Breast cancer (BC) mostly occurs in women over 50 years old, however, around 5% of cases are very young women (BCVY) (age ≤35) being associated to poorer prognosis and shorter survival. Age-associated differences in BC remain poorly studied. Understanding biological differences in BCVY may lead to better therapeutic options for these patients. This work aimed to identify molecular differences between breast tumors from BCVY and old women (BCO). The formalin-fixed samples of a retrospective cohort (N = 49) collecting treatment naïve core needle biopsies from 22 BCVY and 27 BCO (age ≥ 50) were analyzed. A preanalytic sample selection was performed to assess the quality before the RNA sequencing (HiSeq X Series, Illumina). Wilcoxon test was used on normalized RNA-seq data to compare the BCVY and BCO groups. All analyses were performed using R software (v 4.0.2). The BCVY cohort included 12 (55%) luminal, and 10 (45%) non-luminal BCs, while the BCO cohort was 17 (63%) luminal and 10 (37%) non-luminal patients. Principal Components Analysis of mRNA-seq data revealed a higher heterogeneity among BCVY. Age-related gene expression differences were confirmed (40 and 13 genes down- and up-regulated [fold change ± 2]). BCVY presented higher chromosomal instability (CIN70, p= 1e-06) and were more immunoreactive tumors presenting significantly higher scores in immune-related gene signatures (inflammation, cytokines, IFN gamma, macrophages, Th2 cells, B cells, DC, and IGG). Moreover, BCVY tumors were more proliferative as confirmed by higher expression of related gene signatures (p= 5.17e-05) and ki67 (IHC, p= 0.021). These findings were particularly significant in luminal BC. Interestingly, despite there were no differences in ER and PR by IHC, lower expression was found at RNA level in luminal BCVY patients (ESR1: p= 0.0027, PGR: p= 0.0476). Our study provides novel findings on the impact of aging on transcriptional landscape in BC. We found age-related gene expression changes not only in cancer cells but also in the tumor microenvironment. These data suggest that luminal BCVY could be more endocrine-resistant and aggressive than BCO. Therefore, this work highlights age as an important factor to be considered in clinical practice.

Precision oncology research is challenging outside the contexts of oncogenic addiction and/or targeted therapies. We previously showed that phosphoproteomics is a powerful approach to reveal patient subsets of interest characterized by the activity of a few kinases where the underlying genomics is complex. Here, we conduct a phosphoproteomic screening of samples from HER2-negative female breast cancer receiving neoadjuvant paclitaxel ( N = 130), aiming to find candidate biomarkers of paclitaxel sensitivity. Filtering 11 candidate biomarkers through 2 independent patient sets ( N = 218) allowed the identification of a subgroup of patients characterized by high levels of CDK4 and filamin-A who had a 90% chance of achieving a pCR in response to paclitaxel. Mechanistically, CDK4 regulates filamin-A transcription, which in turn forms a complex with tubulin and CLIP-170, which elicits increased binding of paclitaxel to microtubules, microtubule acetylation and stabilization, and mitotic catastrophe. Thus, phosphoproteomics allows the identification of explainable factors for predicting response to paclitaxel.

BACKGROUND:Cancer is the second leading cause of mortality worldwide. Integrating different levels of care by implementing screening programmes, extending diagnostic tools and applying therapeutic advances may increase survival. We implemented a cancer fast-track programme (CFP) to shorten the time between suspected cancer symptoms, diagnosis and therapy initiation.PATIENTS AND METHODS:Descriptive data were collected from the 10 years since the CFP was implemented (2009-2019) at the Clinico-Malvarrosa Health Department in Valencia, Spain. General practitioners (GPs), an oncology coordinator and 11 specialists designed guidelines for GP patient referral to the CFP, including criteria for breast, digestive, gynaecological, lung, urological, dermatological, head and neck, and soft tissue cancers. Patients with enlarged lymph nodes and constitutional symptoms were also considered. On identifying patients with suspected cancer, GPs sent a case proposal to the oncology coordinator. If criteria were met, an appointment was quickly made with the patient. We analysed the timeline of each stage of the process.RESULTS:A total of 4493 suspected cancer cases were submitted to the CFP, of whom 4019 were seen by the corresponding specialist. Cancer was confirmed in 1098 (27.3%) patients: breast cancer in 33%, urological cancers in 22%, gastrointestinal cancer in 19% and lung cancer in 15%. The median time from submission to cancer testing was 11 days, and diagnosis was reached in a median of 19 days. Treatment was started at a median of 34 days from diagnosis.CONCLUSIONS:The findings of this study show that the interval from GP patient referral to specialist testing, cancer diagnosis and start of therapy can be reduced. Implementation of the CFP enabled most patients to begin curative intended treatment, and required only minimal resources in our setting.

Background: Recently, microRNAs have been demonstrated to be potential non-invasive biomarkers for diagnosis, prognosis assessment or prediction of response to treatment in cancer. In this study, we evaluate the potential of miR-30b-5p as a biomarker for early diagnosis of breast cancer (BC) in tissue and plasma. Methods: Expression of miR-30b-5p was determined in a series of 112 BC and 40 normal breast tissues. Circulating miR-30b-5p levels in plasma samples were determined in a discovery cohort of 38 BC patients and 40 healthy donors and in a validation cohort of 83 BC patients and 83 healthy volunteers. miR-30b-5p expression was measured by quantitative real-time PCR and receiver operating characteristics curve analysis was carried out. Results: The miR-30b-Sp expression was significantly lower in BC tissue than in healthy breast samples. In contrast, circulating miR-30b-5p levels were significantly higher in BC patients compared with healthy donors. Furthermore, circulating miR-30b-Sp levels were significantly higher in patients with positive axillary lymph node and de novo metastatic patients. Receiver operating characteristics curve analysis demonstrated a good diagnostic potential of miR-30b-Sp to detect BC even at an early stage of the disease. Conclusion: Thus, we highlight the potential of miR-30b-Sp as a non-invasive, fast, reproducible and cost-effective diagnostic biomarker of BC.

Cancer is the second leading cause of mortality worldwide. Integrating different levels of care by implementing screening programs, extending diagnostic tools and applying therapeutic advances may increase survival. We implemented a cancer fast-track program (CFP) to reduce time from suspected cancer symptoms to confirmation of diagnosis and start of therapy. A descriptive data sample of ten years since implementation of the CFP (2009–2019) were collected at the Clinico-Malvarrosa Health Department in Valencia, Spain. Aiming to shorten time between suspected cancer cases and diagnosis, primary care (PC) physicians, an oncology coordinator and eleven specialists designed guidelines for PC physicians to refer patients through the CFP. Recommendations stablished in the guides included some for potential breast, colorectal, cervical, lung, bladder, dermatological, head and neck and soft tissue cancers. Patients with enlarged lymph nodes and constitutional symptoms were also considered. When the PC physician identified patients with suspected cancer, a proposal was sent for further review to the oncology coordinator. If recommendations were met, a quick appointment was made to attend the patient. We analysed the time lapse between each stage of the process during a ten year follow up. A total of 4574 suspected cancer cases were submitted to the CFP; 4405 were seen by the corresponding specialist. In 1102 (25%) cases, cancer was confirmed. Among these, breast cancer was found in 29%; urological 21%; colorectal 17%; and lung cancer 14%. The median time from submission of a proposal to the specialist assessment was 15 days (1-136); 3 days (1-242) to pathological diagnosis and 34 days (1-333) to treatment. In 832/1102 (74.5%) treatment intention was curative. Interval between patient referral by the PC physician to the specialist, diagnosis of cancer and start of therapy can be reduced. The implementation of CFT allowed most patients to begin curative intended therapy. Our implemented CFP requires a limited amount of resources.

The incidence of Oligometastatic (OM) breast cancer (BC) is ∼ 20-50%. OM is an intermediate state between localized and widely metastatic (met.) disease. Treatment of OM is controversial, the status of no evidence of disease (NED) can be achieved through multidisciplinary management (systemic and local therapies: surgery +/- radiotherapy). The aim was to evaluate the prognostic role of NED status in OM BC patients (pts). A retrospective study was performed in a single institution including 54 non-consecutive OM pts who achieved NED status. Pts were recruited from Jan-88 to Dec-17. Enrolled pts should have the following inclusion criteria: ECOG: 0-1, no relevant comorbidities and no contraindication to surgery. OM disease was defined as < 5 mets. in a single organ. Kaplan–Meier was used to estimate progression-free and overall survival (PFS and OS). Multivariable Cox regression analysis was performed with backward-forward stepwise model selection with AIC. We identified 54 OM pts (Table), 15% were de novo metastatic and 85% relapsed from the early stage (median DFS: 46 mos). 75.9% were luminal, 16.7% HER2+, and 7.4% triple negative. An induction with presurgical treatment was performed in 17% of pts. Radical metastasectomy was performed in 87% and 13% of pts received radical radiotherapy. 68.1% had a single met. lesion and the most frequent met. site was bone (33%). The median PFS and OS were 84 and 130.5 mos. respectivelly (resp.). At 5-year PFS and OS were 57.4% and 78.9% resp. The median OS in the metastatic setting was 54 mos. Worse DFS (< 45 mos.) (log rank p= 0.017) and the presence of visceral mets. (log rank p= 0.006) were related to worse prognosis. Number of mets. and BC subtypes were not prognostic factors. Cox regression highlighted the role of visceral mets. over others in determining prognosis (p= 0.016).Table 319PCharacteristicPatients, n(%)Age (years, median) (range)43 (31 - 71)Metastasis localizationNon-visceralBone1833,3Skin59,2Nodes1018,5VisceralLiver814,8Lung59,2Ovarian59,2Brain35,5Subtypes BCLuminal4175,9HER2+916,6Triple Negative47,4StageI1935,1II814,8II2037,0IV712,9 Open table in a new tab OM BC represents a challenge for therapeutical management. Non-visceral mets. and long DFS confer a better prognosis in this setting.

Novel antibody-drug conjugates against HER2 are showing high activity in clinically HER2-negative (cHER2-) breast cancer (BC) with low HER2 expression. However, the clinical and molecular features of cHER2-/HER2-low BC are yet to be elucidated. We collected retrospective data from 8 multicenter cHER2- BC datasets, including 4 clinical trials. HER2 status in each study was determined using standard FDA-approved antibodies and ISH-techniques and classified according to the ASCO/CAP guidelines. For this study, tumors were regrouped in HER2 0 (0 score) and HER2-low (1+ or 2+ with ISH-negativity). The following variables were compared between the 2 groups in all patients and according to hormone receptor (HR) status: age, grade, ki67, histotype, tumor size, HR, HER2 and nodal status. nCounter-based PAM50 subtypes distribution and the expression of the 50 PAM50 genes, including ERBB2, were also compared. A total of 3,136 patients with cHER2- disease (57.4% HER2-low and 42.6% HER2 0) were evaluated. Overall, 888 (28.3%) tumor samples came from metastatic sites. No statistically significant differences were found regarding clinicopathological variables between HER2-low and HER2 0. Within HR-positive (+) disease (n=2,497), 63.2% and 36.8% of tumors were HER2-low and HER2 0, respectively. Subtype distribution was similar across HR+/HER2-low and HR+/HER2 0. A total of 45/50 PAM50 genes were found differentially expressed between HR+/HER2-low and HR+/HER2 0 (False Discovery Rate [FDR]<5%). High expression of luminal (e.g. ESR1 and FOXA1) and ERBB2, and low expression of proliferation-related genes (e.g. MKI67) was found in HER2-low compared to HER2 0. Within triple negative BC (TNBC) (n=622), 34.2% were HER2-low and 65.8% were HER2 0. Subtype distribution was similar across TNBC/HER2-low and TNBC/HER2 0. No PAM50 gene was found differentially expressed between TNBC/HER2-low and TNBC/HER2 0 (FDR≥5%). Finally, ERBB2 mRNA levels were higher in HER2-low/HR+ tumors than HER2-low/TNBC (p<0.001). HER2-low disease within clinically HER2- BC is frequent. However, significant differences exist according to HR status. Compared to HER2-low/TNBC, HER2-low/HR+ disease is a more distinct biological entity and has higher ERBB2 expression.

CDK4/6 inhibitors (CDK4/6i) in combination with hormone therapy is the standard treatment of hormone-receptor-positive, HER2-negative metastatic breast cancer (mBC). Resistance mechanisms are unknown and constitute an unmet medical need. In PALOMA-3 trial the PIK3CA mutations (PIK3CA mut) were not associated with resistance to palbociclib (p=0.34). The aim is to assess the role of PIK3CA mut in routine clinical practice as a mechanism of resistance to CDK4/6i in patients with luminal mBC. We conducted a retrospective and bi-centric study, between Hospital Clínico Universitario de Valencia and Hospital Universitario ‘12 de Octubre, to evaluate the impact of PIK3CA mut on CDK4/6i treatment in patients with HR+/HER2- mBC. The relationship between PIK3CA mutational status and progression-free survival (PFS) was analyzed by Cox's proportional hazards model and the log rank test. With the aim of homogenizing the sample, patients treated in the first line were also analyzed separately. All patients (n=92) were diagnosed with a luminal mBC. Forty patients (43.5%) presented PIK3CA mut and 52 (56.5%) were wild type (WT). The median PFS was 12.0 months (95% CI 9.3-14.6). No significant difference in PFS was found based on PIK3CA mutational status (10.9 months in PIK3CA mut, 95% CI 7.9-14.0; vs 12.7 months in PIK3CA WT, 95% CI 8.6-16.8) HR 1.05 p=0.84 (logrank test). The incidence of PIK3CA mutations were higher among patients treated at first line for ≤ 6 months (46.67%), however only 26.92% of long-term responders presented PIK3CA mutations. This effect was not identified at second line.Table 57PPatient baseline characteristicsn(%)PIK3CA mut (40)PIK3CA WT (52)Median age (years)51.948.92Menopausal status Premenopausal24 (60.0 %)27 (51.9 %) Postmenopausal16 (40.0 %)25 (48.0 %)Visceral met No24 (60.0%)29 (55.8%) Yes16 (40.0 %)23 (44.2 %)CDK4/6i Palbociclib29 (72.5 %)38 (73.0 %) Ribociclib9 (22.5 %)12 (23.1 %) Abemaciclib2 (5.0 %)2 (3.8 %)Line of therapy 1st Line14 (35.0 %)27 (51.9%) 2nd or more lines26 (65.0 %)25 (48.1%) Open table in a new tab The presence of PIK3CA mutations was not associated with resistance to CDK4/6 inhibitors in terms of PFS. Nevertheless, the frequency of PIK3CA mutations was lower in patients with extended benefit (more than 6 months) at first line of treatment. Future studies to explore the impact of triplet combination therapy (PIK3CA and CDK4/6i plus endocrine treatment) are needed.

Abstract Background: NGS has elucidated the mutational landscape in BC. However, the correlation between mutational landscapes and complex phenotypic traits such as drug response is yet unclear. The genomic and transcriptomic aberrations coalesce into a diverse number of phosphorylation-driven patterns of activation of the proteome. These patterns are and associated to clinical outcomes of interest. The specific nature of the phosphopeptides in a profile versus another can be driven by a small number of activated kinases. In the past we relied on mass-spectrometry-based HTPS to build a kinase-based classification of triple-negative breast cancer (TNBC, Nat. Comms, in press), which is more parsimonious than gene-centered classifications and uncovers new actionable targets. We implemented this methodology to find predictors of response to T in early BC. Methods: Training set: fresh baseline and day+15 tumor biopsies form a trial in NEO HER2-negative BC (N=139) that randomized patients to T (80 mg/m2 weekly x12) plus placebo or nintedanib (150 mg b.i.d) were processed with a hybrid ion trap-orbitrap mass spectrometer after TiO2 phospho-enrichment. Phosphoprofiles were compared pair-wise according to the following factors: day 0 versus day + 15, standard versus experimental arm, pathologic response Symmans/Pusztai (Symmans) 0/1 vs 2/3. Kinases driving each phosphoprofile were solved by an in silico algorithm termed kinase-set enrichment analysis (KSEAS). Validation set: an in-house designed and previously validated mass-spectrometry-to-immunohistochemistry translation algorithm was used to validate the kinases enriched in the profiles from the baseline samples of patients achieving Symmans 0/1 (pCR) or Symmans=3 in the training set. Probes against those kinases were validated in an independent dataset of 160 HER2-negative patients receiving NEO T followed by AC. H-score of each activated kinase was divided in quartiles (Q1 to Q4), and upper-quartiles (Q1) of each kinase were tested in a multivariate logistic regression model to predict pCR adjusted by T, N, age, G, ER/PR and Ki67. Results: >2.5 millions of spectra were captured, identifying >35000 unique phosphopeptides mapping to >2500 unique proteins per sample. Training set: KSEAS revealed that a high activity of CDK4 and pP70S6K drove the baseline phosphoprofiles of the patients obtaining pCR in the T arm, whereas pSTAT3, pSrc and BARD drove that of the patients achieving Symmans= 3. In the validation set, H-score cut-offs for Q1 were 1.21, 0.69, 0.79, 1.64 and 1.54 for pP70S6K, CDK4, pSTAT3, pSrc and BARD. TNBC patients with Q1 pP70S6K or Q1 CDK4 achieved pCR in 100% of the cases (versus 45% in Q2-4). In the HR+ cases, the pCR rate for Q1 patients was 50% and 61% respectively. In the multivariate model (all patients), having Q1 pP70S6K or CDK4 multiplied by 2.3- and 3.6-fold, respectively, the probability of achieving pCR (P<0.05). Regarding Symmans=3, BARD and pSTAT3 lost significance in the validation but not pSrc (2.5-fold less probability of pCR, P<0.005). Conclusions: HTPS is a useful tool to find associations with complex traits. TNBC and HR+ patients with high pP70S6K or CDK4 receiving NEO T-based chemotherapy achieve very high rates of pCR. Citation Format: Quintela-Fandino M, Lluch A, Manso L, Calvo I, Cortes J, Garcia-Saenz JA, Zagorac I, Tapial-Martinez P, Gomez-Lopez G, Fustero C, Muñoz J, Gonzalez-Cortijo L, Mouron S. High-throughput phosphoproteomics (HTPS) in neoadjuvant (NEO) breast cancer (BC) reveals clusters of extreme sensitivity to paclitaxel (T) [abstract]. In: Proceedings of the 2018 San Antonio Breast Cancer Symposium; 2018 Dec 4-8; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2019;79(4 Suppl):Abstract nr PD2-09.

Abstract Background The identification of biomarkers to drive treatment is one of the most important objectives of precision medicine. During last years, the role of PIK3CA mutations have been related to clinical benefit deriving from treatment with PI3K, and mTOR inhibitors. In breast cancer (BC), PIK3CA mutations are widely present and the use, in clinical trials, of selective inhibitors improved clinical outcomes. The aim of this study is to assess the value of a monocentric genomic screening program to select patients for trials with experimental targeted agents. Methods We examined PIK3CA mutation in a cohort of 312 metastatic BC patients diagnosed at Hospital Clinico Valencia-INCLIVA from Jan-13 to Apr-19. The sequencing of hotspot mutations was performed in primary (29.9%) and metastatic tissue (70.1%). We used two different technologies: MassARRAY technology (Sequenom MassARRAY, OncoCarta v1.0) and Iluminia MiSeq System (customised panel, OncoSpot v.1). Hotspots were selected according to databases already published. To be diagnosed as mutated, tumors needed to harbor at least 5% of mutant alleles. 7 clinical trials against PI3K pathway were available. Results PIK3CA analysis was performed in 312 paraffin embedded tumor samples, in which only 5.8% the analysis was not possible due to low quality of DNA. The distribution of BC subtypes were 77.6% Luminal, 13.9% HER2, and 8.5% triple negative (TN). PIK3CA mutations were detected in 96 patients (32.7%). In Luminal, PIK3CA mutations reached 36.8% while only 19.5% in HER2 and 16.0% in TN, respectively. A wide spectrum of PIK3CA mutations was found: H1047R (38.5%), E545K (30.2%), E542K (19.8%), R88Q (3.1%), M1043I (3.1%), N345K (2.1%), P539R (1.0%), K111E (1.0%), C420R (1.0%). One hundred and ninety (64.6%) patients were included in clinical trials, and 74 (38.9%) were treated with PI3K, AKT and mTOR inhibitors. Conclusions PIK3CA mutations were widely present among luminal BC, being actually a specific target for new drugs. PIK3CA mutational analysis was easily and successfully performed in our center. The identification of PIK3CA hotspots mutations lead to the access for our patient to novel drugs into clinical trials, achieving relevant clinical benefits in most of the cases. Legal entity responsible for the study The authors. Funding Has not received any funding. Disclosure A. Lluch: Advisory / Consultancy: Novartis; Advisory / Consultancy: Pfizer; Advisory / Consultancy: Roche/Genentech; Advisory / Consultancy: Eisai; Advisory / Consultancy: Celgene. A. Cervantes: Advisory / Consultancy: Merk serono; Advisory / Consultancy: Roche; Advisory / Consultancy: Beigine; Advisory / Consultancy: Bayer; Advisory / Consultancy: Servier; Advisory / Consultancy: Lilly; Advisory / Consultancy: Novartis; Advisory / Consultancy: Takeda; Advisory / Consultancy: Astelas; Advisory / Consultancy: Pierre Fabre; Speaker Bureau / Expert testimony: Foundation Medicine. All other authors have declared no conflicts of interest.

Abstract Background: Triple negative breast cancers (TNBC) have a greater risk of relapse than non-TNBC. New therapeutic approaches are needed for these patients (pts). CIBOMA/2004-01_GEICAM/2003-11 is a multinational, randomized phase III trial exploring adjuvant capecitabine (X) after completion of standard treatment in early TNBC pts. Materials and Methods: Patients with operable, node-positive (or node-negative with tumor size ≥ 1 cm), centrally confirmed hormone receptor-negative, HER2-negative early BC, who had received 6–8 cycles (cy) of standard anthracycline and/or taxane-containing chemotherapy or 4 cy of doxorubicin-cyclophosphamide (for node-negative disease) in the (neo)adjuvant setting, were eligible. Patients were randomized to either 8 cy of X (1,000 mg/m2 bid, days 1–14, every 3 weeks) or observation. Stratification factors included center, prior taxane-based therapy, number of involved axillary lymph nodes and phenotype (basal vs non-basal, according to cytokeratins 5/6 and/or EGFR positivity). The primary objective was to compare the disease-free survival (DFS) between both treatment arms, and secondary objectives included the comparison in terms of 5-year DFS, overall survival (OS) and safety. Assuming a 30% risk reduction in DFS rate at 5 years (from 64.7% to 73.7%, hazard ratio 0.70) with 80% power and a two-tailed log-rank test at 0.05, 834 evaluable pts were needed. 876 pts had to be finally enrolled considering a drop-out rate of 5%. Results: Recruitment of 876 pts from 8 countries was completed in September 2011. Median age was 49 years; 68.5% of pts were postmenopausal, 55.5% were lymph node negative, 71.7% had a basal phenotype, 67.5% received chemotherapy based on anthracyclines and taxanes. Median follow-up was 7.3 years (range 0.0 to 11.1). DFS was not significantly prolonged with X vs observation (hazard ratio (HR) 0.82; 95% confidence interval (CI), 0.63 to 1.06; P=0.1353). Five-year DFS was 79.6% (95% CI, 75.8% to 83.4%) with X and 76.8% (95% CI, 72.7% to 80.9%) with observation. OS was not statistically different between treatment arms (HR 0.92; 95% CI, 0.66 to 1.28; P=0.6228). In subgroup analysis for DFS, we found no statistically significant interaction between X treatment and different subgroups, with the exception of basal vs non-basal phenotypes (basal HR 0.97, 95% CI 0.72 to 1.32, P=0.8620; non-basal HR 0.51, 95% CI, 0.31 to 0.86, P=0.0101; interaction P=0.0357). Similar results were found for OS (basal HR 1.20, 95% CI 0.81 to 1.77, P=0.3684; non-basal HR 0.48, 95% CI, 0.26 to 0.91, P=0.0205; interaction P=0.0155). 75.2% of pts completed 8 cy of X, with a median relative dose intensity of 86.3%. Grade (G) 3 or higher adverse events (AEs) were observed in 40.4% of pts in X arm. In 9.6% of pts the AEs were related with X. Hand-foot syndrome was the most common AE in X arm (G3 on 18.8% of pts). Conclusions: In our study, the addition of adjuvant X after standard (neo) adjuvant anthracycline and/or taxane-containing chemotherapy was not associated with a statistically significant improvement of DFS or OS compared to observation in pts with early TNBC. However, in a subgroup analysis a significant DFS and OS improvement was observed with X in pts with non-basal phenotype. Sponsor: CIBOMA. Citation Format: Martín M, Barrios CH, Torrecillas L, Ruiz-Borrego M, Bines J, Segalla J, Ruiz A, García-Sáenz JA, Torres R, de la Haba J, García E, Gómez HL, Llombart A, Rodríguez de la Borbolla M, Baena JM, Barnadas A, Calvo L, Pérez-Michel L, Ramos M, Castellanos J, Rodríguez-Lescure A, Cárdenas J, Vinholes J, Martínez de Dueñas E, Godes MJ, Seguí MA, Antón A, López-Álvarez P, Moncayo J, Amorim G, Villar E, Reyes S, Sampaio C, Cardemil B, Escudero MJ, Bezares S, Carrasco E, Lluch A. Efficacy results from CIBOMA/2004-01_GEICAM/2003-11 study: A randomized phase III trial assessing adjuvant capecitabine after standard chemotherapy for patients with early triple negative breast cancer [abstract]. In: Proceedings of the 2018 San Antonio Breast Cancer Symposium; 2018 Dec 4-8; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2019;79(4 Suppl):Abstract nr GS2-04.

Abstract Background: The expression of ER, PR and HER2 in breast cancer is determined by routine pathology assessment to indicate systemic treatment. Here, we investigated the ability of a standardized mRNA-based assay to predict ER, PR and HER2 status and treatment response. Methods: ESR1, PGR and ERBB2 expression was analyzed from the standardized nCounter-based PAM50 assay in 1,544 FFPE breast tumors obtained from 13 independent studies (NeoEribulin, GEICAM 2012-09, TBCRC023/006, LPT109096, EGF117165, PAMELA, PerELISA, GEICAM 2003-11, VENTANA, IBIMA and HCB). All immunohistochemical and in situ hybridization analyses followed ASCO/CAP criteria and were performed in central labs except for Neoeribulin and VENTANA studies. To explore the best cutoff for each gene, we used Monte Carlo cross validation (repeated 1000 times, 2/3 training, 1/3 testing) to achieve the highest kappa values when gene expression was compared to pathology assessment. Receiver operating characteristic analysis and area under the ROC curve (AUC) was used to evaluate the performance of each gene to predict ER, PR and HER2 status. Finally, the association of each gene (using the pre-established cutoffs) with pathological complete response (pCR) was evaluated using univariate logistic regression analyses in 2 neoadjuvant cohorts: 1) A combined HER2+ cohort using 191 tumor samples from PAMELA/PerELISA phase II trials, where patients received neoadjuvant dual HER2 blockade without chemotherapy for 15-18 weeks and 2) a combined cohort of hormone receptor-positive/HER2-negative using 205 tumor samples from IBIMA/HCB consecutive series, where patients received anthracycline/taxane-based chemotherapy. Results: Concordance between ESR1 and ER was 95.3% (95% confidence interval [95CI] 94.0-96.4%; AUC=0.98). ER+ and ER- cases were classified as ESR1- and ESR1+ in 5.5% and 4.8% of the cases, respectively. Concordance between PR and PGR was 85.2% (95CI 83.1-87.1%; AUC=0.95), and between ERBB2 and HER2 was 92.8% (95CI 91.4-94.1%; AUC=0.95). HER2+ and HER2- cases were classified ERBB2- and ERBB2+ in 16.7% and 2.9% of the cases, respectively. In the neoadjuvant HER2+ cohort, the pCR rates were 44.6% in ESR1- and 18.8% in ESR1+ (odds ratio [OR] 3.9; 95CI 1.9-8.4; p<0.001), 38.5 in PGR- and 15.8% in PGR+ (OR 3.2; 95CI 1.6-6.7; p=0.001), 2.6% in ERBB2- and 35.3% in ERBB2+ (OR 19.7; 95CI 2.6-149.1; p<0.001). In the neoadjuvant HR+/HER2- cohort, the pCR rates were 40% in ESR1- and 5.12% in ESR1+ (OR 11.5; 95IC 2.7-48.5; p<0.001), 15.5% in PGR- and 4.3% in PGR+ (OR 3.8; 95IC 1.3-11.7; p=0.019), 7.36% in ERBB2- and 0% in ERBB2+ (OR NA). Finally, ESR1, PGR and ERBB2 expression as continuous variables were found significantly associated with pCR in both cohorts. Conclusion: ESR1, PGR and ERBB2 standardized mRNA levels show high concordance with ER, PR and HER2, and might provide a more objective and quantitative prediction of response to chemotherapy and anti-HER2 therapy. The level of concordance between GE and pathology-based assessments is similar to 2 FDA 510(k) cleared pathology-based ER assays (e.g. SP1 versus 6F11 antibody clones), local versus central HER2 testing, and ER or PR status when determined by the ligand-binding assay versus immunohistochemistry. Citation Format: Pascual T, Tsai YS, Martín M, Lluch A, Cortes J, Llombart A, Conte P, Guarneri V, Rimawi MF, Alba E, Ruiz-Borrego M, Rojo F, de la Haba J, Schiff R, Adamo B, Vidal M, Paré L, Chic N, Muñoz M, Galvan P, Gonzalez-Farre B, Brauer HA, Sullivan A, Nuciforo P, Parker JS, Prat A. Standardized nCounter-based determination of estrogen receptor (ER), progesterone receptor (PR) and HER2 receptor status in breast cancer [abstract]. In: Proceedings of the 2018 San Antonio Breast Cancer Symposium; 2018 Dec 4-8; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2019;79(4 Suppl):Abstract nr P2-08-06.

Abstract Background Copy Number Alterations (CNAs) represent changes in the copy number of genomic segments of somatic cells due to chromosomal instability. CNAs include gene amplifications or deletions and can be involved in tumorigenesis. We analyzed CNAs data in pre- and post-treatment (ttm) tumors from patients (pts) with early breast cancer (BC) in the neoadjuvant trials GEICAM/2006-03 and GEICAM/2006-14, with the aim to identify CNAs in particular genomic regions (genetic entropy) associated with treatment response. Methods GEICAM/2006-03 (NCT00432172) HER2-negative pts were selectively treated according to clinical subtypes: triple negative (TN) pts were treated with standard anthracycline/taxane -based chemotherapy (AT-CT) +/- carboplatin, while luminal patients were randomized to AT-CT vs. hormonotherapy; GEICAM/2006-14 (NCT00841828) HER2+ pts received AT-CT plus anti-HER2 therapy.Shallow-whole genome Illumina sequencing DNA data from 204 paraffin-embedded tumors (100 pre- and 104 post-ttm) were segmented to obtain CNAs and recurrent altered genomic regions were defined. We used Wilcoxon test to analyze the frequency of altered regions and logistic regression analyses to explore their association with tumor response, in terms of pathological complete response (pCR) in breast and axilla. Validation of altered genes associated with therapy response was performed in the microarray gene expression-based Hatzis dataset (GSE25066) from pts receiving neoadjuvant AT-CT (1). Results A total of 672 regions covering the whole genome were identified upon analysis of CNAs data. Regions were categorized according to their alteration status as amplified, normal and lost. Comparative analysis of alterations revealed 11 regions significantly different (p<0.05) in pre- vs post-ttm tumors. Logistic regression analysis showed that in pre-ttm tumors specific alterations of 8 regions localized in 3 different genomic loci (11q12, 16q22 and 21q22) were significantly associated with pCR (p<0.05). Independent analyses of CNAs data with “CGH regions” and “GISTIC2.0” tools confirmed the special relevance of 2 of these 8 regions (#653 and #654), amplified in the locus 21q22.12. This locus contains 20 genes whose expression was tested in Hatzis dataset (1) (GSE25066): the analysis showed that overexpression of 5 of these 20 genes (CHAF1B, CBR1, CBR3, RCAN1 and SLC5A3) turned out to be significantly higher in the cohort of pts who reached pCR, in agreement with our findings. Some of these genes have already been described as proliferation markers (CHAF1B) or involved in treatment response (CBR1) in BC. Other genes related to BC in this genomic region are the transcription factor RUNX1 and the Lysine Methyltransferase SETD4. Conclusions According to our results, neoadjuvant therapy can modulate genomic aberrations landscape in BC. Our data suggest that amplification of specific genes in the genomic locus (21q22.12) is involved in the neoadjuvant therapy response in early BC. (1): Hatzis et al., JAMA 2011, 305(18) 1873-81 Citation Format: Alba E, Rueda OM, Lluch A, Albanell J, Chin S-F, Chacón JI, Calvo L, De la Haba-Rodriguez J, Bermejo B, Ribelles N, Sánchez-Rovira P, Plazaola A, Barnadas A, Cirauqul B, Ramos M, Arcusa A, Carrasco E, Herranz J, Chiesa M, Caballero R, Santonja A, Rojo F, Caldas C. Genome copy number entropy as predictor of response for neoadjuvant therapy in early breast cancer [abstract]. In: Proceedings of the 2018 San Antonio Breast Cancer Symposium; 2018 Dec 4-8; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2019;79(4 Suppl):Abstract nr P5-12-03.

Abstract Background: Therapeutic targets in TNBC remain a challenge. We have observed that some Luminal A primary breast tumors give rise to HER2-enriched (HER2E) subtype metastases but remain clinically HER2 negative (HER2E/cHER2-). Molecular features that drive these HER2E/cHER2- tumors may represent key targets of metastatic progression. Methods: A comparative genetic and transcriptomic analysis in TCGA (1100 patients) related to the FGFR family was performed. We focused on FGFR4, in part, due to its unique association with the HER2E expression subtype and we developed a robust FGFR4-signature based upon a supervised analysis of a HER2E/cHER2- PDX (WHIM11) treated with a FGFR4 inhibitor (BLU9931). We also constructed a new Luminal Tumor Score (LTS) to identify the optimal axis of separation between Luminal A versus HER2E tumors (higher scores represent greater Luminal A phenotype). Univariate and multivariate analyses were performed using TCGA and METABRIC (1971 samples). Finally, we performed RNA-seq on a cohort of 77 matched primary breast cancer and metastatic tissues pairs from the GEICAM/2009-03 and Hospital Clinic of Barcelona study, and did multiple analyses on these cohorts using our FGFR4-signatures. Results: FGFR4 was significantly higher in HER2E subtype (P<0.0001), independent of HER2 clinical status. FGFR4 amplification/deletions and mutations were rare and did not correlate with FGFR4 high expression. In vivo, BLU9931 treatment of WHIM11 showed a significant tumor growth inhibition (P=0.01), prolonged survival, and a significant higher LTS (P=0.016). We also identified 745 up-regulated genes called FGFR4-repressed (FGFR4-rep) and 427 down regulated genes called FGFR4-induced (FGFR4-ind), after BLU9931 treatment. Gene set enrichment analysis revealed that FGFR4-ind genes were enriched for STAT3, PI3K/AKT/mTOR pathway and KRAS activation, proliferation, hypoxia, glycolysis and metastasis. FGFR4-rep genes were involved with KRAS inhibition, cell polarity, p53 pathway and upregulation of IFNγ response. In the METABRIC cohort, FGFR4-ind and FGFR4-rep each predicted OS (HR=6.30, P<0.0001; HR=0.33; P<0.0001, respectively). Multivariate analysis showed FGFR4-ind (HR=2.34, P=0.014) as a significant independent prognostic factor beyond subtype for OS. Supervised analysis of the 77 primary-met cohort revealed that the FGFR4-ind was significantly higher in luminal metastases compared with their primaries counterparts (P<0.001), along with proliferation, angiogenesis, and a M2 macrophage signature (with most other immune features being unchanged). Finally, univariate and multivariate analysis demonstrated that the FGFR4-related signatures predicted site-specific metastasis for lung, liver and brain, but not for bone and lymph nodes. Conclusion: FGFR4 is one of the drivers of HER2-enriched subtype tumors, including those that are clinically HER2-. The FGFR4-ind signature was predictive of worse survival, progression in the metastatic setting, and site-specific metastasis. Treatment options in HER2-enriched TNBC, and for HER2E/cHER2+ patients, may benefit from targeting FGFR4, whose high expression is not based upon genomic or genetic features. Citation Format: Garcia Recio S, Parker JS, Fan C, Mott K, He X, Cejalvo JM, Brasó Maristany F, Galván P, Lluch A, Albanell J, Rojo F, Martinez de Dueñas E, Prat A, Perou CM. FGFR4 as a key regulator of HER2E subtype in the primary and metastatic setting [abstract]. In: Proceedings of the 2018 San Antonio Breast Cancer Symposium; 2018 Dec 4-8; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2019;79(4 Suppl):Abstract nr PD9-04.

Abstract Background dNLR is a novel biomarker associated with clinical outcome in solid tumors including early stage breast cancer (BC). Here we report the association of dNLR with pCR in triple-negative (TN) and luminal BC patients (pts) treated with neoadjuvant CT. Methods This was a retrospective analysis of two randomized studies (GEICAM/2006-03 - NCT00432172 and ETNA - NCT01822314) involving 821 pts with early stage (>2cm) or locally advanced TN or luminal BC receiving anthracycline/taxane-based CT +/- carboplatin (GEICAM/2006-03) or nab-paclitaxel/paclitaxel followed by anthracycline regimen (ETNA). dNLR was calculated as the ratio of neutrophils and the difference between total leukocytes and neutrophils in peripheral blood before CT (baseline) and at the end of treatment (EOT). Logistic regression analyses were used to explore dNLR association with pCR at the two time points by molecular subtype. Results 684 and 137 pts were from ETNA and GEICAM/2006-03 study respectively (TN n = 308; Luminal n = 513). In TN subgroup median baseline dNLR was 1.61 [interquartile range (IQR): 1.25-2.04] and at EOT 1.65 (IQR: 0.96-2.22). In luminal BC median baseline dNLR was 1.68 (IQR: 1.22-1.96) and at EOT 1.48 (IQR: 0.86-1.93). No association with pCR was found in luminal BC. Baseline dNLR was associated with pCR in TNBC in univariate analysis (OR: 0.709, CI: 0.5-1.006, p = 0.0406). At EOT significant association of dNLR as continuous variable with pCR was observed in TNBC both in univariate and multivariate analysis (OR: 0.665, CI: 0.501-0.884, p = 0.0034, n = 255 and OR: 0.62, CI: 0.406-0.946, p = 0.0231, n = 228 respectively). Quartiles distribution of EOT dNLR was also associated with pCR in univariate and multivariate analysis (Q1 vs combined Q2/Q3/Q4 group: OR: 0.32, CI: 0.17-0.64, p = 5e-04 and OR: 0.26, CI: 0.1-0.63, p = 0.002 respectively). Also a ROC curve - based optimal cut off method revealed significant association of EOT dNLR with pCR in TNBC (>2.231, OR: 0.43, CI: 0.25-0.74, p = 0.0035). Conclusions No association was found between dNLR and pCR in luminal BC. High dNLR levels at baseline and especially EOT seem to be associated with worse clinical outcome in TN tumors after neoadjuvant CT. Clinical trial identification GEICAM/2006-03 - NCT00432172: 07 Feb 2007 ETNA - NCT01822314: 02 Abr 2013. Legal entity responsible for the study GEICAM Spanish Breast Cancer Group. Funding Has not received any funding. Disclosure M. Mansutti: Advisory / Consultancy: Celgene; Advisory / Consultancy: Novartis; Advisory / Consultancy: Pfizer; Travel / Accommodation / Expenses: Roche; Travel / Accommodation / Expenses: AstraZeneca. A. Lluch: Research grant / Funding (self), Research grant / Funding (institution): Amgen; Research grant / Funding (self), Research grant / Funding (institution): AstraZeneca; Research grant / Funding (self), Research grant / Funding (institution): Boehringer-Ingelheim; Research grant / Funding (self), Research grant / Funding (institution): GSK; Advisory / Consultancy, Research grant / Funding (self), Research grant / Funding (institution): Novartis; Advisory / Consultancy, Research grant / Funding (self), Research grant / Funding (institution): Pfizer; Advisory / Consultancy, Research grant / Funding (self), Research grant / Funding (institution): Roche/Genentech; Advisory / Consultancy, Research grant / Funding (self), Research grant / Funding (institution): Eisai; Advisory / Consultancy, Research grant / Funding (self), Research grant / Funding (institution): Celgene; Research grant / Funding (self), Research grant / Funding (institution): Pierre-Fabre. M. Thill: Honoraria (self), Advisory / Consultancy, Research grant / Funding (institution), Travel / Accommodation / Expenses: Genomic Health; Honoraria (self), Advisory / Consultancy, Travel / Accommodation / Expenses: Amgen; Honoraria (self), Advisory / Consultancy, Travel / Accommodation / Expenses: AstraZeneca; Honoraria (self), Advisory / Consultancy, Travel / Accommodation / Expenses: Celgene; Honoraria (self), Advisory / Consultancy, Travel / Accommodation / Expenses: Eisai; Honoraria (self): Hexal; Honoraria (self), Travel / Accommodation / Expenses: Medtronic; Honoraria (self), Advisory / Consultancy, Travel / Accommodation / Expenses: MSD; Honoraria (self), Advisory / Consultancy, Travel / Accommodation / Expenses: Myriad; Honoraria (self), Advisory / Consultancy, Travel / Accommodation / Expenses: Novartis; Honoraria (self), Advisory / Consultancy, Travel / Accommodation / Expenses: Pfizer; Honoraria (self), Advisory / Consultancy, Travel / Accommodation / Expenses: PFM Medical; Honoraria (self), Advisory / Consultancy, Travel / Accommodation / Expenses: Roche; Honoraria (self), Advisory / Consultancy, Travel / Accommodation / Expenses: RTI Surgical; Honoraria (self), Advisory / Consultancy, Travel / Accommodation / Expenses: Teva; Advisory / Consultancy, Travel / Accommodation / Expenses: Biom-up; Advisory / Consultancy, Travel / Accommodation / Expenses: Lilly; Advisory / Consultancy, Travel / Accommodation / Expenses: Neodynamics; Advisory / Consultancy, Travel / Accommodation / Expenses: Norgine; Advisory / Consultancy, Travel / Accommodation / Expenses: Tesaro. L. Gianni: Advisory / Consultancy, MetIS, Novartis, Odonate Therapeutics, Revolution Medicine, Synaffix, Zymeworks: CONSULTANCY: ADC Therapeutics,; Advisory / Consultancy, RESEARCH FUNDING (institution): Zymeworks, Daiichi SankyoZymeworks, Daiichi Sankyo: AstraZeneca; Advisory / Consultancy, PATENTS, ROYALTIES: European Patent Application N. 12195182.6 and 12196177.5’, Roche: Celgene; Advisory / Consultancy: Lilly; Advisory / Consultancy: G1 Therapeutics; Advisory / Consultancy: Genentech; Advisory / Consultancy: Genomic Health; Advisory / Consultancy: MSD; Advisory / Consultancy: Oncolytics Biotech; Advisory / Consultancy: Odonate Therapeutics; Advisory / Consultancy: Onkaido; Advisory / Consultancy: Roche; Advisory / Consultancy: Pfizer; Advisory / Consultancy: Tahio Pharmaceutical; Advisory / Consultancy: Sandoz; Advisory / Consultancy: SeaGen,; Advisory / Consultancy: Synthon; Advisory / Consultancy: Zymeworks,; Advisory / Consultancy: CD47; Advisory / Consultancy: GENENTA. All other authors have declared no conflicts of interest.

Abstract Background: Metastatic breast cancer is the second leading cause of death among women globally. A better understanding of tumor biology, and the availability of high-throughput technologies, have enabled the emergence of precision medicine bringing new expectations and giving rise to molecular screening programs (MSP). SOLTI, as a collaborative Spanish network, designed AGATA, the first multi-institutional MSP ever implemented in this country. Here, we report both the primary and some of the secondary results of the pilot study. Methods: A total of 10 sites within SOLTI network in Spain participated. DNA-sequencing of 56 cancer related genes was performed using FFPE tumor samples (primary or metastatic). Each clinical case was reviewed by a multidisciplinary advisory board (MAB), which recommended, in a prospective manner, potential experimental treatments, mainly in the context of clinical trials. The primary objective was to determine the success rate of matching a DNA alteration to an experimental drug or drug class. Secondary objectives included a comprehensive molecular characterization of tumor samples by PAM50 subtyping and quantification of protein expression levels by MASS-SPEC (70 proteins panel). Results: 305 patients (pts) were screened from September 2014 to July 2017 and 260 (85.3%) were finally evaluated by the MAB. Pts characteristics were: mean age 54 years (29-80), ER+/HER2- (n=192; 74%), HER2+ (n=30; 11.5%) and TNBC (n=38; 14.5%). 163 primary tumors and 97 metastatic samples were profiled. Regarding the primary objective, 116 pts (45%) presented at least one mutation (range 1-6) that could be matched to a drug or drug class. Of these, 13 pts (11.2%) received therapy matched to their molecular profile according to the MAB recommendation and their follow-up is still on-going. No mutation was detected in 97 (37%) pts (WT), and 47 patients (18.1%) presented a mutation but no match was possible. The most common mutations were PIK3CA (34%), TP53 (22%), AKT1 (5%), ESR1 (3%) and ERBB2 (3%). Intrinsic subtype distribution in 177 samples was as follows: 34% Luminal A (n=60); 21% Luminal B (n=36); 13% HER2E (n=22); 19% Basal-like (n=34) and 13% Normal-like (n=23). Compared to primary tumors (n=110), the proportion of HER2-enriched disease in metastatic tumors (n=63) was significantly higher (6% vs 20%; p=0.005). Protein expression analysis was performed in 146 samples (94 primary and 57 metastasis). In 19 cases (13%), the outlier expression of some targetable proteins (FGFR1 [n=4, 2.7%], IGF1R [n=4, 2.7%], EGFR [n=1, 0.7%], CEACAM5 [n=6, 4.1%], IDO1 [n=2, 1.37%], TROP2 [n=2, 1.37%]) were identified. Of note, HER2 overexpression (>740 amol/μg) was observed in 4 HER2- cases. Finally, among WT tumors, 21% presented a potential drug-matched protein target. Conclusions: Nationwide molecular screening in Spain is feasible. Nearly half of patients had tumors with mutation(s), mostly PIK3CA, that could be matched to a potential drug or drug class. PAM50 profile might be helpful to navigate towards a therapeutic decision making, although the MAB could not make any targeted-driven recommendation yet with this data. More clinical evidence is needed to use MASS-SPEC as a diagnostic tool. Citation Format: Pernas S, Villagrasa P, Nuciforo PG, Vivancos A, Scaltriti M, Rodón J, Burgués O, Canes J, Dueñas M, Cecchi F, Vidal M, Lluch A, Perelló A, Llombart A, Dorca J, Montaño A, Oliveira M, Ribas G, Rapado I, Paré L, Prat A, Ciruelos E. Primary and secondary results of the first nationwide molecular screening program in Spain for patients with advanced breast cancer (AGATA SOLTI-1301 study) [abstract]. In: Proceedings of the 2018 San Antonio Breast Cancer Symposium; 2018 Dec 4-8; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2019;79(4 Suppl):Abstract nr P6-18-02.