), we considered distant-relapse free interval (DRFI) as the main study endpoint. Cox regression analyses were adjusted for histology, chemotherapy (yes/no), type of local therapy, in addition to previously reported clinico-pathological variables (PMID: 29158011). Then, we conducted a stratified case-cohort study consisting of 311 patients (97 with distant relapse-cases) with successful digital assessment of adipocytes on normal mammary adipose tissue sample collected at surgery, as previously described (PMID: 33120083). Stratification factors were treatment arm and type of prior ET. Weighted Cox regression models (PMID: 17554753, 18712477) were used to evaluate prognostic association with adipocyte size (area in µm²) categorized in quartile of the 75th percentile (Q4 vs Q1-3), as done previously (PMID: 33753731). Results: 1392 (39%) patients were normal weight, 1315 (36%) were overweight and 899 (25%) were obese. Older age at diagnosis, lymph node involvement (≥4), large tumor size and menopausal status were associated with overweight and obesity. After a median follow-up of 84 months, no associations were found between DRFI and BMI in the univariable and multivariable analysis (adjusted HR overweight vs normal=0.99, 95%CI: 0.76 – 1.29, p=0.977 and HR obese vs normal=1.11, 95%CI: 0.84 - 1.47, p=0.425). In the case-cohort study, patients with larger adipocytes had an increased hazard of distant recurrence in the univariable and multivariable analysis (see table 1). Conclusion: In this study, we did not detect any association between BMI and survival outcomes. We however found that larger mammary adipocytes were independently associated with an increased hazard of distant recurrence. These results suggest that mammary adipocytes, which can be easily evaluated on H&E slides using digital pathology, should be considered as a novel marker to evaluate the risk of late recurrence in patients with breast cancer. Citation Format: Edoardo Isnaldi, François Richard, Giuseppe Marano, Patrizia Boracchi, Marion Maetens, Giuseppe Floris, Guy Jerusalem, Andrea Gombos, Alastair M. Thompson, Erika Hitre, Stefan Aebi, Marco Colleoni, Patrizia Dell’Orto, Roswitha Kammler, Patrick Neven, Giuseppe Viale, Meredith Regan, Elia Biganzoli, Christine Desmedt. Impact of increased adiposity on extended aromatase inhibitor treatment in postmenopausal patients with estrogen receptor positive (ER+) breast cancer: a retrospective analysis of the SOLE trial [abstract]. In: Proceedings of the 2022 San Antonio Breast Cancer Symposium; 2022 Dec 6-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2023;83(5 Suppl):Abstract nr P3-05-28.

Clinico-pathologic characteristics of patients according to NGS results (success/failure).

Weighted Kaplan-Meier (KM) estimates of breast cancer-free interval (BCFI) and distant recurrence-free interval (DRFI) according to tumor subtype, separately by PIK3CA mutational status (mutated, MUT vs wild-type, WT). The 5-year event-free estimates (95% CI) are provided. Hazard ratio (HR) estimates (95% CI) and log-rank tests from weighted univariate models.

Purpose:Women with hormone receptor–positive early breast cancers have a persistent risk of relapse and biomarkers for late recurrence are needed. We sought to identify tumor genomic aberrations associated with increased late-recurrence risk.Experimental Design:In a secondary analysis of Study of Letrozole Extension trial, a case-cohort–like sampling selected 598 primary breast cancers for targeted next-generation sequencing analysis of gene mutations and copy-number gains (CNGs). Correlations of genomic aberrations with clinicopathologic factors and breast and distant recurrence-free intervals (BCFIs and DRFIs) were analyzed using weighted Cox models.Results:Analysis of mutations and CNGs was successfully performed for 403 and 350 samples, including 148 and 134 patients with breast cancer recurrences (median follow-up time, 5.2 years), respectively. The most frequent alterations were PIK3CA mutations (42%) and CNGs of CCND1 (15%), ERBB2 (10%), FGFR1 (8%), and MYC (8%). PIK3CA mutations and MYC CNGs were associated with lower (P = 0.03) and higher (P = 0.004) tumor grade, respectively; a higher Ki-67 was seen in tumor with CCND1, ERBB2, and MYC CNGs (P = 0.01, P < 0.001, and P = 0.03, respectively). FGFR1 CNG was associated with an increased risk of late events in univariate analyses [17/29 patients; BCFI: HR, 3.2; 95% confidence interval (CI), 1.48–6.92; P = 0.003 and DRFI: HR, 3.5; 95% CI, 1.61–7.75; P = 0.002) and in multivariable models adjusted for clinicopathologic factors.Conclusions:Postmenopausal women with hormone receptor–positive early breast cancer harboring FGFR1 CNG had an increased risk of late recurrence despite extended therapy. FGFR1 CNG may represent a useful prognostic biomarker for late recurrence and a therapeutic target.

Pleural mesothelioma (PM) is an aggressive malignancy with poor prognosis. Although histology and pathologic stage are important prognostic factors, better prognostic biomarkers are needed. The ribosomal protein S6 is a downstream target of the phosphatidylinositol 3-kinase (PI3K) pathway involved in protein synthesis and cell proliferation. In previous studies, low phosphorylated S6 (pS6) immunoreactivity was significantly correlated with longer progression-free survival (PFS) and overall survival (OS) in PM patients. We aimed to correlate pS6 expression to clinical data in a large multi-centre PM cohort as part of the European Thoracic Oncology Platform (ETOP) Mesoscape project. Tissue Micro Arrays (TMAs) of PM were constructed and expression of pS6 was evaluated by a semi-quantitatively aggregate H-score. Expression results were correlated to patient characteristics as well as OS/PFS. pS6 IHC results of 364 patients from 9 centres, diagnosed between 1999 and 2017 were available. The primary histology of included tumours was epithelioid (70.3%), followed by biphasic (24.2%) and sarcomatoid (5.5%). TMAs included both treatment-naïve and tumour tissue taken after induction chemotherapy. High pS6 expression (181 patients with H-score>1.41) was significantly associated with less complete resection. In the overall cohort, OS/PFS were not significantly different between pS6-low and pS6-high patients. In a subgroup analysis non-epithelioid (biphasic and sarcomatoid) patients with high pS6 expression showed a significantly shorter OS (p

The primary objective of this study is to evaluate the clinical significance of RANK/L expression, in both a retrospective cohort of surgically resected stage I-III NSCLC (Lungscape) and a randomized clinical trial-cohort (SPLENDOUR) of advanced NSCLC treated with chemotherapy alone or in combination with denosumab.RANK-L expression was assessed on tissue microarrays (TMAs) in Lungscape and whole sections in SPLENDOUR, using immunohistochemistry, with H-scores values 0 indicating positivity. Prevalence of RANK positivity and its association with clinicopathological characteristics, and patient outcome was explored in a subset of the ETOP Lungscape cohort and in SPLENDOUR. Also investigated were the prevalence of RANK overexpression (proportion of positive cancer cells ≥ 50%) in the Lungscape cohort, and RANK-L in the SPLENDOUR trial.In the Lungscape cohort, RANK expression was assessed at a median follow-up of 46 months (N = 488 patients; 4 centers); 35% were female, 44/49/6% adenocarcinomas (AC)/squamous cell carcinomas (SCC)/other, 48/27/25% with stage I/II/III. Median RFS/TTR/OS were 58/Not reached/74 months. Prevalence of RANK expression was 31% (95%CI:27%-35%); significantly higher in AC: 50% (95%CI:43%-57%) vs SCC: 12% (95%CI:8%-16%) (p 0.001); more frequent in females (42% vs 25%, p 0.001) and tumors ≤ 4 cm (35.3% vs 23.3%, p = 0.0065). No association with outcome was found. In the SPLENDOUR trial (463 patients), the prevalence of membranous and cytoplasmic RANK positivity was 34% (95%CI:30%-38%) and 9% (95%CI:7%-12%), respectively, while prevalence for RANK-L was 5% (95%CI:3%-7%) and 36% (95%CI:31%-40%), respectively. Cytoplasmic RANK-L positivity was more common among females (47% vs 31%, p = 0.001) and in non-SCC histology (45% vs 10%, p 0.0001). At the pre-specified 1% significance level, no prognostic or predictive effect was found.Both cohorts indicate that RANK expression is more common in adenocarcinoma/non-squamous NSCLC and in female patients. No prognostic effect is found, and in the clinical trial involving addition of denosumab to chemotherapy no predictive effect is detected.

Tamoxifen (T) is a pro-drug that undergoes CYP2D6-mediated metabolic activation to metabolites that more potently inhibit estrogen stimulated growth compared to the parent drug. While many studies have examined the role of CYP2D6 genotype in T-treated postmenopausal women, the role of CYP2D6 metabolism in premenopausal women (pre-MW) receiving T, with or without ovarian function suppression (OFS) or exemestane (E) and OFS is unknown. Methods: SOFT randomized 3066 (pre-MW) from 2003-2011 in 27 countries, stratified according to prior receipt or nonreceipt of chemotherapy and nodal status, to receive 5 years of T, T+OFS, or E+OFS. We designed a pharmacogenetics substudy (activated October 2010) to collect blood DNA from North American (NA) patients (pts) or to extract non-tumor DNA from available formalin fixed paraffin embedded (FFPE) tissue blocks. For pts with a blood sample, CYP2D6 was genotyped beginning with the Luminex Tag-It Mutation Detection Kit and when needed, with a copy number variation assay and/or sequencing assays. For pts with FFPE-derived DNA, CYP2D6 genotyping for *3, *4, *6, *9, *10, *17 and *41 was performed using a Taqman Allelic Discrimination Assay. CYP2D6 phenotypes were called by classifying pts on the basis of a combination of poor (PM: *3, *4, *5, *6, *7, *8), slow (SM: *10), intermediate (IM: *9, *17, *29, *41) and extensive metabolizer alleles (EM; all others). Activity scores (AS) from phenotypes assigned for each allele: 0 if PM, 0.25 if SM, 0.5 if IM and 1 if EM allele, and multiplied x2 or x3 if duplicate or triplicate. With concomitant use of potent CYP2D6 inhibitor, AS=0; use of weak inhibitor subtracted 0.5. Metabolizer status was defined by CYP2D6 genotype alone or in combination with CYP2D6 inhibitor use at randomization from the AS: extensive (AS 1.25 to 3), intermediate (AS >0.5 to 0.3 ng/ml in 1053, and successfully derived CYP2D6 genotypes for 765/3047 pts (25%). 182 (15%) pts had DFS events after 8 yrs median follow-up. Metabolizer status from genotype was 57% extensive, 29% intermediate, 15% slow/poor. Metabolizer status was not associated with DFS in pts assigned T alone (P=0.60; Table), nor in pts assigned T+OFS (P=0.41) or E+OFS (P=0.30). 11% of pts used CYP2D6 inhibitors concomitantly at randomization; for 8% it changed the metabolizer status. The results using this definition were consistent. Conclusion: This retrospective-prospective SOFT pharmacogenetics substudy found no relation of CYP2D6 metabolizer status with DFS in premenopausal pts receiving T, T + OFS, or E + OFS. Given that 50% were pretreated with chemotherapy, further study is needed regarding the role of CYP2D6 metabolism in patients treated with T monotherapy. TableTreatment GroupN pts (N events)Comparison (N pts)Hazard Ratio95% CITamoxifen324 (56)Intermediate (114) vs Extensive (210)0.780.43-1.39Tamoxifen265 (52)Slow/Poor (55) vs Extensive (210)1.110.58-2.13Tamoxifen + OFS357 (46)Intermediate (122) vs Extensive (235)0.730.38-1.40Tamoxifen + OFS299 (45)Slow/Poor (64) vs Extensive (235)1.250.64-2.43Exemestane + OFS344 (46)Intermediate (107) vs Extensive (237)0.590.28-1.22Exemestane + OFS293 (47)Slow/Poor (56) vs Extensive (237)1.130.56-2.27 Citation Format: Matthew P. Goetz, Gini F. Fleming, Mary Kuffel, John R. Hawse, John L. Black, Richard Weinshilboum, James N. Ingle, Patrizia dell’Orto, Olivia Biasi, Roswitha Kammler, Sherene Loi, Marco Colleoni, Giuseppe Viale, Prudence A Francis, Meredith M Regan. The role of CYP2D6 mediated tamoxifen metabolism in the suppression of ovarian function trial (SOFT) [abstract]. In: Proceedings of the 2020 San Antonio Breast Cancer Virtual Symposium; 2020 Dec 8-11; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2021;81(4 Suppl):Abstract nr PD2-09.

Purpose:Women with hormone receptor–positive early breast cancers have a persistent risk of relapse and biomarkers for late recurrence are needed. We sought to identify tumor genomic aberrations associated with increased late-recurrence risk.Experimental Design:In a secondary analysis of Study of Letrozole Extension trial, a case-cohort–like sampling selected 598 primary breast cancers for targeted next-generation sequencing analysis of gene mutations and copy-number gains (CNGs). Correlations of genomic aberrations with clinicopathologic factors and breast and distant recurrence-free intervals (BCFIs and DRFIs) were analyzed using weighted Cox models.Results:Analysis of mutations and CNGs was successfully performed for 403 and 350 samples, including 148 and 134 patients with breast cancer recurrences (median follow-up time, 5.2 years), respectively. The most frequent alterations were PIK3CA mutations (42%) and CNGs of CCND1 (15%), ERBB2 (10%), FGFR1 (8%), and MYC (8%). PIK3CA mutations and MYC CNGs were associated with lower (P = 0.03) and higher (P = 0.004) tumor grade, respectively; a higher Ki-67 was seen in tumor with CCND1, ERBB2, and MYC CNGs (P = 0.01, P < 0.001, and P = 0.03, respectively). FGFR1 CNG was associated with an increased risk of late events in univariate analyses [17/29 patients; BCFI: HR, 3.2; 95% confidence interval (CI), 1.48–6.92; P = 0.003 and DRFI: HR, 3.5; 95% CI, 1.61–7.75; P = 0.002) and in multivariable models adjusted for clinicopathologic factors.Conclusions:Postmenopausal women with hormone receptor–positive early breast cancer harboring FGFR1 CNG had an increased risk of late recurrence despite extended therapy. FGFR1 CNG may represent a useful prognostic biomarker for late recurrence and a therapeutic target.

BACKGROUND: Although the triple-negative breast cancer (TNBC) tumor microenvironment (TME) has been deeply characterized, much remains unknown about pathways attributing to an immunosuppressive TME in this disease. The phosphorylation of JNK (c-Jun N-terminal kinase) pathway has been associated with promoting an immunosuppressive phenotype, enhancing TNBC aggressiveness. Here, we aimed to explore the role of the JNK pathway in TNBC using a gene signature inferring the level of JNK phosphorylation. For this purpose, we used the TNBC cohort from the phase III adjuvant IBCSG 22-00 trial, which evaluated low-dose cyclophosphamide and methotrexate (CM) chemotherapy showing no clinical benefit in the overall population. METHODS: JNK gene signature was developed in TNBC samples by integrating RNA-seq gene expression data and phospho-JNK-targeted proteomic data in The Cancer Genome Atlas. Stochastic subsampling was performed to select the candidate markers. LASSO regression was performed to determine the final transcriptional signature to reflect JNK phosphorylation. The signature was then applied in a cohort composed of 498 TNBCs selected using stratified 1:3 relapse cases and non-relapse subcohort ratio from the IBCSG 22-00 trial. RNA-seq data from FFPE tumor samples were available for 347 patients. The JNK signature was calculated as the mean of the products between gene expression and signature coefficients, defining high and low levels using the median cut-off. Immune hot tumors were defined according to TNBC molecular subtypes or tumor-infiltrating lymphocytes (TILs) levels higher than 30%. Multivariable Cox models were used for disease-free survival (DFS) analysis. Wilcoxon test was used to evaluate the association between gene signatures and the levels of JNK. RESULTS: Tumors with either immunomodulatory (IM) phenotype or high TILs showed better DFS when presenting low levels of JNK compared to high levels (HR = 0.75, 95% CI, 0.57 to 0.99; P-value = 0.024 and HR = 0.62, 95% CI, 0.43 to 0.89, P-value = 0.0013). No significant differences in DFS were observed in other TNBC subtypes or tumors with low TILs, further highlighting the relevance of JNK signaling pathway in tumors presenting immune infiltration. Moreover, immune hot tumors with high levels of JNK were enriched for angiogenesis and eosinophils signatures and linked to immunosuppression. The immune targets B7-H3, CSF1R, GITR, and GARP were significantly associated with high levels of JNK. Of note, these genes are involved in the immune escape, activation of macrophages, and regulation of Tregs population. On the other hand, low levels of JNK were associated with higher levels of activated CD8+ T cells, pointing to an anti-tumor immune response, as well as with higher levels of the immune targets PDL-1, CTLA4, CD47, DCIR, and TIGIT. Of interest, a significant DFS benefit was found in IM tumors with high levels of JNK when treated with low-dose CM, compared to those who were not (HR = 0.52, 95% CI, 0.28 to 0.99; P interaction = 0.045). CONCLUSIONS: We developed a JNK gene signature to estimate the phosphorylation level of JNK from gene expression data in TNBC, and validated the associated biological and prognostic value in the IBCSG 22-00 trial. Our results are in line with the immunosuppressive effect described by the JNK gene signature and highlight the heterogeneity of immune response in immune hot TNBCs. Of note, high levels of JNK were associated with worse DFS, as well as with a benefit from low-dose CM potentially related to the immunomodulatory effect described for metronomic regimens. Overall, our findings suggest a potential role of the JNK signature in identifying TNBCs with an immunosuppressive TME and provide the rationale to explore its role as a biomarker for immunotherapy. Further validation of these findings is required. Citation Format: Andrea Joaquin Garcia, Takashi Semba, Mattia Rediti, Daniel J. McGrail, Xuemei Xie, Xiaoping Wang, Dileep R. Rampa, David Venet, Samira Majjaj, Roswitha Kammler, Marco Colleoni, Sherene Loi, Giuseppe Viale, Meredith Regan, Françoise Rothé, Christos Sotiriou, Naoto T. Ueno. Role of immunosuppressive JNK pathway in the tumor microenvironment among Triple Negative Breast Cancer subtypes in IBCSG Trial 22-00 [abstract]. In: Proceedings of the 2022 San Antonio Breast Cancer Symposium; 2022 Dec 6-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2023;83(5 Suppl):Abstract nr PD9-07.

Ki-67 has been clinically validated for risk assessment in breast cancer, but the analytical validation and cutpoint-definition remain a challenge. Intraclass correlation coefficients (ICCs) are a statistical parameter for Ki-67 interobserver performance. However, the maximum degree of variance among pathologists allowed for meaningful biomarker results has not been defined. Different amounts of variance were added to central pathology Ki-67 data (n = 9069) from three cohorts (IBCSGVIII + IX, BIG1-98, GeparTrio) by simulation of 4500 evaluations for each cohort, which were grouped by ICCs, ranging from excellent (ICC = 0.9) to poor concordance (ICC = 0.1). Endpoints were disease-free survival (DFS) and pathological complete response (pCR, GeparTrio). Ki-67 was a significant continuous prognostic marker for DFS over a wide range of cutpoints between 8% and 30% in all three cohorts. In our modelling approach, Ki-67 was a stable prognostic marker despite increased interpathologist variance. Even for a poor ICC of 0.5, one or more significant Ki-67 cutoffs were detected in 86.8% (GeparTrio), 92.4% (IBCSGVIII + IX) and 100% of analyses (BIG1-98). Similarly, in GeparTrio, even with an extremely low ICC of 0.2, 99.6% of analyses were significant for pCR. Our study shows that Ki-67 is a continuous marker which is extremely robust to pathologist variation. Even if only 50% of variance is attributable to true Ki-67-based proliferation (ICC = 0.5), this information is sufficient to obtain statistically significant differences in clinical cohorts. This stable performance explains the observation that many Ki-67 studies achieve significant results despite relevant interobserver variance and points to a high clinical validity of this biomarker. For clinical decisions based on analysis of individual patient data, ongoing efforts to further reduce interobserver variability, including ring trials and standardized guidelines as well as image analysis approaches, should be continued.

Summary Background HER2-positive breast cancers usually contain large amounts of T-cell infiltrate. We hypothesised that trastuzumab resistance in HER2-positive breast cancer could be mediated by immune mechanisms. We assessed the safety and anti-tumour activity of pembrolizumab, a programmed cell death protein 1 (PD-1) inhibitor, added to trastuzumab in trastuzumab-resistant, advanced HER2-positive breast cancer. Methods We did this single-arm, multicentre, phase 1b–2 trial in 11 centres based in five countries. Eligible participants were women aged 18 years or older, who had advanced, histologically confirmed, HER2-positive breast cancer; documented progression during previous trastuzumab-based therapy; an Eastern Cooperative Oncology Group performance status of 0 or 1; and a formalin-fixed, paraffin-embedded metastatic tumour biopsy for central assessment of programmed cell death 1 ligand 1 (PD-L1) status. In phase 1b, we enrolled patients with PD-L1-positive tumours in a 3 + 3 dose-escalation of intravenous pembrolizumab (2 mg/kg and 10 mg/kg, every 3 weeks) plus 6 mg/kg of intravenous trastuzumab. The primary endpoint of the phase 1b study was the incidence of dose-limiting toxicity and recommended phase 2 dose; however, a protocol amendment on Aug 28, 2015, stipulated a flat dose of pembrolizumab of 200 mg every 3 weeks in all Merck-sponsored trials. In phase 2, patients with PD-L1-positive and PD-L1-negative tumours were enrolled in parallel cohorts and received the flat dose of pembrolizumab plus standard trastuzumab. The primary endpoint of the phase 2 study was the proportion of PD-L1-positive patients achieving an objective response. This trial is registered in ClinicalTrials.gov, number NCT02129556, and with EudraCT, number 2013-004770-10, and is closed. Findings Between Feb 2, 2015, and April 5, 2017, six patients were enrolled in phase 1b (n=3 received 2 mg/kg pembrolizumab, n=3 received 10 mg/kg pembrolizumab) and 52 patients in phase 2 (n=40 had PD-L1-positive tumours, n=12 had PD-L1-negative tumours). The data cutoff for this analysis was Aug 7, 2017. During phase 1b, there were no dose-limiting toxicities in the dose cohorts tested. Median follow-up for the phase 2 cohort was 13·6 months (IQR 11·6–18·4) for patients with PD-L1-positive tumours, and 12·2 months (7·9–12·2) for patients with PD-L1-negative tumours. Six (15%, 90% CI 7–29) of 40 PD-L1-positive patients achieved an objective response. There were no objective responders among the PD-L1-negative patients. The most common treatment-related adverse event of any grade was fatigue (12 [21%] of 58 patients). Grade 3–5 adverse events occurred in 29 (50%) of patients, treatment-related grade 3–5 adverse events occurred in 17 (29%), and serious adverse events occurred in 29 (50%) patients. The most commonly occurring serious adverse events were dyspnoea (n=3 [5%]), pneumonitis (n=3 [5%]), pericardial effusion (n=2 [3%]), and upper respiratory infection (n=2 [3%]). There was one treatment-related death due to Lambert-Eaton syndrome in a PD-L1-negative patient during phase 2. Interpretation Pembrolizumab plus trastuzumab was safe and showed activity and durable clinical benefit in patients with PD-L1-positive, trastuzumab-resistant, advanced, HER2-positive breast cancer. Further studies in this breast cancer subtype should focus on a PD-L1-positive population and be done in less heavily pretreated patients. Funding Merck, International Breast Cancer Study Group.

INTRODUCTION: KRAS mutations, the most frequent gain-of-function alterations in NSCLC, are currently emerging as potential predictive therapeutic targets. The role of KRAS-G12C (Kr_G12C) is of special interest after the recent discovery and preclinical analyses of two different Kr_G12C covalent inhibitors (AMG-510, MRTX849). METHODS: KRAS mutations were evaluated in formalin-fixed, paraffin-embedded tissue sections by a microfluidic-based multiplex polymerase chain reaction platform as a component of the previously published European Thoracic Oncology Platform Lungscape 003 Multiplex Mutation study, of clinically annotated, resected, stage I to III NSCLC. In this study, -Kr_G12C mutation prevalence and its association with clinicopathologic characteristics, molecular profiles, and postoperative patient outcome (overall survival, relapse-free survival, time-to-relapse) were explored. RESULTS: KRAS gene was tested in 2055 Lungscape cases (adenocarcinomas: 1014 [49%]) with I or II or III stage respective distribution of 53% or 24% or 22% and median follow-up of 57 months. KRAS mutation prevalence in the adenocarcinoma cohort was 38.0% (95% confidence interval (CI): 35.0% to 41.0%), with Kr_G12C mutation representing 17.0% (95% CI: 14.7% to 19.4%). In the "histologic-subtype" cohort, Kr_G12C prevalence was 10.5% (95% CI: 9.2% to 11.9%). When adjusting for clinicopathologic characteristics, a significant negative prognostic effect of Kr_G12C presence versus other KRAS mutations or nonexistence of KRAS mutation was identified in the adenocarcinoma cohort alone and in the "histologic-subtype" cohort. For overall survival in adenocarcinomas, hazard ratio (HR)G12C versus other KRAS is equal to 1.39 (95% CI: 1.03 to 1.89, p= 0.031) and HRG12C versus no KRAS is equal to 1.32 (95% CI: 1.03 to 1.69, p= 0.028) (both also significant in the "histologic-subtype" cohort). For time-to-relapse, HRG12C versus other KRAS is equal to 1.41 (95% CI: 1.03 to 1.92, p= 0.030). In addition, among all patients, for relapse-free survival, HRG12C versus no KRAS is equal to 1.27 (95% CI: 1.04 to 1.54, p= 0.017). CONCLUSIONS: In this large, clinically annotated stage I to III NSCLC cohort, the specific Kr_G12C mutation is significantly associated with poorer prognosis (adjusting for clinicopathologic characteristics) among adenocarcinomas and in unselected NSCLCs.

PD-L1, as assessed by immunohistochemistry, is a predictive biomarker for immuno-oncology treatment in lung cancer. Different scoring methods have been used to assess its status, resulting in a wide range of positivity rates. We use the European Thoracic Oncology Platform Lungscape non-small cell lung carcinoma cohort to explore this issue. PD-L1 expression was assessed via immunohistochemistry on tissue microarrays (up to four cores per case), using the DAKO 28-8 immunohistochemistry assay, following a two-round external quality assessment procedure. All samples were analyzed under the same protocol. Cross-validation of scoring between tissue microarray and whole sections was performed in 10% randomly selected samples. Cutoff points considered: ≥1, 50 (primarily), and 25%. At the two external quality assessment rounds, tissue microarray scoring agreement rates between pathologists were: 73% and 81%. There were 2008 cases with valid immunohistochemistry tissue microarray results (50% all cores evaluable). Concordant cases at 1, 25, and 50% were: 85, 91, and 93%. Tissue microarray core results were identical for 70% of cases. Sensitivity of the tissue microarray method for 1, 25, and 50% was: 80, 78, and 79% (specificity: 90, 95, 98%). Complete agreement between tissue microarrays and whole sections was achieved for 60% of the cases. Highest sensitivity rates for 1% and 50% cutoffs were detected for higher number of cores. Underestimation of PD-L1 expression on small samples is more common than overestimation. We demonstrated that classification of PD-L1 on small biopsy samples does not represent the overall expression of PD-L1 in all non-small cell cancer carcinoma cases, although the majority of cases are ‘correctly’ classified. In future studies, sampling more and larger biopsies, recording the biopsy size and tumor load may permit further refinement, increasing predictive accuracy.

In postmenopausal, estrogen receptor-positive, human epidermal growth factor receptor 2 (HER2)-negative early breast cancer, the risk for distant recurrence can extend beyond 5 years of adjuvant endocrine therapy. This study aims to identify genomic driver alterations associated with late distant recurrence.Next generation sequencing was used to characterize driver alterations in primary tumors from a subset of 764 postmenopausal estrogen receptor-positive/HER2-negative patients from the BIG 1-98 randomized trial. Late distant recurrence events were defined as ≥5 years from time of randomization). The association of driver alterations with distant recurrence-free interval in early and late time periods was assessed using Cox regression models. Multivariable analyses were carried out to adjust for clinicopathological factors. Weighted analysis methods were used in order to correct for over-sampling of distant recurrences.A total of 538 of 764 (70%) samples were successfully sequenced including 88 (63%) early and 52 (37%) late distant recurrence events after a median follow up of 8.1 years. In univariable analysis for late distant recurrence, PIK3CA mutations (58.8%) were significantly associated with reduced risk [hazard ratio (HR) 0.40, 95% confidence interval (CI) 0.20-0.82, P = 0.012], whereas amplifications on chromosome 8p11 (10.9%) (HR 4.79, 95% CI 2.30-9.97, P0.001) and BRCA2 mutations (2.3%) (HR 5.39, 95% CI 1.51-19.29, P = 0.010) were significantly associated with an increased risk. In multivariable analysis, only amplifications on 8p11 (P = 0.002) and BRCA2 mutations (P = 0.013) remained significant predictors.In estrogen receptor-positive/HER2-negative postmenopausal early breast cancer, PIK3CA mutations were associated with reduced risk of late distant recurrence, whereas amplifications on 8p11 and BRCA2 mutations were associated with increased risk of late distant recurrence. The characterization of oncogenic driver alterations may aid in refining treatment choices in the late disease setting, and help identify potential drug targets for testing in future trials.

Introduction: Longitudinal evaluation of mutations in blood samples was a prespecified secondary objective in the BELIEF trial of erlotinib and bevacizumab in advanced EGFR-positive NSCLC. Here, we report the testing results and explore the correlation of EGFR status in blood with clinical outcomes. Methods: Blood samples were prospectively collected from patients at baseline, at response evaluation, and at progression and sent to a central laboratory. Circulating free DNA was purified and EGFR mutations were analyzed with a validated real-time quantitative polymerase chain reaction assay. Results: EGFR exon 19/21 mutations were detected in 55 of 91 baseline blood samples (60.4%) and correlated with a significantly worse progression-free survival: 11.4 months (95% confidence interval [CI]: 9.0-14.8 mo) for the patients who were positive versus 22.9 months (95% CI: 9.5-33.9 mo) for those who were negative (log-rank p = 0.0020). Among the 74 samples at response, exon 19/21 mutations were detected only in three samples (4.1%). In contrast, 29 of 58 patients (50.0%) were exon 19/21 positive at progression and showed a significantly worse median overall survival of 21.7 months (95% CI: 17.0-30.9 mo) compared with 37.4 months (95% CI: 22.6-53.1 mo) for those who were negative (log-rank p = 0.011). Blood samples at the three time points were available for 48 patients. Of those, among 14 exon 19/21 EGFR-negative at presentation, 13 (93%) were persistently negative for the sensitizing mutations after progression and the p.T790M could only be detected in the blood of two patients. Conclusions: Longitudinal testing of EGFR mutations in blood can offer valuable clinical information. In patients of the BELIEF study, detection of EGFR mutations in circulating free DNA at presentation was associated with shorter progression-free survival, whereas positivity at progression correlated with shorter overall survival. Finally, patients negative in blood at presentation were almost invariably negative at relapse. (C) 2019 Published by Elsevier Inc. on behalf of International Association for the Study of Lung Cancer.

Background Preclinical and clinical data suggest that HER2-positive (HER2+) breast cancer (BC) will be amendable to immunotherapeutic approaches. We evaluated pembrolizumab with trastuzumab in patients (pts) with trastuzumab-resistant HER2+, PD-L1 positive (PD-L1pos), unresectable loco-regional or metastatic BC and a parallel cohort of pts with HER2+, PD-L1 negative (PD-L1neg) BC during the phase II study. Methods: Pts with advanced BC and progression on prior trastuzumab-based therapies, ECOG 0-1, and a metastatic tumor biopsy in the last year were eligible. HER2 positivity and quantity of tumor-infiltrating lymphocytes (TILs) on H&E slide were centrally evaluated. PD-L1 score was assessed by Merck central lab. Tumor imaging was performed at weeks 12, 18, 24 and every 12 weeks, thereafter. Primary endpoints were safety of the combination (phase Ib) and objective response rate (ORR) per RECIST 1.1 (phase II). Secondary endpoints were PFS, duration of response, and OS. Phase Ib was a 3+3 dose-escalation of 2 pembrolizumab doses (2mg/kg, 10mg/kg) Q3W. In phase II, pts received pembrolizumab 200mg Q3W for 24 months or until disease progression. Clinically stable pts with progression were allowed to continue pembrolizumab until confirmation on subsequent assessment. Pts with isolated CNS progression were also allowed to continue pembrolizumab after local treatment. Planned total enrollment was 61 pts. For the phase II PD-L1pos cohort, a Simon two-stage design (N=40; proceed if ≥2/17 respond) was used which had 85% power to compare ORR of 7% vs. 22% (1-sided α=0.05). For the PD-L1neg cohort, a single-stage design with 15 pts had >95% power to compare ORR of 1% vs. 20% (1-sided α=0.14). Clinicaltrials.gov: NCT02129556. Results: 6 pts enrolled in phase Ib between April and July 2015; no DLTs were observed. The PD-L1pos cohort enrolled 40 pts between August 2015 and September 2016. The PD-L1neg cohort enrolled May 2016 to April 2017, stopping after 12 pts due to low rate of PD-L1 negativity, maintaining >90% power to detect the target difference in ORR. PD-L1 testing labs changed in April 2016. Prior to this time, QualTek PD-L1 positive was defined as ≥1% on tumor or TILs. Using the Dako 22C3 antibody, positive was defined as tumor PD-L1 combined positive score (CPS)≥1%. 146 pts were screened to enroll 58 pts. Of screened pts, median stromal TILs was 1% (mean: 4.8%, SD: 9.1%, range: 0 to 60%; n=127); 52% of pts were PD-L1pos, with higher positivity rates while using the Dako assay compared with Qualtek (65% vs. 43%, p=0.009). Median TILs of pts in the PD-L1pos cohort was 2% (mean: 8.1%, SD: 11.2%, range: 0 to 40%) and 0% (mean: 1.2%, SD: 2.2%, range: 0 to 5%) in the PD-L1neg cohort. Of enrolled pts, median age was 51yrs (range: 28-72), 69% had visceral metastases. 29% of pts received prior pertuzumab, 72% had prior T-DM1, 40% prior lapatinib. 38% of pts were ER-positive, 62% were ER-negative. Median TILs in enrolled ER pos and ER neg pts were 1.5% and 2.0%, respectively. PD-L1 positivity rates were also not significantly different by ER status (p=0.5). Final safety data and efficacy results will be presented at the meeting. Citation Format: Loi S, Giobbe-Hurder A, Gombos A, Bachelot T, Hui R, Curigliano G, Campone M, Biganzoli L, Bonnefoi H, Jerusalem G, Bartsch R, Rabaglio-Poretti M, Kammler R, Maibach R, Smyth MJ, Di Leo A, Colleoni M, Viale G, Regan MM, Andre F. Phase Ib/II study evaluating safety and efficacy of pembrolizumab and trastuzumab in patients with trastuzumab-resistant HER2-positive metastatic breast cancer: Results from the PANACEA (IBCSG 45-13/BIG 4-13/KEYNOTE-014) study [abstract]. In: Proceedings of the 2017 San Antonio Breast Cancer Symposium; 2017 Dec 5-9; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2018;78(4 Suppl):Abstract nr GS2-06.

Background: Neoadjuvant endocrine therapy (NET) with gonadotropin-releasing hormone (GnRH) agonist and aromatase inhibitors is effective in selected premenopausal patients (pts). Degarelix, an antagonist of GnRH, has immediate onset of action through binding to GnRH receptors in the pituitary gland and thereby suppressing the production of LH and FSH. Its suppressing activity in premenopausal women might be faster and free of estrodial breakthrough on continued treatment compared with a GnRH angonist, and thereby provide significant clinical value for pts who are candidates for short-term NET. Methods: Eligible pts were premenopausal women with cT2-4b, any nodal stage, ER and PgR >50%, HER2-negative (by IHC and/or ISH) breast cancer who were not candidates for breast conserving surgery. Premenopausal status was determined locally with estradiol (E2) levels >54 pg/mL (or >198 pmol/L), measured within 14 days prior to randomization. Pts were randomized 1:1 to Triptorelin (T) 3.75 mg i.m. on day 1 of every cycle or Degarelix (D) 240 mg s.c. given as two injections of 120 mg on day 1 of cycle 1, then 80 mg s.c. on day 1 of cycles 2-6 with letrozole (L) 2.5 mg/day for 6 cycles. Each cycle was 28 days. Definitive surgery was performed within 2-3 weeks after the last administration of T or D. Serum was collected prior to the first injection (baseline), 24 and 72 hours, 7 and 14 days, then prior to injection on day 1 of cycles 2-6. The primary endpoint was time to optimal ovarian function suppression (OFS) calculated as time from the first injection of D or T to the first assessment of centrally assessed 17-β-estradiol (E2) level in the range of optimal OFS (≤2.72 pg/mL or ≤10 pmol/L) during the 6 cycles of NET. The trial had 90% power to detect a difference using a logrank test, 2-sided α=0.05. Secondary endpoints included tolerability, Ki67changes, PEPI score, best overall response. NCT02005887 Results: TREND completed accrual of 51 pts in January 2017. A preliminary analysis based on the first 45 pts is reported here. 89% of patients were ≥40 yrs, 76% had T1-2 and 22% T3 tumors, and 51% were node-positive. Dominant histology type was ductal (93%). The table summarizes centrally-assessed E2 according to treatment at baseline and for the first 5 assessment time points indicating immediate suppression for the D+L arm. E2 levels on day 1 of cycles 2-6 were all below the limit of quantification (0.625 pg/mL) for the D+L arm. For the T+L arm continued OFS was not maintained in 4 pts. BaselineCycle 1Cycle 2Day:01371429No. Pts D+L222221212221T+L232321232222Median (IQR) D+L96.2 (64.2,206.8)10.1 (4.0,21.8)0.6 (0.6,1.0)0.6 (0.6,0.6)0.6 (0.6,0.6)0.6 (0.6, 0.6)T+L85.1 (49.7,118.0)37.4 (17.9,59.2)12.8 (7.7,23.8)9.0 (1.2,29.7)0.6 (0.6,1.4)0.6 (0.6, 0.6) Conclusion: Evidence from this first analysis demonstrates rapid and maintained OFS with the combination of D+L as a NET in premenopausal breast cancer patients. The final analysis of the total population, including secondary endpoints, will be presented at the symposium. Citation Format: Colleoni M, Gray K, Munzone E, Dellapasqua S, Zamagni C, Gianni L, Johansson H, Viale G, Kammler R, Maibach R, Rabaglio-Poretti M, Di Leo A, Coates AS, Gelber RD, Regan MM, Goldhirsch A. A randomized phase II trial evaluating the endocrine activity and efficacy of neoadjuvant degarelix versus triptorelin in premenopausal patients receiving letrozole for primary endocrine responsive breast cancer (TREND; IBCSG 41-13) [abstract]. In: Proceedings of the 2017 San Antonio Breast Cancer Symposium; 2017 Dec 5-9; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2018;78(4 Suppl):Abstract nr P1-10-06.

[EN] Background Reported prevalence of driver gene mutations in non-small-cell lung cancer (NSCLC) is highly variable and clinical correlations are emerging. Using NSCLC biomaterial and clinical data from the European Thoracic Oncology Platform Lungscape iBiobank, we explore the epidemiology of mutations and association to clinicopathologic features and patient outcome (relapse-free survival, time-to-relapse, overall survival). Methods Clinically annotated, resected stage I¿III NSCLC FFPE tissue was assessed for gene mutation using a microfluidics-based multiplex PCR platform. Mutant-allele detection sensitivity is¿>1% for most of the ~150 (13 genes) mutations covered in the multiplex test. Results Multiplex testing has been carried out in 2063 (76.2%) of the 2709 Lungscape cases (median follow-up 4.8¿years). FFPE samples mostly date from 2005 to 2008, yet recently extracted DNA quality and quantity was generally good. Average DNA yield/case was 2.63¿µg; 38 cases (1.4%) failed QC and were excluded from study; 95.1% of included cases allowed the complete panel of mutations to be tested. Most common were KRAS, MET, EGFR and PIK3CA mutations with overall prevalence of 23.0%, 6.8%, 5.4% and 4.9%, respectively. KRAS and EGFR mutations were significantly more frequent in adenocarcinomas: PIK3CA in squamous cell carcinomas. MET mutation prevalence did not differ between histology groups. EGFR mutations were found predominantly in never smokers; KRAS in current/former smokers. For all the above mutations, there was no difference in outcome between mutated and non-mutated cases. Conclusion Archival FFPE NSCLC material is adequate for multiplex mutation analysis. In this large, predominantly European, clinically annotated stage I¿III NSCLC cohort, none of the mutations characterized showed prognostic significance.

Purpose Identification of single-gene biomarkers that are prognostic of outcome can shed new insights on the molecular mechanisms that drive breast cancer and other cancers. Methods Exploratory analysis of 20,464 single-gene messenger RNAs (mRNAs) in the Molecular Taxonomy of Breast Cancer International Consortium (METABRIC) discovery cohort indicates that low expression of FGD3 mRNA is prognostic for poor outcome. Prognostic significance of faciogenital dysplasia 3 (FGD3), SUSD3, and other single-gene proliferation markers was evaluated in breast cancer and The Cancer Genome Atlas (TCGA) cohorts. Results A meta-analysis of Cox regression of FGD3 mRNA as a continuous variable for overall survival of estrogen receptor (ER)–positive samples in METABRIC discovery, METABRIC validation, TCGA breast cancer, and Combination Chemotherapy in Treating Women With Breast Cancer (E2197) cohorts resulted in a combined hazard ratio (HR) of 0.69 (95% CI, 0.63 to 0.75), indicating better outcome with high expression. In the ER-negative samples, the combined meta-analysis HR was 0.72 (95% CI, 0.63 to 0.82), suggesting that FGD3 is prognostic regardless of ER status. The potential of FGD3 as a biomarker for freedom from recurrence was evaluated in the Breast International Group 1-98 (BIG 1-98; Letrozole or Tamoxifen in Treating Postmenopausal Women With Breast Cancer) study (HR, 0.85; 95% CI, 0.76 to 0.93) for breast cancer–free interval. In the Hungarian Academy of Science (HAS) breast cancer cohort, splitting on the median had an HR of 0.49 (95% CI, 0.42 to 0.58) for recurrence-free survival. A comparison of the Stouffer P value in five ER-positive cohorts showed that FGD3 ( P = 3.8E-14) outperformed MKI67 ( P = 1.06E-8) and AURKA ( P = 2.61E-5). A comparison of the Stouffer P value in four ER-negative cohorts showed that FGD3 ( P = 3.88E-5) outperformed MKI67 ( P = .477) and AURKA ( P = .820). Conclusion FGD3 was previously shown to inhibit cell migration. FGD3 mRNA is regulated by ESR1 and is associated with favorable outcome in six distinct breast cancer cohorts and four TCGA cancer cohorts. This suggests that FGD3 is an important clinical biomarker.

Introduction: In a well-defined NSCLC cohort of the ETOP Lungscape program, we explored the epidemiology of IHC MET overexpression and amplification, their inter-correlation, and their association to outcome.Methods: Resected NSCLC were assessed for MET gene copy number (GCN) and expression using silver in-situ hybridization (SISH) and immunohistochemistry (IHC) on TMAs in a multicenter setting. MET amplification was defined as MET/centromere ratio >= 2 (with average MET GCN >= 4), high MET GCN as CGN >= 5 and MET IHC + as >= 2 + intensity in >= 50% of tumor cells. A total of 182 MET IHC + and EGFR/KRAS WT tumors were analyzed for METex14 skipping mutation.Results: MET IHC+ was found in 23.8% of 2432 patients, significantly associated with female gender, small tumor size, and adenocarcinoma histology. We observed a high inter-laboratory variability in IHC and SISH analysis. MET amplification prevailed in 4.6% and MET GCN >= 5 in 4.1% of 1572 patients. MET amplification and MET GCN >= 5 were not significantly associated with any tumor characteristics or stage. Both were significantly associated with IHC MET positivity (p Conclusion: MET overexpression is found in 23.8% of surgically resected NSCLC. MET amplification prevails in 4.6% and is associated with MET overexpression. Both have no influence on prognosis. The large inter-laboratory variability in IHC highlights the challenge of MET IHC analysis in routine practice.

Background: EarlyR is a prognostic gene signature score in ER+ breast cancer (BC) computed from the expression values of ESPL1, SPAG5, MKI67, PLK1 and PGR using a novel algorithm. EarlyR has been validated in multiple cohorts profiled on Affymetrix and Illumina microarrays. This study sought to verify prognostic features of EarlyR in a cohort of BIG 1-98. Patients and Methods: Illumina DASL assay was used to measure gene expression in FFPE tissue of primary BC from a case-cohort sampling subset of postmenopausal women in BIG 1-98 treated with adjuvant endocrine therapy (letrozole or tamoxifen). Chemotherapy treatment was at the discretion of individual physicians and patients. Among the 1218 patients centrally reviewed with sufficient RNA material for the DASL assay, 1174 with ER+ status and assessed for EarlyR are included in the analytic cohort. EarlyR scores and pre-specified risk strata (≤25=low, 26-75=intermediate, >75=high) were computed, while blinded to clinical data. The analysis endpoints included distant recurrence free interval (DRFI) defined as time from randomization to BC recurrence at a distant site within 8 years and BC free-interval (BCFI) defined as time from randomization to first invasive BC recurrence at a local, regional or distant site or invasive contralateral BC within 8 years. Weighted proportional hazards models (univariate and multivariate, stratified by treatment assignment) were used to adjust for Kaplan-Meier, hazard ratio estimates and Wald test statistics to obtain unbiased analyses and to give consistent estimates. Results: The distribution of the EarlyR risk groups was 67% low, 19% intermediate and 14% high risk in this ER+ cohort. EarlyR was prognostic for 8-year DRFI (P-trend=0.008). Patients with high EarlyR risk score (>75) had significantly increased risk of distant recurrence within 8 years (univariate HR=1.73, 95%CI: 1.14-2.64) compared to low EarlyR risk group (≤25). The estimated 8-year DRFI (95%CI) is 84%(80%-88%) for high risk vs. 91%( 89%-92%) for low risk, corresponding to an absolute DRFI risk reduction of 7% (low vs high). EarlyR is also prognostic of 8-year BCFI in ER+ (P-trend=0.002) with the estimated 8-year BCFI (95%CI) 79%(75%-84%) for high risk vs. 88%( 86%-89%) for low risk. Consistent results were observed in ER+, HER2- (P-trend=0.01 for DRFI, P-trend=0.004 for BCFI), in ER+, LN- (P-trend=0.05 for DRFI, P-trend=0.03 for BCFI) and ER+, LN+ (P-trend=0.08 for DRFI, P-trend=0.03 for BCFI) subsets. Conclusions: This study confirmed the prognostic significance of EarlyR using FFPE tissue from the BIG 1-98 trial. In analyses of all ER+ patients and subsets LN-, LN+ and HER2-, EarlyR classifies 65%-70% of patients as low risk, 11-16% as high risk, and < 20% as intermediate risk. In these subsets, the size of the low risk group is larger and the size of the intermediate risk group is smaller than those reported for commercially available signatures. EarlyR identifies a set of high-risk patients with relatively poor prognosis who may be considered for additional treatment. The clinical utility of EarlyR requires further study. Citation Format: Buechler S, Gray KP, Gökmen-Polar Y, Willis S, Thürlimann B, Kammler R, Leyland-Jones B, Badve SS, Regan MM. Independent validation of EarlyR gene signature in BIG 1-98: A randomized, double-blind, phase III trial comparing letrozole and tamoxifen as adjuvant endocrine therapy for postmenopausal women with hormone receptor-positive, early breast cancer [abstract]. In: Proceedings of the 2016 San Antonio Breast Cancer Symposium; 2016 Dec 6-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2017;77(4 Suppl):Abstract nr P4-12-01.

Background Next generation sequencing (NGS) has revealed that ER+/HER2- BCs have diverse somatic copy number and mutation profiles but thus far the clinical relevance of such findings is unknown. We characterized the molecular alterations in post-menopausal primary BC patients treated in the BIG 1-98 adjuvant letrozole and tamoxifen study and evaluated their associations with prognosis. Materials and Methods NGS was used to genotype DNA from archival primary tumor blocks for 286 cancer-related genes. From 2706 available eligible samples (confirmed ER+, excluding HER2-positivity or neoadjuvant treatment, adequate DNA quality/quantity), a case-cohort design selected 764 samples: all distant relapses and a stratified sampling of non-relapses after 8yr median follow-up. Mutation prevalence and associations with clinicopathological factors (CP) as well as distant recurrence-free interval (DRFS) were analyzed using weighted tests and Cox regression models, with sampling weights to represent the ER+/HER2-negative trial population. Multivariable analyses were adjusted for tumor size, nodal status, grade and age. Results NGS data was available from 538/764 samples (70%), there were 140 (26%) distant relapses. Median sequencing depth was 483x. There was a mean of 11 mutations (1-46) per sample, with 25% having 13 or more mutations and no tumors without a mutation. Overall 28 genes were altered at a frequency of >10%. The most commonly mutated genes were PIK3CA (49.3%), NCOR1 (27.2%), MAP3K1 (23.8%) TP53 (16.6%), CCND1 (17.8%) and GATA3 (17.1%). Alterations that were significantly associated with both Luminal B (Ki67 >14%) and grade 3 included TP53 mutations (p Gene alterations that were significantly associated with shorter DRFS included TP53 (HR:2.16), ARID1A (HR:2.43), CHEK2 (HR:2.54), BRCA2 (HR:1.93), PTEN (HR:2.03), CCND1 (HR:1.82) and FGFR1 (HR:1.78). PIK3CA was significantly associated with lower risk of distant relapse (HR:0.64; 0.43-0.97). Increasing number of total mutations was significantly associated with shorter DRFS (HR:1.04; 95% CI 1.01-1.07; p=0.006). In the multivariable model adjusted for CP factors, ARID1A, BRCA2, CCND1, CHEK2 and PTEN remained independent for shorter DRFS. Greater than 90% of PIK3CA mutations co-existed with another alteration, most common being NCOR1 (29%), MAP3K1 (24%), CDH1 (16%), GATA3 (16%), TP53 (14%) and CCND1 (13%). Patients with a PIK3CA mutation had greater benefit with letrozole over tamoxifen monotherapy (HR 0.32; 0.13-0.8) than those without (HR:0.70; 0.33-1.48) (Pint=0.06). This effect was strongest in the subgroup of PIK3CA mutant patients who were CCND1 and TP53 wild-type (HR:0.24, 0.12-0.48; Pint=0.02) with only 1% relapsing at 5 years. Conclusion: For the first time, we report the prognostic relevance of oncogenic mutations in ER+/HER2- postmenopausal early-stage BCs from a clinical trial. Tumors with PIK3CA mutations derived greater benefit from letrozole over tamoxifen monotherapy, especially if wild-type for CCND1 and TP53. These findings could significantly improve prognostic risk classification and guide future clinical trials of targeted therapies in ER+/HER2- BCs. Citation Format: Loi S, Asher R, Lee CK, Luen S, Savas P, Kammler R, Dell'Orto P, Blasi OM, Demanse D, JeBailley L, Dolan S, Hackl W, Thuerlimann B, Viale G, Regan M, Colleoni MA. Clinical implications of somatic mutations in post-menopausal early-stage estrogen receptor (ER)-positive HER2-negative breast cancer (BC): Results from the BIG 1-98 study [abstract]. In: Proceedings of the 2016 San Antonio Breast Cancer Symposium; 2016 Dec 6-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2017;77(4 Suppl):Abstract nr S1-10.

Introduction Mutations in erb-b2 receptor tyrosine kinase 2 (HER2) oncogene are observed in approximately 3% of lung adenocarcinomas or mixed tumors with adenocarcinoma component. Activity of various biologically distinct HER2 inhibitors, including the pan-HER inhibitor afatinib, has been reported in several retrospective trials or small series in advanced pretreated NSCLC with HER2 mutations. We report the first prospective evaluation of afatinib for the treatment of this molecularly defined entity. Methods NICHE, a single-arm phase II trial using a two-stage Simon's design, explored the potential of afatinib to control disease in pretreated patients with advanced NSCLC harboring HER2 exon 20 mutations. A total of 13 patients entered the trial and were treated with afatinib 40 mg/day until tumor progression or lack of tolerability. Results The first-stage stopping boundary was crossed when five of nine patients did not achieve disease control at 12 weeks. The accrual into the trial was stopped with total 13 patients enrolled, with 7 (53.8%) achieving disease control at 12 weeks. Except for 1 patient with early death, progression was documented for all patients, with median progression-free survival of 15.9 weeks (95% confidence interval: 6.0–35.4), and median overall survival of 56.0 weeks (95% confidence interval: 16.3– upper limit not estimable). The toxicity profile was in the expected range. Conclusions Afatinib did not show the expected potential for disease control in NSCLC. However, more than half of the patients in the full cohort achieved disease control at 12 weeks.

Background The androgen receptor (AR) is an emerging prognostic marker and therapeutic target in breast cancer. AR is expressed in 60–80% of breast cancers, with higher prevalence among estrogen receptor-positive (ER+) tumors. Androgen treatment inhibits ER signaling in ER+/AR+ breast cancer cell lines, and AR expression is associated with improved survival for this subtype in epidemiologic studies. However, whether AR expression modifies the efficacy of selective ER modulators or aromatase inhibitors for ER+ cancers remains unclear. Methods We evaluated the prognostic and predictive value of AR expression among 3021 postmenopausal ER+ breast cancer patients in the Breast International Group (BIG) trial 1–98. The BIG 1–98 study was a four-armed, double-blind, phase III randomized clinical trial that compared 5 years of tamoxifen or letrozole monotherapy, or sequences of 2 years and 3 years treatment with one drug and then the other. AR expression was measured by immunohistochemistry and the percentage of AR-positive nuclei was quantified. The association between AR expression and prognosis was evaluated using Cox proportional hazards models. Continuous AR-by-treatment interactions were assessed using Subpopulation Treatment Effect Pattern Plots (STEPP). Results Eighty-two percent of patients had AR+ (≥ 1%) tumors. Patients with AR+ cancers were more likely to have smaller, lower-grade tumors, with higher expression of ER and PR. AR expression was not associated with breast cancer-free interval (BCFI) (415 events) over a median 8.0 years of follow-up (p = 0.12, log-rank test). In multivariable-adjusted models, AR expression was not associated with BCFI (HR = 1.07, 95% CI 0.83–1.36, p = 0.60). The letrozole versus tamoxifen monotherapy treatment effect did not significantly differ for AR+ tumors (HR = 0.63, 95% CI 0.44–0.75, p = 0.003) and AR− tumors (HR = 0.39, 95% CI 0.21–0.72, p = 0.002) (p-heterogeneity = 0.16). STEPP analysis also suggested no heterogeneity of the treatment effect across the continuum of AR expression. Conclusions AR expression was not associated with prognosis, nor was there heterogeneity of the letrozole versus tamoxifen treatment effect by AR expression. These findings suggest that AR expression may not be an informative biomarker for the selection of adjuvant endocrine therapy for postmenopausal women with ER+ breast cancers. Trial registration ClinicalTrials.gov, NCT00004205, Registered 27 January 2003—Retrospectively registered, https://clinicaltrials.gov/ct2/show/study/NCT00004205. Electronic supplementary material The online version of this article (10.1186/s13058-019-1118-z) contains supplementary material, which is available to authorized users.

The purpose of this study was to assess the prognostic and predictive value of tumor-infiltrating lymphocytes (TILs) in the triple-negative breast cancer (TNBC) cohort of the phase III IBCSG trial 22-00, comparing low-dose oral ‘metronomic’ cyclophosphamide-methotrexate maintenance chemotherapy (CM-maintenance) to no-CM-maintenance in early breast cancer. TILs were evaluated in full-face hematoxylin-and-eosin-stained sections of tumor samples confirmed centrally as TNBC (

Background: The prognostic significance of increased levels of CD8+ tumor infiltrating lymphocytes(TILs) in ER- breast cancer has been described. We sought to identify possible immune-related biomarkers for predicting benefit from letrozol(LET) or tamoxifen(TAM) for recurrence in ER+ breast cancer. Patient and Methods: We used Illumina DASL Assay to measure gene expression in FFPE primary breast cancers from a subset of postmenopausal patients enrolled in the BIG 1-98 randomized phase 3 trial comparing 5 years LET (n=344) vs TAM (n=381) as adjuvant endocrine therapy. Gene sets (n=1910) that represent cell states and perturbations within the immune system from the Human Immunology Project Consortium were used in an exploratory analysis to identify possible predictive signatures. Results: We identified five distinct gene signatures from previously reported laboratory experiments associated with immune cell differentiation that are highly predictive of benefit (reduced breast cancer recurrence risk) of LET over TAM, each with gene signature p-values Conclusion: The role of selective estrogen receptor modulators on immune response has been well described, where TAM has been shown to prevent differentiation and activation of dendritic cells (Naibandian 2005). Similarly, it has been shown that MET inhibitors negatively regulate neutrophils suggesting that anti-MET drugs in cancer could impact immune response (Finisquerra 2015). These findings suggest that if TAM is a negative regulator of immune response why in the ATAC clinical trial, the combination therapy of anastrozole plus TAM were not significantly different from TAM alone were anastrozole was superior. With the increasing importance of understanding the role of immune response on outcome and the use of combination therapies the assessment of TILs in the neoadjuvant setting will be critical for guiding therapy. Citation Format: Willis S, Gray KP, Regan MM, Rae JM, Kammler R, Young B, Ditzel HJ, Lyng MB, Colleoni M, Viale G, Leyland-Jones B. Immune related gene expression signatures predict benefit of letrozole over tamoxifen in BIG 1-98. [abstract]. In: Proceedings of the Thirty-Eighth Annual CTRC-AACR San Antonio Breast Cancer Symposium: 2015 Dec 8-12; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2016;76(4 Suppl):Abstract nr P3-07-36.

PURPOSE To evaluate endocrine activity in terms of ovarian function suppression (OFS) of degarelix (a gonadotropin-releasing hormone [GnRH] antagonist) versus triptorelin (a GnRH agonist) in premenopausal patients receiving letrozole as neoadjuvant endocrine therapy for breast cancer. PATIENTS AND METHODS Premenopausal women with stage cT2 to 4b, any N, M0; estrogen receptor and progesterone receptor greater than 50%; human epidermal growth factor receptor 2–negative breast cancer were randomly assigned to triptorelin 3.75 mg administered intramuscularly on day 1 of every cycle or degarelix 240 mg administered subcutaneously (SC) on day 1 of cycle 1 then 80 mg SC on day 1 of cycles 2 through 6, both with letrozole 2.5 mg/day for six 28-day cycles. Surgery was performed 2 to 3 weeks after the last injection. Serum was collected at baseline, after 24 and 72 hours, at 7 and 14 days, and then before injections on cycles 2 through 6. The primary end point was time to optimal OFS (time from the first injection to first assessment of centrally assessed estradiol level ≤ 2.72 pg/mL [≤ 10 pmol/L] during neoadjuvant therapy). The trial had 90% power to detect a difference using a log-rank test with a two-sided α of .05. Secondary end points included response, tolerability, and patient-reported endocrine symptoms. RESULTS Between February 2014 and January 2017, 51 patients were enrolled (n = 26 received triptorelin plus letrozole; n = 25 received degarelix plus letrozole). Time to optimal OFS was three times faster for patients assigned to degarelix and letrozole than to triptorelin and letrozole (median, 3 v 14 days; hazard ratio, 3.05; 95% CI, 1.65 to 5.65; P < .001). Furthermore, OFS was maintained during subsequent cycles for all patients assigned to receive degarelix and letrozole, whereas 15.4% of patients assigned to receive triptorelin and letrozole had suboptimal OFS after cycle 1 (six events during 127 measurements). Adverse events as a result of both degarelix plus letrozole and triptorelin plus letrozole were as expected. CONCLUSION In premenopausal women receiving letrozole for neoadjuvant endocrine therapy, OFS was achieved more quickly and maintained more effectively with degarelix than with triptorelin.

Introduction: Phosphatase and tensin homolog (PTEN) loss is frequently observed in NSCLC and associated with both phosphoinositide 3-kinase activation and tumoral immunosuppression. PTEN immunohistochemistry is a valuable readout, but lacks standardized staining protocol and cutoff value. Methods: After an external quality assessment using SP218, 138G6 and 6H2.1 anti-PTEN antibodies, scored on webbook and tissue microarray, the European Thoracic Oncology Platform cohort samples (n = 2245 NSCLC patients, 8980 tissue microarray cores) were stained with SP218. All cores were H-scored by pathologists and by computerized pixel-based intensity measurements calibrated by pathologists. Results: All three antibodies differentiated six PTEN+ versus six PTEN- cases on external quality assessment. For 138G6 and SP218, high sensitivity and specificity was found for all H-score threshold values including prospectively defined 0, calculated 8 (pathologists), and calculated 5 (computer). High concordance among pathologists in setting computer-based intensities and between pathologists and computer in H-scoring was observed. Because of over-integration of the human eye, pixel-based computer H-scores were overall 54% lower. For all cutoff values, PTEN-was associated with smoking history, squamous cell histology, and higher tumor stage (p < 0.001). In adenocarcinomas, PTEN-was associated with poor survival. Conclusion: Calibration of immunoreactivity intensities by pathologists following computerized H-score measurements has the potential to improve reproducibility and homogeneity of biomarker detection regarding epitope validation in multicenter studies. (C) 2018 International Association for the Study of Lung Cancer. Published by Elsevier Inc. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/).

A range of somatic driver alterations has been described in estrogen receptor-positive, HER2-negative (ER+/HER2-) early breast cancer (BC); however, the clinical relevance is unknown.To investigate associations of driver alterations with prognosis and the role of PIK3CA mutations in prediction of benefit associated with endocrine therapy in postmenopausal patients with ER+/HER2- early BC treated with tamoxifen or letrozole.The Breast International Group (BIG) 1-98 trial randomized 8010 postmenopausal patients with hormone receptor-positive, operable, invasive BC to monotherapy with letrozole, tamoxifen, or a sequential strategy for 5 years. Driver alterations were characterized using next-generation sequencing in primary tumors from a subset of 764 patients from 7329 eligible patients with ER+/HER2- BC, with 841 distant recurrences after a median of 8.1 years of follow-up. To correct for the oversampling of distant recurrences, weighted analysis methods were used. This analysis was conducted from April 4, 2016, to November 30, 2016.The prevalence of driver alterations, associations with clinicopathologic factors, distant recurrence-free interval, and treatment interactions were analyzed. Multivariable analyses were performed to adjust for clinicopathologic factors.Of 764 samples, 538 (70.4%), including 140 distant recurrence events, were successfully sequenced. Nineteen driver alterations were observed with 5% or greater frequency, with a mean of 4 alterations (range, 0-15) per tumor. PIK3CA mutations were the most common (49%) and were significantly associated with reduction in the risk for distant recurrence (hazard ratio [HR], 0.57; 95% CI, 0.38-0.85; P = .006). TP53 mutations (HR, 1.92; 95% CI, 1.21-3.04; P = .006), amplifications on 11q13 (HR, 2.14; 95% CI, 1.36-3.37; P = .001) and 8p11 (HR, 3.02; 95% CI, 1.88-4.84; P .001), and increasing number of driver alterations (HR per additional alteration, 1.18; 95% CI, 1.11-1.25; P .001) were associated with significantly greater risk. Amplifications on 11q13 and 8p11 remained significant predictors in multivariable analysis, but not PIK3CA and TP53 mutations. Patients with tumors harboring kinase or helical domain PIK3CA mutations derived significantly greater benefit from letrozole over tamoxifen than patients whose tumors did not (P interaction = .002).In ER+/HER2- postmenopausal, early-stage BC, amplifications on 11q13 and 8p11 were significantly associated with increased risk for distant recurrence and PIK3CA mutations were predictive of greater magnitude of benefit from letrozole. With these findings, DNA-based classification may aid adjuvant treatment decision making in this setting.ClinicalTrials.gov Identifier: NCT00004205.

Summary Background We previously reported the 5-year results of the phase 3 IBCSG 23-01 trial comparing disease-free survival in patients with breast cancer with one or more micrometastatic (≤2 mm) sentinel nodes randomly assigned to either axillary dissection or no axillary dissection. The results showed no difference in disease-free survival between the groups and showed non-inferiority of no axillary dissection relative to axillary dissection. The current analysis presents the results of the study after a median follow-up of 9·7 years (IQR 7·8–12·7). Methods In this multicentre, randomised, controlled, open-label, non-inferiority, phase 3 trial, participants were recruited from 27 hospitals and cancer centres in nine countries. Eligible women could be of any age with clinical, mammographic, ultrasonographic, or pathological diagnosis of breast cancer with largest lesion diameter of 5 cm or smaller, and one or more metastatic sentinel nodes, all of which were 2 mm or smaller and with no extracapsular extension. Patients were randomly assigned (1:1) before surgery (mastectomy or breast-conserving surgery) to no axillary dissection or axillary dissection using permuted blocks generated by a web-based congruence algorithm, with stratification by centre and menopausal status. The protocol-specified primary endpoint was disease-free survival, analysed in the intention-to-treat population (as randomly assigned). Safety was assessed in all randomly assigned patients who received their allocated treatment (as treated). We did a one-sided test for non-inferiority of no axillary dissection by comparing the observed hazard ratios (HRs) for disease-free survival with a margin of 1·25. This 10-year follow-up analysis was not prespecified in the trial's protocol and thus was not adjusted for multiple, sequential testing. This trial is registered with ClinicalTrials.gov, number NCT00072293. Findings Between April 1, 2001, and Feb 8, 2010, 6681 patients were screened and 934 randomly assigned to no axillary dissection (n=469) or axillary dissection (n=465). Three patients were ineligible and were excluded from the trial after randomisation. Disease-free survival at 10 years was 76·8% (95% CI 72·5–81·0) in the no axillary dissection group, compared with 74·9% (70·5–79·3) in the axillary dissection group (HR 0·85, 95% CI 0·65–1·11; log-rank p=0·24; p=0·0024 for non-inferiority). Long-term surgical complications included lymphoedema of any grade in 16 (4%) of 453 patients in the no axillary dissection group and 60 (13%) of 447 in the axillary dissection group, sensory neuropathy of any grade in 57 (13%) in the no axillary dissection group versus 85 (19%) in the axillary dissection group, and motor neuropathy of any grade (14 [3%] in the no axillary dissection group vs 40 [9%] in the axillary dissection group). One serious adverse event (postoperative infection and inflamed axilla requiring hospital admission) was attributed to axillary dissection; the event resolved without sequelae. Interpretation The findings of the IBCSG 23-01 trial after a median follow-up of 9·7 years (IQR 7·8–12·7) corroborate those obtained at 5 years and are consistent with those of the 10-year follow-up analysis of the Z0011 trial. Together, these findings support the current practice of not doing an axillary dissection when the tumour burden in the sentinel nodes is minimal or moderate in patients with early breast cancer. Funding International Breast Cancer Study Group.

Background: ALK rearrangement is documented in 2%-7% of NSCLC, depending on the population studied and detection method used. Although the reverse transcriptasepolymerase chain reaction (RT-PCR) was the first used and published method, fluorescence in situ hybridization (FISH) has become the primary standard diagnostic method. Recently, immunohistochemistry (IHC) has also proven to be a reproducible, faster and sensitive technique. This is one of the first studies concurrently comparing all three techniques in resected lung adenocarcinomas from the large ETOP Lungscape cohort. Methods: 95 cases from the ETOP Lungscape iBiobank, selected based on any degree of IHC staining (clone 5A4 antibody, Novocastra, UK), were examined by ALK FISH (Abbott Molecular, Inc.; Blackhall, JCO 2014) and central RT-PCR. For the latter, formalin-fixed, paraffin-embedded (FFPE) unstained slides were collected from participating centers. Slides were de-paraffinized, Toluidine Blue stained, and tumors macro-dissected. Tissue digestion and RNA extraction were performed (Qiagen RNeasy FFPE Kit). Using primers described in the literature covering most of ALK known translocations, RT-PCR (Superscript One-Step RT-PCR with Platinum Taq - 40 loops) was performed, followed by capillary electrophoresis in two separate mixes. Co-amplification of B-actin was done to validate the procedure and RNA quality. All tests were duplicated. Results: 76 of 95 RT-PCR had adequate RNA quality (B-actin co-amplification present). Among these, 18 were FISH positive, 16 were RT-PCR positive, including EML4-ALK V3a/b in 7, V1 in 5, V2 in one, and undetermined variants in 3 cases. 53 of 54 FISH negative cases were also RT-PCR negative (98%). 15 of 18 FISH positives harbored a translocation by RT-PCR (83%). Among the 4 discrepant cases, 2 FISH+/ RT-PCR- cases had IHC H-scores of 180 and 260, and 98.3% and 95% of rearranged cells by FISH, probably corresponding to variants not covered by the RT-PCR. One had an IHC H-score of 5, and 16% cells rearranged on FISH, most probably corresponding to a FISH false positive case. The last had an IHC H-score of 200, 13% rearranged cells by FISH, and, thus is defined as a false negative FISH result. Provided IHC is defined as positive by an H-score above 120, all but one case (H-Score 20, FISH and RT-PCR positive) gave concordant results by a combination of FISH and RT-PCR. Overall, using as true negative or true positive the concordant result of two of the methods, the third method is characterized by high specificity and sensitivity with corresponding values of 100/98/100% and 94/94/89% for IHC/FISH/RT-PCR, respectively. Conclusion: RTPCR is a very good tool for sorting discordant IHC/FISH cases, however, we do not recommend using this technique as single method due to the lower sensitivity of RT-PCR, as not all variants are covered, and also due to the limitations with RNA preservation.

Background: Single nucleotide polymorphisms (SNPs) of the aromatase enzyme (CYP19A1) and estrogen receptor alpha (ESR1) may be associated with breast cancer susceptibility and endocrine-mediated side effects. The IBCSG Tamoxifen (Tam) and Exemestane (Exe) Trial (TEXT) includes a translational research project to assess whether selected SNPs may influence treatment efficacy and toxicity. We report on early-onset hot flashes and sweating (HF/S) and musculoskeletal symptoms (MS; myalgia, arthralgia, stiffness) with respect to CYP19A1 and ESR1 SNPs under combined endocrine therapy. Patients and Methods: 2,672 premenopausal women with HR+ early breast cancer were randomized to treatment with the GnRH-agonist triptorelin (Trip)+Tam or Trip+Exe for 5 years. Randomization was stratified according to intended use of chemotherapy (yes/no) and lymph node status (N- vs N+). Estrogen-depletion side effects (HF/S and MS) were recorded at baseline, 3-monthly during the first year and 6-monthly thereafter using the NCI CTCAE v3.0. DNA was centrally extracted from whole blood with Qiagen kits. SNPs of CYP19A1 (rs4646 and rs10046) and ESR1 (rs207764, rs2234693 and rs9340799) were analyzed by a pyrosequencing method (Diatech Pharmacogenetics S.r.l., Jesi, Italy). Control genotypes (wt/wt; wt/v; v/v) for all SNPs were processed in each run. Logistic regression was used to analyze two endpoints: presence or absence of grade (gr) 2-3 HF/S during first 6 months and gr 2-4 MS during first 12 months. Four genetic models were used to explore associations with side effects: genetic (wt/wt; wt/v; v/v), additive, dominant and recessive. Results: DNA was isolated and genotyped for 1970 (74%) consenting women. Clinical characteristics and outcomes of this cohort were consistent with the overall trial. At baseline median age was 44, median BMI 24 kg/m2 and 86% had regular menses. During follow-up, 43% reported gr 2-3 HF/S and 27% reported gr 2-4 MS. The 5 SNPs did not deviate from Hardy-Weinberg equilibrium (p>0.30) and minor allele frequency ranged from 29%-48%. The CYP19A1 SNP rs10046 (C>T) was associated with gr 2-3 HF/S. Specifically, women with variant homozygote genotype (T/T) had a reduced risk of HF/S (39% T/T vs 45%). The univariate odds ratio (OR) was 0.77 (95%CI: 0.62-0.96; p=0.02) compared with other variant groups. The multivariate model showed consistent results after adjusting for age, BMI, menstrual status, chemo use, treatment allocation (Tam vs Exe) and baseline HF/S. The other 4 SNPs were not associated with the selected side effects. Conclusions: Our analysis indicates association of HF/S with CYP19A1 rs10046 genetic variants. The lack of association between early-onset of selected estrogen-depletion side effects and the other 4 SNPs may be masked by the concurrent Trip. Ovarian reserve before treatment may also influence the impact of SNP genotypes on early-onset side effects. Further analysis with details of concurrent medications and treatment adherence will contribute to the interpretation of these results. Funded by Susan G. Komen for the Cure. Citation Format: Harriet Johansson, Kathryn P Gray, Olivia Pagani, Meredith M Regan, Giuseppe Viale, Valentina Aristarco, Debora Macis, Antonella Puccio, Susanne Roux, Rudolf Maibach, Marco Colleoni, Manuela Rabaglio-Poretti, Alan S Coates, Richard D Gelber, Aron Goldhirsch, Roswitha Kammler, Bernardo Bonanni, Barbara A Walley. CYP19A1 and ESR1 polymorphisms and selected early-onset side effects during combined endocrine therapy in the IBCSG TEXT trial for premenopausal women with hormone receptor-positive (HR+) early breast cancer [abstract]. In: Proceedings of the Thirty-Seventh Annual CTRC-AACR San Antonio Breast Cancer Symposium: 2014 Dec 9-13; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2015;75(9 Suppl):Abstract nr P1-12-01.

Background: Ki67 has been suggested as a marker for diagnosis of luminal A and B breast carcinomas. Interestingly, on one hand a multitude of studies have described significant results for Ki67 as a prognostic marker, while on the other hand the analytical validation and standardization of this marker has been a challenge. The best parameter for Ki67 interobserver performance is the interclass correlation coefficient (ICC). ICC values between 0.59 and 0.92 have been reported. Recently a minimum ICC of 0.8 has been suggested as a goal for the international ring trial and as a prerequisite for introduction of Ki67 into clinical practice. However, this suggested ICC is not derived from analysis of data, and the amount of pathologist variance that is allowed for meaningful biomarker results is still not defined. Methods: This study is based on a total of 9069 tumor samples from three large clinical cohorts (IBCSG VIII+IX, BIG1-98, and GeparTrio). In a systematic modeling approach, we introduced different amounts of variance to previously generated central pathology Ki67 datasets by simulation of a total of 1800 different pathologist evaluations for each study cohort. These evaluations were grouped into groups with defined ICCs, ranging from very good concordance (ICC=0.9) to extremely poor concordance (ICC=0.1). For each of the simulated pathologist evaluations, all possible Ki67 cutoffs were systematically evaluated using the web-based software Cutoff Finder (http://molpath.charite.de/cutoff/). As endpoints, we used DFS for all three study cohorts as well as pCR for the neoadjuvant cohort. Results: For the neoadjuvant GeparTrio study, the different groups with ICCs of 0.8, 0.6 and 0.4 showed a very similar performance resulting in significant analyses for prediction of pCR across a wide range of cutoffs. The odd ratios for pCR were slightly lower with lower ICC. Even with an extremely low ICC of 0.2, 99% of the analyses had one or more significant cutpoints. The survival endpoint DFS was shown to be very stable despite increased interpathologist variance in all three clinical cohorts. Even with a poor ICC of 0.4, the majority of cutpoints were significant for DFS. For IBCSG VIII+IX 85% of the analyses with an ICC of 0.4 had one or more significant cutpoints for Ki67. In the large BIG 1-98 dataset (n=6090) even an ICC of 0.2 resulted in one or more significant DFS cutpoints in 100% of the analyses. Comparable results were obtained if the analysis was restricted to luminal tumors. Conclusion: Our results suggest that Ki67 is extremely robust to pathologist variation. Even if less than 40% of the variance is attributable to true Ki67-based proliferation (ICC Citation Format: Denkert C, Budczies J, Regan M, Loibl S, Dell'Orto P, von Minckwitz G, Mastropasqua M, Mehta K, Müller V, Kammler R, Pfitzner BM, Fasching PA, Viale G. Systematic analysis and modulation of Ki67 interobserver variance in 9069 patients from three clinical trials – How much pathologist concordance is needed for meaningful biomarker results?. [abstract]. In: Proceedings of the Thirty-Eighth Annual CTRC-AACR San Antonio Breast Cancer Symposium: 2015 Dec 8-12; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2016;76(4 Suppl):Abstract nr P5-07-02.

We investigated the occurrence and the prognostic and predictive relationship of a selected number of somatic mutations in triple-negative breast cancer (TNBC) patients having known clinical outcomes treated within the IBCSG Trial 22-00. A matched case–control sampling selected patients enrolled in the IBCSG Trial 22-00 who had TNBC tumors, based on local assessment. Cases had invasive breast cancer recurrence (at local, regional, or distant site) according to the protocol definition. Matched controls had not recurred. Mutational analysis was performed with OncoCarta panel v1.0 using Mass Array System. The panel includes 19 genes belonging to different functional pathways as PI3K pathway, receptor tyrosine kinase, and cell cycle-metabolic group. Conditional logistic regression assessed the association of mutation status with breast cancer recurrence. Mutation assessment was successful for 135 patients (49 cases, 86 controls). A total of 37 (27.4%) of the 135 patients had at least one mutation in the selected genes. PIK3CA was the most common mutated gene (18/135; 13.3%), followed by BRAF, KIT and PDGFRA (each 4/135, 3.0%) and AKT1 (3/135; 2.2%). TNBC patients with at least one mutation had increased odds of recurrence compared with those with wild-type tumors (odds ratio (OR) 2.28; 95% CI 0.88–5.92), though this difference was not statistically significant (p = 0.09). We found no evidence that these mutations were predictive for the value of maintenance metronomic chemotherapy. Mutations in the tested oncogenes were not associated with breast cancer recurrence in this TNBC subset of patients. The question of whether any of these mutated genes (e.g., PIK3CA) may represent a useful therapeutic target in TNBC may be answered by ongoing clinical trials and/or larger dataset analysis.

Summary Background The tyrosine kinase inhibitor erlotinib improves the outcomes of patients with advanced non-small-cell lung carcinoma (NSCLC) harbouring epidermal growth factor receptor ( EGFR ) mutations. The coexistence of the T790M resistance mutation with another EGFR mutation in treatment-naive patients has been associated with a shorter progression-free survival to EGFR inhibition than in the absence of the T790M mutation. To test this hypothesis clinically, we developed a proof-of-concept study, in which patients with EGFR -mutant NSCLC were treated with the combination of erlotinib and bevacizumab, stratified by the presence of the pretreatment T790M mutation. Methods BELIEF was an international, multicentre, single-arm, phase 2 trial done at 29 centres in eight European countries. Eligible patients were aged 18 years or older and had treatment-naive, pathologically confirmed stage IIIB or stage IV lung adenocarcinoma with a confirmed, activating EGFR mutation (exon 19 deletion or L858R mutation). Patients received oral erlotinib 150 mg per day and intravenous bevacizumab 15 mg/kg every 21 days and were tested centrally for the pretreatment T790M resistance mutation with a peptide nucleic acid probe-based real-time PCR. The primary endpoint was progression-free survival. The primary efficacy analysis was done in the intention-to-treat population and was stratified into two parallel substudies according to the centrally confirmed pretreatment T790M mutation status of enrolled patients (T790M positive or negative). The safety analysis was done in all patients that have received at least one dose of trial treatment. This trial was registered with ClinicalTrials.gov, number NCT01562028. Findings Between June 11, 2012, and Oct 28, 2014, 109 patients were enrolled and included in the efficacy analysis. 37 patients were T790M mutation positive and 72 negative. The overall median progression-free survival was 13·2 months (95% CI 10·3–15·5), with a 12 month progression-free survival of 55% (95% CI 45–64). The primary endpoint was met only in substudy one (T790M-positive patients). In the T790M-positive group, median progression-free survival was 16·0 months (12·7 to not estimable), with a 12 month progression-free survival of 68% (50–81), whereas in the T790M-negative group, median progression-free survival was 10·5 months (9·4–14·2), with a 12 month progression-free survival of 48% (36–59). Of 106 patients included in the safety analysis, five had grade 4 adverse events (one acute coronary syndrome, one biliary tract infection, one other neoplasms, and two colonic perforations) and one died due to sepsis. Interpretation The BELIEF trial provides further evidence of benefit for the combined use of erlotinib and bevacizumab in patients with NSCLC harbouring activating EGFR mutations. Funding European Thoracic Oncology Platform, Roche.

Background: While representing the largest fraction of women diagnosed with primary breast cancer, older postmenopausal women with ER+, HER2− tumors are less responsive to chemoendocrine therapy than younger women and have been underrepresented in molecular profiling of randomized trials. IBCSG Trial IX, a randomized controlled trial in postmenopausal women, median age 61y, with node negative disease, failed to demonstrate the benefit of preceding tamoxifen (T) by 3 cycles of CMF for ER+ tumors. We sought to determine if MS, a proliferation score, could identify a subset of women who differentially benefit from addition of chemotherapy to T in this trial. Methods: From 1988–1999, 1669 eligible patients (1040 with ER+, HER2− tumors) were randomized to CMF→T vs T. Disease-free survival (DFS) was the primary trial endpoint; breast cancer-free interval (BCFI) which excludes second (non-breast) malignancies and censors deaths without prior cancer event was also evaluated. Analysis was limited to the first 7 years of follow-up. From 671 (ER+, HER2−) available subjects, 568 were successfully profiled by RT-PCR. The mRNA expression levels of 14 equally-weighted proliferation genes and 3 normalization genes were used to generate MS; predetermined binary categorization of MS was used. Analysis of this post hoc, pre-specified study used results from centralized laboratory IHC and Cox models to assess the predictive value of MS on DFS and BCFI, adjusting for traditional risk factors of local treatment, age, ER, PR, Ki67, tumor size and grade. Results: Subgroups of MS (low, 169 samples (30%) and high, 399 samples (70%)) were identified. MS by treatment interaction was significant for DFS and BCFI (each p ≤ 0.004). Among patients with low MS, CMF→T improved DFS (HR 0.19, 95% CI 0.06–0.59) and BCFI (HR 0.19, 95% CI 0.05–0.72) vs T; 7y DFS was 95% vs 83% with CMF→T vs T. Among patients with high MS, CMF→T did not improve DFS (HR 1.27, 95% CI 0.79–2.05) or BCFI (HR 1.37, 95% CI 0.80–2.33) and 7y DFS of 81% for CMF→T and T. Continuous MS was moderately correlated with log Ki67 (r = 0.47) but not correlated with ER or PR. The MS by treatment interaction remained significant with Ki67 in the model. Conclusions: Low MS was associated with differential benefit favoring those women receiving CMF→T vs T alone for both DFS and BCFI in the first 7 years. The effect was independent of traditional risk factors including Ki67. Hence this study, which is unconfounded by chemotherapy-induced ovarian ablation in younger women, identifies a subset of postmenopausal women with ER+, HER2− tumors that benefit from CMF chemotherapy. This seemingly incongruous observation is consistent with a) the prior observation that only the low-proliferation subgroup by PAM50 11-gene signature benefits from the addition of weekly paclitaxel to adjuvant FEC (GEICAM/9906), b) the ability of MS to identify a subset of women with tumors with disseminated luminal progenitor cells activated through the agonistic activity of tamoxifen, and c) the repetitive dosing of cyclophosphamide and taxol being hypothesized to act via tumor stroma/anti-angiogenesis. The relative contribution of these factors is under investigation. Citation Information: Cancer Res 2012;72(24 Suppl):Abstract nr PD10-03.

Background: Available endocrine therapies, tamoxifen or aromatase inhibitors (AIs), are effective on average for the large population of postmenopausal women with steroid hormone-receptor positive early breast cancer, but many patients still relapse and die of their disease. Treatment selection may be improved by determining patient and disease features that suggest one or another endocrine therapy regimen is better for the individual patient. Recent clinical studies have presented conflicting evidence about whether postmenopausal women receiving adjuvant tamoxifen who carry genetic variants associated with low or absent CYP2D6 activity have a worse disease outcome. BIG 1-98 is a double-blind randomized trial comparing five years of the AI letrozole (Letx5), tamoxifen (Tamx5) and sequences of Let and Tam as adjuvant endocrine therapy for postmenopausal women with endocrine-responsive breast cancer. BIG 1-98 enrolled 8010 patients between 1998 and 2003. Results at 6 years median follow-up showed that, on average, Let was superior to Tam for disease-free and overall survival, and for time to distant recurrence. The sequential arms (Tamx2 → Letx3; Letx2 → Tamx3 had similar results compared with Let. Genotyping is underway using DNA extracted from tumor blocks of patients in trial BIG 1-98 to elucidate genetic determinants of interindividual variability in efficacy and side effects of adjuvant endocrine therapies. We hypothesize that variants of CYP2D6 are associated with shorter breast cancer-free interval on tamoxifen monotherapy, and that the relation will not be observed with letrozole monotherapy as a control. The association with sequential therapies is unknown and will be explored. Methods: Formalin-fixed, paraffin-embedded primary tumor specimens were reviewed in the IBCSG Central Pathology Laboratory and DNA isolated from one or two 1mm cores. Single nucleotide polymorphisms (SNPs) of CYP2D6 {including CYP2D6*4, *2, *3A, *6, *7, *10, *17, *41} were genotyped on the Beckman SNPstream or the ABI 7900 TaqMan system. The association of CYP2D6 variants with breast cancer-free interval and onset of hot flashes/night sweats will be investigated. The role of adjuvant chemotherapy, which was administered in about one-third of trial patients prior to randomization to adjuvant endocrine therapy, will also be investigated. Results: At this time, CYP2D6*4 has been genotyped from FFPE material of 2000 patients, with a success rate greater than 75%. The frequency of homozygous and heterozygous CYP2D6*4 was 8% and 18%, respectively, which is in line with anticipated allele frequency in a predominantly Caucasian population. Genotyping of additional CYP2D6 alleles is ongoing for these patients, as well as full genotyping for up to another 2000 patients. Conclusions: Trial BIG 1-98 provides a large, homogeneously-treated and followed cohort of patients treated with tamoxifen and other endocrine therapy regimens with available material for genotyping. Results of CYP2D6 genotyping (8 alleles) for all available patients will be presented at the meeting. Citation Information: Cancer Res 2010;70(24 Suppl):Abstract nr S1-8.

Estrogen receptor 1 (ESR1) and ESR2 gene polymorphisms have been associated with endocrine-mediated physiological mechanisms, and inconsistently with breast cancer risk and outcomes, bone mineral density changes, and hot flushes/night sweats. DNA was isolated and genotyped for six ESR1 and two ESR2 single-nucleotide polymorphisms (SNPs) from tumor specimens from 3691 postmenopausal women with hormone receptor-positive breast cancer enrolled in the BIG 1-98 trial to receive tamoxifen and/or letrozole for 5 years. Associations with recurrence and adverse events (AEs) were assessed using Cox proportional hazards models. 3401 samples were successfully genotyped for five SNPs. ESR1 rs9340799(XbaI) (T>C) variants CC or TC were associated with reduced breast cancer risk (HR = 0.82,95% CI = 0.67-1.0), and ESR1 rs2077647 (T>C) variants CC or TC was associated with reduced distant recurrence risk (HR = 0.69, 95% CI = 0.53-0.90), both regardless of the treatments. No differential treatment effects (letrozole vs. tamoxifen) were observed for the association of outcome with any of the SNPs. Letrozole-treated patients with rs2077647 (T>C) variants CC and TC had a reduced risk of bone AE (HR = 0.75, 95% CI = 0.58-0.98, P interaction = 0.08), whereas patients with rs4986938 (G>A) genotype variants AA and AG had an increased risk of bone AE (HR = 1.37, 95% CI = 1.01-1.84, P interaction = 0.07). We observed that (1) rare ESR1 homozygous polymorphisms were associated with lower recurrence, and (2) ESR1 and ESR2 SNPs were associated with bone AEs in letrozole-treated patients. Genes that are involved in estrogen signaling and synthesis have the potential to affect both breast cancer recurrence and side effects, suggesting that individual treatment strategies can incorporate not only oncogenic drivers but also SNPs related to estrogen activity.

To determine whether CYP19A1 polymorphisms are associated with abnormal activity of aromatase and with musculoskeletal and bone side effects of aromatase inhibitors. DNA was isolated from tumor specimens of 4861 postmenopausal women with hormone receptor-positive breast cancer enrolled in the BIG 1-98 trial to receive tamoxifen and/or letrozole for 5 years. Tumors were genotyped for six CYP19A1 polymorphisms using PCR-based methods. Associations with breast cancer-free interval (BCFI), distant recurrence-free interval (DRFI), musculoskeletal and bone adverse events (AEs) were assessed using Cox proportional hazards models. All statistical tests were two-sided. No association between the CYP19A1 genotypes and BCFI or DRFI was observed overall. A reduced risk of a breast cancer event for tamoxifen-treated patients with rs700518 variants was observed (BCFI CC/TC vs. TT: HR 0.53, 95 % CI 0.34-0.82, interaction P = 0.08), but not observed for letrozole-treated patients. There was an increased risk of musculoskeletal AEs for patients with rs700518 variants CC/TC versus TT (HR 1.22, 95 % CI 1.03-1.45, P = 0.02), regardless of treatment. Tamoxifen-treated patients with rs4646 variants had a reduced risk of bone AEs (AA/CA vs. CC: HR 0.76, 95 % CI 0.59-0.98), whereas an increase of minor allele (C) of rs10046 was associated with an increased risk of bone AEs (HR 1.28, 95 % CI 1.07-1.52). rs936308 variants were associated with a reduced risk of bone AEs in letrozole-treated patients (GG/GC vs. CC: HR 0.73, 95 % CI 0.54-0.99), different from in tamoxifen-treated patients (GG/GC vs. CC: HR 1.32, 95 % CI 0.92-1.90, interaction P = 0.01). CYP19A1 rs700518 variants showed associations with BCFI, DRFI, in tamoxifen treated patients and musculoskeletal AEs regardless of treatment. SNPs rs4646, rs10046, and rs936308 were associated with bone AEs.

The BELIEF trial (NCT01562028) examined the efficacy of E+B for the 1st line treatment of European p with advanced NSCLC harboring exon 19 deletion or exon 21 L858R epidermal growth factor receptor (EGFR) mutations with or without pretreatment T790M. Median progression-free survival (mPFS) was 13.8 months (m) (95% CI 10.3-16.2) for the 109 p enrolled in the study. mPFS was 16m (95% CI 13.1-not estimable [NE]) and 10.4m (95% CI 9.2-15.6) for the 37 T790M(+) p and 72 T790M(-) p, respectively (P = 0.089). We have previously shown that low BRCA1 levels neutralize the negative effect of pretreatment T790M and are associated with longer PFS to erlotinib. Low astrocyte elevated gene 1 (AEG1) levels are associated with longer PFS to erlotinib. A secondary objective of the BELIEF trial was the assessment of the prognostic impact of BRCA1 and AEG1 mRNA expression in the baseline tumor tissue. We assessed the baseline mRNA expression levels of BRCA1 and AEG1 and correlated them with PFS and response in the 109 EGFR-mutant NSCLC p of the BELIEF trial. BRCA1 and AEG1 mRNA levels were estimable by quantitative-real time PCR in 46 and 61 p respectively. Expression levels were divided into two groups according to their median. No statistically significant associations were found among the expression of the two biomarkers and gender, smoking status, histology, PS or type of EGFR mutation. The association of BRCA1 and AEG1 mRNA with PFS did not differ according to T790M status (interaction P = 0.81 and 0.42, respectively). Among the T790M (+) p, those with low BRCA1 mRNA had a longer mPFS of 24.6m (95% CI: 4.9-NE) compared to 15.4 m (95% CI: 2.7-NE) for p with high BRCA1 mRNA; P = 0.63. For all and T790M (-) p, BRCA1 levels did not differentiate mPFS to E+B (low vs high BRCA1 mRNA, 11.0 m [95% CI: 6.0-NE] vs 9.5 m [95% CI: 7.2-NE]; P = 0.99 and 6.9 m [95% CI: 2.1-NE] vs 9.4 m [95% CI: 4.1-NE]; P = 0.71, respectively). Similarly, among the T790M (+) p, those with low AEG1 expression had a longer mPFS of 24.6m (95% CI: 4.9-NE) compared to 15.4 m (95% CI: 5.2-NE) for those with high AEG1 mRNA; P = 0.93. For all and T790M(-) p, AEG1 levels did not differentiate mPFS to E+B (low vs high AEG1 mRNA, 10.3 m [95% CI: 8.4-24.6] vs 15.4 m [95% CI: 6.0-33.9]; P = 0.90 and 10.3m [95% CI: 8.4-NE] vs 13.3m [95% CI: 4.7-33.9] P = 0.67). No statistically or clinically significant associations were found among BRCA1, AEG1 mRNA levels and response to E+B. The BELIEF trial reconfirms our previous findings that pretreatment EGFR T790M is present in 34% of the patients. E+B is associated with a prolonged mPFS of 24.6m for EGFR-mutant p with both pretreatment EGFR T790M and low BRCA1 mRNA expression, as observed in our previous study with erlotinib alone. Similar results were observed for AEG1 levels though the interaction was not statistically significant for either biomarker. Citation Format: Rafael Rosell, Niki Karachaliou, Ana Giménez-Capitán, Carles Codony-Servat, Oliver Gautschi, Enriqueta Felip, Alessandra Curioni-Fontecedro, Solange Peters, Santiago Ponce-Aix, Martin Früh, Miklos Pless, Sanjay Popat, Sinead Cuffe, Paolo Bidoli, Adolfo Favaretto, Roswitha Kammler, Urania Dafni, Zoi Tsourti, Miguel Angel Molina-Vila, Rolf A. Stahel. The role of BRCA1 and AEG1 mRNA expression in advanced non-small-cell lung cancer (NSCLC) patients (p) with EGFR activating and pretreatment T790M mutations receiving the combination of erlotinib plus bevacizumab (E+B) in the BELIEF trial. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 269.