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Background: HER2DX is a 27-gene prognostic (risk-score) and predictive (pathological complete response [pCR]-score) assay in early-stage HER2+ breast cancer (BC) based on clinical data and the expression of 4 gene signatures (immune, proliferation, luminal differentiation, and HER2 amplicon). Here we aim to evaluate, for the first time, the ability of HER2DX to predict pCR following neoadjuvant THP in HER2+ BC. Methods: Standardized HER2DX was evaluated centrally on baseline pre-treatment FFPE tumor biopsies from the DAPHNe phase II trial (Waks et al. NPJ Breast 2022; NCT03716180), in which patients (pts) with newly diagnosed stage II-III HER2+ BC were treated with neoadjuvant weekly paclitaxel × 12 and HP every 3 weeks × 4. Primary aim was to test the ability of HER2DX pCR-score to predict pCR (ypT0/isN0). Secondary objectives were to test the ability of HER2DX pCR-score to predict pCR independent of clinical-pathological variables and PAM50 subtype (HER2-enriched vs not) and to evaluate the association of HER2DX pCR-score with HER2DX risk-score. Five patients who received additional neoadjuvant chemotherapy after THP were excluded from this analysis. Logistic regression and receiver-operator curve (ROC) analysis were assessed. Statistical analyses were performed in R code 4.0.5. Results: HER2DX was evaluated in 80 of 97 pts (82.5%) enrolled in the DAPHNe trial who received study treatment. Clinical T2-4 disease represented 81.3% of cases (n=65), clinical node-negative disease (cN0) represented 65.0% of cases (n=52), and 70.0% of tumors (n=56) were hormone receptor-positive. The overall pCR rate was 60.0% (95% confidence interval [CI] 49.3-70.7): 87.0% (95% CI 79.6-94.4) in hormone receptor-negative disease and 48.2% (95% CI 37.2-59.1) in hormone receptor-positive disease. The proportion of HER2DX low-, medium- and high-pCR groups was 38.8%, 27.5% and 33.7%, respectively. HER2DX pCR-score (as a continuous variable from 0 to 100) was significantly associated with pCR (odds ratio [OR]=1.05, p< 0.0001). In the overall population, the pCR rates in HER2DX pCR-high, pCR-med and pCR-low groups were 92.6%, 63.6% and 29.0% (pCR-high vs pCR-low OR=30.6, p< 0.0001), respectively. The AUC ROC of HER2DX pCR score (as a continuous variable) and pCR status was 0.835. In the ER-negative population, the pCR rates in HER2DX pCR-high, pCR-med and pCR-low groups were 94.7%, 66.7%, and 0%, respectively (Table 1). HER2DX pCR-score was significantly associated with pCR independent of hormone receptor status, HER2 immunohistochemistry (IHC) score, clinical stage, and PAM50 HER2-enriched subtype. The correlation between HER2DX pCR-score and HER2DX risk-score was weak (Pearson coefficient=-0.12), as previously described (Prat et al. EBiomedicine 2022). 51.3% of patients were categorized as HER2DX low-risk. Conclusion: The 27-gene HER2DX genomic test predicts pCR following neoadjuvant THP in newly diagnosed stage II-III HER2+ BC. Patients with HER2DX pCR-low score and HER2DX high-risk score, representing 22.5% of pts, warrant further attention in order to optimize therapeutic strategies in this subset. The combination of HER2DX pCR-score and risk-score might guide therapeutic decisions by identifying patients who are ideal candidates for de-escalated or escalated systemic and locoregional treatments. Table 1 Citation Format: Adrienne Waks, Esther R. Ogayo, Laia Paré, Mercedes Marín-Aguilera, Fara Brasó-Maristany, Patricia Galván, Oleguer Castillo, Olga Martínez-Sáez, Ana Vivancos, Patricia Villagrasa, Paolo Tarantino, Neelam Desai, Jennifer Guerriero, Otto Metzger, Nadine Tung, Ian Krop, Joel S Parker, Charles M. Perou, Aleix Prat, Eric Winer, Sara Tolaney, Elizabeth A. Mittendorf. Independent validation of the HER2DX genomic test in HER2-positive breast cancer treated with neoadjuvant paclitaxel, trastuzumab and pertuzumab (THP): a correlative analysis from the DAPHNe phase II clinical 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 P1-04-05.

Background: Palbociclib might be less effective than ribociclib in HR+/HER2- and HER2-enriched BC. This hypothesis is currently being tested in the HARMONIA prospective phase III clinical trial (NCT05207709). Here, we evaluated the downstream biological effects of both drugs using cell lines and patient tumor samples. Methods: Three HR+ BC cell lines (i.e., MCF7, T47D and BT474) were treated at 2 different dose levels of palbociclib or ribociclib (i.e., 100 nM and 500 nM) +/- fulvestrant (1 nM) for 72 hours (h). PAM50 gene signatures were determined on the nCounter, as well as phosphorylation of RB (p-RB) by Western Blot and senescence-associated β-galactosidase activity by FACS. In vitro experiments were performed in triplicates. PAM50 gene signatures were obtained from 49 paired baseline versus week-2 samples and 49 paired baseline versus surgery samples of the CORALLEEN phase II study (Prat, Lancet Oncol. 2020), which treated 49 women with PAM50 Luminal B HR+/HER2- early BC with neoadjuvant ribociclib (600 mg daily) plus endocrine therapy (ET). PAM50 signature scores were also evaluated in publicly available data from 23 paired baseline versus week-2 samples and 16 paired baseline versus surgery samples of the NEOPALANA phase II trial (Ma, Clin Cancer Res. 2017) which treated 50 patients with HR+/HER2- early BC with palbociclib (125 mg daily) plus anastrozole. Changes in PAM50 signatures upon CDK4/6i were determined by paired t-tests and significant analysis of microarray (SAM). Results: Across all cell lines, both palbociclib and ribociclib statistically significantly reduced p-RB at 72h with both doses (i.e., 100 and 500 nM) compared to non-treated cells (p< 0.001). Senescence was also observed at 72h with both doses. Both drugs +/- ET significantly increased the Luminal A signature and decreased Luminal B and proliferation signatures with both doses. However, the HER2-enriched signature was only significantly reduced when both CDK4/6 inhibitors were given at 500 nM. In tumor samples from the CORALLEEN and NEOPALANA phase II studies, a similar change in PAM50 biology was observed with both drugs, namely an increase in Luminal A signature and a decrease in Luminal B and proliferation signatures after 2 weeks of treatment and at surgery. At 2 weeks of treatment, the HER2-enriched signature was significantly decreased in both studies with ribociclib (p< 0.001) and palbociclib (p< 0.001). However, the decrease in the HER2-enriched signature was only observed in surgical samples of patients treated with ribociclib (p< 0.001), but not palbociclib (p=0.194). A difference in sample size could explain this result. Nevertheless, in CORALLEEN, the median number of days between the last dose of ribociclib and surgery was 13.1 days (range: 1-78). In NEOPALANA, the median number of days between the last dose of palbociclib and surgery was 29 days (range: 8-49), except for 8 patients who received additional 10-12 days of palbociclib immediately before surgery (Ma, Clin Cancer Res. 2017). In patients who underwent surgery at 8 days or before, the HER2-enriched signature was significantly decreased for both ribociclib (p< 0.001) and palbociclib (p=0.013). Interestingly, in patients that underwent surgery after >8 days from the last dose, a significant reduction of the HER2-enriched signature was only observed with ribociclib (p< 0.001), but not with palbociclib (p=0.500). Conclusions: Both palbociclib and ribociclib have similar effects on PAM50 biology when given at the same dose. However, in clinical practice, palbociclib is given at a lower dose than ribociclib, and although HER2-enriched signature is significantly decreased in tumors after 2 weeks of CDK4/6i+ET, this effect is only maintained at later time points with ribociclib, indicating a dose-dependent efficacy of CDK4/6i in this biologically aggressive subtype. Citation Format: Natàlia Lorman-Carbó, Olga Martínez-Sáez, Aranzazu Fernandez-Martinez, Patricia Galván, Nuria Chic, Barbara Adamo, Maria Vidal, Montserrat Muñoz, Charles M. Perou, Joaquín Gavilá, Tomás Pascual, Aleix Prat, Fara Brasó-Maristany. Dissecting the biological activity of different CDK4/6 inhibitors (CDK4/6i) in hormone receptor-positive/HER2-negative (HR+/HER2-) breast cancer (BC) [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 P1-13-16.

Background Within HR+/HER2- disease, patients with non-luminal subtypes of breast cancer (HER2-enriched [HER2-E] and Basal-like) have poorer prognosis than those with luminal subtypes, may be more sensitive to chemotherapy, and have higher expression of immune-related genes and tumor infiltrating lymphocytes (TILs). Here, we report the interim efficacy and safety data of the TATEN trial (NCT04251169), the first study designed to evaluate pembrolizumab and paclitaxel in HR+/HER2-negative, PAM50 non-luminal, metastatic breast cancer (MBC). Methods TATEN is a single-arm, multicenter phase II study evaluating pembrolizumab in combination with paclitaxel in patients with HR+/HER2-, PAM50 non-luminal, MBC. Key inclusion criteria include progression to prior CDK4/6 inhibitors, presence of measurable disease by RECIST V1.1, no prior chemotherapy for MBC, and ECOG 0-1. Patients receive pembrolizumab at 200 mg every 3 weeks (on D1 of each 21-day cycle, beginning at cycle 1) in combination with weekly paclitaxel at 80 mg/m2, beginning at cycle 2. The primary endpoint is to evaluate overall response rate (ORR), defined as the rate of complete (CR) and partial response (PR) according to RECIST V1.1. Secondary endpoints include clinical benefit rate (CBR; CR + PR + stable disease >24 weeks), progression free survival, overall survival, safety, and predictive biomarkers. Tumor samples collected during advanced/metastatic disease are mandatory to assess PAM50 and other translational endpoints. This study had a planned interim analysis after 15 patients were evaluable for ORR based on a Simon’s two stage design with 80% power and a type I error rate of 0.05. Stage I of the trial would be considered successful if at least 6 patients achieved a PR and/or CR. In that case, the trial would recruit up to 46 evaluable patients for a target ORR ≥ 41. Here we report results from the patients who received at least one dose of combination treatment and had a first, post-baseline, tumor assessment according to RECIST v1.1 (evaluable population). Results From July 2020 to December 2021, 119 patients were screened, and 25 PAM50 non-luminal tumors were identified (21%). From these, 17 (68%) patients were recruited, and 15 were evaluable for primary endpoint. Two patients discontinued the trial before the first dose of pembrolizumab and paclitaxel, because of clinical progressive disease (PD). Baseline patient characteristics were as follows: median age 53 years (range: 40-77), ECOG 0 52.9%, de novo MBC at diagnosis 29.4%, and visceral disease 64.7% (including 53.3% with liver metastasis). Ten patients had received paclitaxel treatment in the adjuvant setting. Regarding PAM50 intrinsic subtype, two patients had basal-like and 13 HER2-E tumors. At the time of data cut-off (May 17,2022), 8 patients (53.3%) had stopped their treatment because of PD and 2 (13.3%) due to toxicity. Five patients (33.3%) were still on treatment. The ORR was 53.3 % (8 of 15, 95% CI 26.6-78.7), meeting the pre-specified ORR for the first stage of the trial. The CBR was 86.6% (13 of 15, 95%CI 59.5-98.34), and median PFS was 7.5 months (95% CI: 5.6 – 10.2). Overall, all patients experienced treatment-related adverse events (TRAEs) of any grade, while 53.3% of patients experienced grade 3 TRAEs. No grade 4 or 5 TRAEs were reported in the evaluable population. Correlative analysis including gene expression analysis and centralized TILs scoring and PD-L1 IHC will be presented. Conclusions The first stage of TATEN combining pembrolizumab with paclitaxel after progression to CDK4/6 inhibitors in patients with HR+/HER2-negative, PAM50 non-luminal MBC met its pre-specified endpoint. Completion of the stage II part of the trial with the inclusion of up to 46 patients is warranted to assess the activity of this combination in this group of patients. Correlative studies to find predictive biomarkers of response to this regimen are ongoing and will be presented at the meeting. This study was funded by MSD. Citation Format: Aleix Prat, Tomás Pascual, Montserrat Muñoz, Cristina Hernando, Silvia Vazquez, Salvador Blanch, Manuel Alva, Mafalda Oliveira, Esther Sanfeliu, Lorea Villanueva, Fara Brasó-Maristany, Nuria Chic, Eva Ciruelos. TATEN TRIAL (SOLTI-1716) Targeting non-Luminal disease by PAM50 with pembrolizumab + paclitaxel in Hormone Receptor-positive/HER2-negative (HR+/HER2-) metastatic breast cancer (MBC): interim analysis [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 P3-06-02.

INTRODUCTION: Breast cancer (BC) is a highly prevalent and heterogeneous disease, entailing different so-called intrinsic subtypes (IS) according to gene expression, namely Luminal A, Luminal B, HER2-Enriched (HER2E) and Basal-like, as well as a normal breast-like group. The HER2E is still a poorly understood entity, which needs further biologic characterization to improve therapeutic management. MATERIAL AND METHODS: Patients (pts) treated at Hospital Clinic (Barcelona, Spain) over 18 years with a diagnosis of metastatic BC, with available matched primary and metastatic tumor samples for gene expression analyses were retrospectively recruited. Using the nCounter® Breast Cancer 360 panel, we studied the expression of 776 genes pertaining to different tumor and immune pathway-related signatures, with a focus on HER2E vs. non-HER2E tumors. Significant changes were considered at a false discovery rate (FDR) < 5%. Main clinicopathological features were also compared. IS changes from primary to metastatic disease were assessed. RESULTS: Ninety-one pts with paired tumor samples were included. Briefly, primary tumors were 67.0% hormone receptor-positive (HR+)/HER2-negative (HER2-), 14.8% were HER2-positive (HER2+) and 18.2% were triple-negative (TNBC). IS distribution in primary tumors was the following: 25 Luminal A (28%), 22 Luminal B (24%), 24 HER2E (26%), 12 Basal-like (13%), and 8 Normal-like (9%). In contrast, IS distribution in metastatic tumors was the following: 13 Luminal A (14%), 22 Luminal B (24%), 34 HER2-E (37%), 16 Basal-like (18%), and 6 Normal-like (7%). The HER2E disease proved to be a relatively stable subtype, with 16 (66.7%) tumors not changing IS in the transition to metastatic disease (p=0.078). Particularly, within HR+/HER2-, HER2+ and TNBC, 8/12 (66.7%), 8/10 (80.0%) and 1/2 (50.0%) tumors remained HER2E. Conversely, an overall significant switch from Luminal to non-Luminal tumors at the metastatic progression was observed (p=0.031), with 14/19 (73.7%) new non-Luminal BC being HER2E. When considering all primary and metastatic tumors, HER2E were observed to be less frequently estrogen receptor (ER) positive than non-HER2E tumors (55.8% vs. 78.7%; p=0.002), with lower mean progesterone receptor (PR) levels (15.3% vs. 25.8%; p=0.027). Compared to the other subtypes (as a group), the PAM50 risk of recurrence score (ROR-P) was higher for HER2E tumors (59.1 vs. 41.7; p < 0.001), which were also more frequently HER2+ (36.5% vs. 5.8%) and less likely HR+/HER2- (50.0% vs. 73.6%) and TNBC (13.5% vs. 20.7%), compared to non-HER2E (p < 0.001). No significant differences in grade, TILs, Ki67 and histotype were observed. Overall, 140/776 genes were significantly downregulated in HER2E vs. non-HER2E tumors, including genes related to cell adhesion, migration, DNA damage repair, apoptosis, estrogen signalling pathway, senescence. Conversely, 178/776 genes were significantly upregulated, including the tyrosine-kinase receptor FGFR4, genes involved in nuclear activity, DNA transcription regulation, MAP-kinase signaling pathways, proliferation, cytokine response, epithelial-to-mesenchymal transition (EMT), cell cycle progression, and immune-related genes such as CD274 (PD-L1 gene). DISCUSSION: A switch towards more aggressive IS from primary to advanced disease was observed, with an increase in HER2E prevalence. HER2E tumors, which tend to maintain their IS at the metastatic disease, showed upregulation of genes related to proliferation, survival and EMT, coherently with their well-known worse prognosis. FGFR4 and CD274 upregulation, along with downregulation of genes involved in DNA damage repair suggest these tumors might benefit from targeted therapeutic approaches. Genomic higher risk was not convoyed by consistent clinicopathological features, except for lower PR levels and HER2 overexpression at IHC. Further characterization according to primary/metastatic and HR status is ongoing. Intrinsic subtype distribution across primary and metastatic breast tumors Citation Format: Francisco Javier Muñoz-Carrillo, Laia Paré, Benedetta Conte, Adela Rodríguez, Patricia Galván, Esther Sanfeliu, Blanca González-Farré, Claudette Falato, Isabel Garcia-Fructuoso, Barbara Adamo, Nuria Chic, Olga Martínez-Sáez, Tomás Pascual, Reinaldo Moreno, Montserrat Muñoz, Fara Brasó-Maristany, Aleix Prat, Francesco Schettini, Maria Vidal. Clinico-Pathological and Molecular Characterization of HER2-Enriched Breast Tumors Independently of HER2 Status [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 P2-23-16.

Background: All intrinsic molecular subtypes are represented among HER2-positive breast cancer, with implications on clinical outcome and treatment sensitivity. The impact of molecular subtypes on the pattern and site of relapse is largely unexplored. Methods: 677 patients with HER2-positive early breast cancer from the Shorther trial (n=437), the CherLOB trial (n=84) and two Institutional cohorts (Istituto Oncologico Veneto IRCCS Padova n=39 and Hospital Clinic Barcelona n=117) were included. Only patients with available PAM50 intrinsic molecular subtyping were considered. We analyzed the incidence of distant relapse (at any site and at specific sites) as the first event. Cumulative incidence was estimated according to competing risk analysis (Fine and Gray’s method). Competing risk regression was used to calculate the subdistribution Hazard Ratios (subHR) and their 95% Confidence Interval (CI). Results: The distribution of molecular subtypes was: 130 LumA (19%), 75 LumB (11%), 347 HER2-e (51%), 46 Basal (7%), 79 Normal (12%). Median follow up was 8.4 years (95%CI 8.2-8.6). The 10-yr cumulative incidence rates of distant relapse as first event were: LumA 7.9%, LumB 14.8%, HER2-e 14.7%, Basal 15.5%, Normal 10.4% (HER2-e vs LumA: SubHR 2.21, 95%CI 1.05-4.64, p=0.037). Table 1 shows the 5-yr and 10-yr cumulative incidence rates of distant metastases at specific sites (as first event) according to intrinsic subtype. HER2-e enriched and Basal tumors were more prone as compared to other subtypes to develop brain and lung metastasis as first event, respectively. Isolated brain metastases without extracranial disease occurred only in patients with HER2-e tumors. All brain metastases as first event occurred within 5 years from diagnosis. Bone-only disease as first event was less frequent in HER2-e and Basal subtype (subHR HER2-e vs LumA: 0.32, 95%CI 0.10-10.4. p=0.058). Next, we analyzed the frequency of site-specific first metastasis among patients who experienced a distant metastasis as first event (n=77). Lung metastases were more frequent in Basal tumors (LumA 25.0%, LumB 20.0%, HER2-e 24.4%, Basal 71.4%, Normal 0.0%, p=0.037) and bone metastases were more frequent in Luminal tumors (LumA 100.0%, LumB 60.0%, HER2-e 31.1%, Basal 42.9%, Normal 57.1%, p=0.006). Among 45 HER2-e patients with a first distant relapse, 25.6% were diagnosed with a brain metastasis and 15.6% had brain-only disease. Conclusions: Molecular subtypes influence the metastatic behaviour of clinically HER2-positive breast cancer. These results, if further validated, may have implication in planning personalized monitoring strategies. Table 1. 5-yr and 10-yr cumulative incidence rates of distant metastasis at specific sites (as first event) according to intrinsic subtype. Citation Format: Maria Vittoria Dieci, Giancarlo Bisagni, Stefania Bartolini, Antonio Frassoldati, Daniele Giulio Generali, Federico Piacentini, Gaia Griguolo, Enrico Tagliafico, Fara Brasó-Maristany, Nuria Chic, Francesca Porra, Roberto Vicini, Roberto D’Amico, Sara Balduzzi, Aleix Prat, PierFranco Conte, Valentina Guarneri. Pattern of distant relapse according to intrinsic molecular subtype in patients with HER2-positive breast cancer: a combined analysis of ShortHER, CherLOB, and two institutional cohorts. [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 P2-11-12.

Introduction: The TNBC-DX risk score includes the 14-gene immunoglobulin (IGG) immune signature, tumor size, and nodal status and has shown prognostic value for survival in early-stage TNBC (B. Conte et al., ESMO Breast 2021). However, currently unknown are the value of the TNBC-DX risk score and IGG immune signature in 1) predicting pathologic complete response (pCR) following neoadjuvant therapy, and 2) predicting outcomes the context of neoadjuvant anti-PD1 treatment. Here, we assessed the IGG signature and the TNBC-DX risk score in patients with TNBC treated with neoadjuvant chemoimmunotherapy (NeoPACT; NCT03639948) and neoadjuvant chemotherapy without immunotherapy (NeoSTOP; NCT02413320). Methods: NeoPACT trial enrolled 120 patients with stage I-III TNBC who received carboplatin (AUC 6) + docetaxel (75 mg/m2) + pembrolizumab (200 mg) every 21 days x 6 cycles. NeoSTOP randomized 100 patients with stage I-III TNBC to two chemotherapy regimens; Arm B of NeoSTOP was included in this correlative study as the chemotherapy regimen was identical to NeoPACT. RNA isolated from pretreatment tumor tissue was subjected to next-generation sequencing. The 14-gene IGG immune signature and TNBC-DX risk score were calculated in silico as previously described. Evaluation of stromal tumor-infiltrating lymphocytes (sTILs) was performed as previously described. Markers were tested for prediction of pCR. Logistic regression analysis was used to examine the effect of multiple variables. Event-free survival (EFS) curves were assessed by the Kaplan-Meier method and groups compared by the log-rank test, followed by Cox regression analysis. Results: In this analysis, 112 patients were treated with chemoimmunotherapy on NeoPACT (node-positive = 38%, pCR rate = 58%). In the NeoPACT trial, the 14-gene IGG signature (as a continuous variable) was significantly associated with improved pCR (odds ratio [OR]=1.105, 95% CI 1.019-1.197, P=0.015 for every 0.2 increment). The pCR rates in IGG-high (≥ median) and IGG-low (< median) groups were 71% and 44%, respectively (OR=3.152, 95% CI 1.420-6.996, P=0.005). In terms of EFS, the 14-gene IGG signature was not prognostic (hazard ratio [HR]=0.507, 95% CI 0.148-1.735, p=0.269). In contrast, TNBC-DX risk score was strongly associated with EFS (HR=5.684, 95% CI 1.226-26.356, P=0.012), even when adjusted for sTILs and pCR status (HR=8.415, 95% CI 1.054-67.169, P=0.044). Estimated 3-year EFS rates in TNBC-DX high and low risk groups (above and below median) were 77% and 89%, respectively (P=0.012). In 43 NeoSTOP patients treated with neoadjuvant chemotherapy only (node-positive = 33%, pCR rate = 53%), no association of IGG signature with pCR or TNBC-DX score with EFS was observed. Finally, we observed a moderate correlation between IGG signature and sTILs in both trial datasets combined (r=0.642, P< 0.001). Conclusions: High expression of the 14-gene IGG immune signature in baseline pretreatment tumor samples in early-stage TNBC is significantly associated with pCR following pembrolizumab-based neoadjuvant chemotherapy. The combination of this signature with tumor burden as assessed by TNBC-DX is prognostic for long-term outcomes. Availability of biomarkers that can predict both pathological response and survival with chemoimmunotherapy can optimize this therapy, and evaluation of this biomarker in larger studies is warranted. Citation Format: Priyanka Sharma, Shane R. Stecklein, Rachel Yoder, Joshua M. Staley, Roberto Salgado, Laia Paré, Benedetta Conte, Fara Brasó-Maristany, Anne O’Dea, Lauren Nye, Manana Elia, Deepti Satelli, Gregory Crane, Richard McKittrick, Qamar Khan, Andrew K. Godwin, Aleix Prat. PD11-07 Association of TNBC-DX scores with outcomes in triple-negative breast cancer (TNBC) treated with neoadjuvant pembrolizumab and chemotherapy: a correlative analysis from NeoPACT and NeoSTOP trials [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 PD11-07.

In advanced HER2-positive (HER2+) breast cancer, the new antibody-drug conjugate trastuzumab deruxtecan is more effective compared with trastuzumab emtansine (T-DM1). However, trastuzumab deruxtecan can have considerable toxicities, and the right treatment sequence is unknown. Biomarkers to guide the use of anti-HER2 therapies beyond HER2 status are needed. Here, we evaluated if preestablished levels of ERBB2 mRNA expression according to the HER2DX standardized assay are associated with response and survival following T-DM1. In ERBB2 low, medium, and high groups, the overall response rate was 0%, 29%, and 56%, respectively (P

ImportanceBiomarkers to guide the use of pertuzumab in the treatment of early-stage ERBB2 (formerly HER2)-positive breast cancer beyond simple ERBB2 status are needed.ObjectiveTo determine if use of the HER2DX genomic assay (Reveal Genomics) in pretreatment baseline tissue samples of patients with ERBB2-positive breast cancer is associated with response to neoadjuvant trastuzumab-based chemotherapy with or without pertuzumab.Design, Setting, and ParticipantsThis is a retrospective diagnostic/prognostic analysis of a multicenter academic observational study in Spain performed during 2018 to 2022 (GOM-HGUGM-2018-05). In addition, a combined analysis with 2 previously reported trials of neoadjuvant cohorts with results from the assay (DAPHNe and I-SPY2) was performed. All patients had stage I to III ERBB2-positive breast cancer, signed informed consent, and had available formalin-fixed paraffin-embedded tumor specimens obtained prior to starting therapy.ExposuresPatients received intravenous trastuzumab, 8 mg/kg, loading dose, followed by 6 mg/kg every 3 weeks in combination with intravenous docetaxel, 75 mg/m2, every 3 weeks and intravenous carboplatin area under the curve of 6 every 3 weeks for 6 cycles, or this regimen plus intravenous pertuzumab, 840 mg, loading dose, followed by an intravenous 420-mg dose every 3 weeks for 6 cycles.Main Outcome and MeasuresAssociation of baseline assay-reported pathologic complete response (pCR) score with pCR in the breast and axilla, as well as association of baseline assay-reported pCR score with response to pertuzumab.ResultsThe assay was evaluated in 155 patients with ERBB2-positive breast cancer (mean [range] age, 50.3 [26-78] years). Clinical T1 to T2 and node-positive disease was present in 113 (72.9%) and 99 (63.9%) patients, respectively, and 105 (67.7%) tumors were hormone receptor positive. The overall pCR rate was 57.4% (95% CI, 49.2%-65.2%). The proportion of patients in the assay-reported pCR-low, pCR-medium, and pCR-high groups was 53 (34.2%), 54 (34.8%), and 48 (31.0%), respectively. In the multivariable analysis, the assay-reported pCR score (as a continuous variable from 0-100) showed a statistically significant association with pCR (odds ratio [OR] per 10-unit increase, 1.43; 95% CI, 1.22-1.70; P P P P = .77). A statistically significant interaction between the assay-reported pCR score and the effect of pertuzumab in pCR was observed.Conclusions and RelevanceThis diagnostic/prognostic study demonstrated that the genomic assay predicted pCR following neoadjuvant trastuzumab-based chemotherapy with or without pertuzumab. This assay could guide therapeutic decisions regarding the use of neoadjuvant pertuzumab.

Breast cancer; Heterodimerization Cáncer de mama; Heterodimerización Càncer de mama; Heterodimerització Anti-HER2 therapies have markedly improved prognosis of HER2-positive breast cancer. However, different mechanisms play a role in treatment resistance. Here, we identified AXL overexpression as an essential mechanism of trastuzumab resistance. AXL orchestrates epithelial-to-mesenchymal transition and heterodimerizes with HER2, leading to activation of PI3K/AKT and MAPK pathways in a ligand-independent manner. Genetic depletion and pharmacological inhibition of AXL restored trastuzumab response in vitro and in vivo. AXL inhibitor plus trastuzumab achieved complete regression in trastuzumab-resistant patient-derived xenograft models. Moreover, AXL expression in HER2-positive primary tumors was able to predict prognosis. Data from the PAMELA trial showed a change in AXL expression during neoadjuvant dual HER2 blockade, supporting its role in resistance. Therefore, our study highlights the importance of targeting AXL in combination with anti-HER2 drugs across HER2-amplified breast cancer patients with high AXL expression. Furthermore, it unveils the potential value of AXL as a druggable prognostic biomarker in HER2-positive breast cancer. A.A.-A., E.J.A., and F.B.-M. were supported by Asociación Española contra el Cáncer AECC (PRDVA18013LLUC to A.A.-A., POSTD211413AREN to E.J.A., and AECC_Postdoctoral17-1062 to F.B.-M.). A.M.-S. was funded by the Spanish Government (PFIS FI20/00188). J.Ar. is supported by Breast Cancer Research Foundation (BCRF-20-08), Instituto de Salud Carlos III Project reference number AC15/00062, and the EC under the framework of the ERA-NET TRANSCAN-2 initiative cofinanced by FEDER, Instituto de Salud Carlos III (CB16/12/00449 and PI19/01181), and Asociación Española Contra el Cáncer (AECC). A.P. was supported by Instituto de Salud Carlos III—PI19/01846, Breast Cancer Now—2018NOVPCC1294. P.E. and A.L. were funded by Instituto de Salud Carlos III and cofinanced by FEDER (PI18/01219 to P.E. and CB16/12/00481 to A.L.). J.M.C. was funded by Sociedad Española de Oncología Médica (Rio Hortega-SEOM) and Compromiso ADAMED.

Survival outcomes based on the PAM50MET score in the BOLERO-2 trial. (A) Progression-free survival using a predefined cutoff in patients treated with placebo. (B) Progression-free survival using a predefined cutoff in patients treated with everolimus. (C) Overall survival using a predefined cutoff in patients treated with placebo. (D) Overall survival using a predefined cutoff in patients treated with everolimus. Interaction tests between PAM50 and treatment arm for PFS and OS were statistically non-significant (all p-values >0.05). PAM50MET, final model.

RPPA data analysis

Purpose:FGFR1 amplification (FGFR1amp) is recurrent in metastatic breast cancer (MBC) and is associated with resistance to endocrine therapy and CDK4/6 inhibitors (CDK4/6is). Multi-tyrosine kinase inhibitors (MTKIs) and selective pan-FGFR inhibitors (FGFRis) are being developed for FGFR1amp breast cancer. High-level FGFR amplification and protein expression by IHC have identified breast cancer responders to FGFRis or MTKIs, respectively.Experimental Design:Here, we used preclinical models and patient samples to identify predictive biomarkers to these drugs. We evaluated the antitumor activity of an FGFRi and an MTKI in a collection of 17 breast cancer patient–derived xenografts (PDXs) harboring amplification in FGFR1/2/3/4 and in 10 patients receiving either an FGFRi/MTKI. mRNA levels were measured on FFPE tumor samples using two commercial strategies. Proliferation and angiogenesis were evaluated by detecting Ki-67 and CD31 in viable areas by immunofluorescence.Results:High FGFR1–4 mRNA levels but not copy-number alteration (CNA) is associated with FGFRi response. Treatment with MTKIs showed higher response rates than with FGFRis (86% vs. 53%), regardless of the FGFR1–4 mRNA levels. FGFR-addicted PDXs exhibited an antiproliferative response to either FGFRis or MTKIs, and PDXs exclusively sensitive to MTKI exhibited an additional antiangiogenic response. Consistently, the clinical benefit of MTKIs was not associated with high FGFR1–4 mRNA levels and was observed in patients previously treated with antiangiogenic drugs.Conclusions:Tailored therapy with FGFRis in molecularly selected MBC based on high FGFR1–4 mRNA levels warrants prospective validation in patients with CDK4/6i-resistant luminal breast cancer and in patients with TNBC without targeted therapeutic options.

Purpose:AZD5363/capivasertib is a pan-AKT catalytic inhibitor with promising activity in combination with paclitaxel in triple-negative metastatic breast cancer harboring PI3K/AKT-pathway alterations and in estrogen receptor–positive breast cancer in combination with fulvestrant. Here, we aimed to identify response biomarkers and uncover mechanisms of resistance to AZD5363 and its combination with paclitaxel.Experimental Design:Genetic and proteomic markers were analyzed in 28 HER2-negative patient-derived xenografts (PDXs) and in patient samples, and correlated to AZD5363 sensitivity as single agent and in combination with paclitaxel.Results:Four PDX were derived from patients receiving AZD5363 in the clinic which exhibited concordant treatment response. Mutations in PIK3CA/AKT1 and absence of mTOR complex 1 (mTORC1)-activating alterations, for example, in MTOR or TSC1, were associated with sensitivity to AZD5363 monotherapy. Interestingly, excluding PTEN from the composite biomarker increased its accuracy from 64% to 89%. Moreover, resistant PDXs exhibited low baseline pAKT S473 and residual pS6 S235 upon treatment, suggesting that parallel pathways bypass AKT/S6K1 signaling in these models. We identified two mechanisms of acquired resistance to AZD5363: cyclin D1 overexpression and loss of AKT1 p.E17K.Conclusions:This study provides insight into putative predictive biomarkers of response and acquired resistance to AZD5363 in HER2-negative metastatic breast cancer.

Supplementary Figure Legends

Supplementary Table S2 HTG OBP panel raw data

Low pAkt S473 is associated with resistance to AZD5363 monotherapy

Clinical-pathological and genomic variable (n = 21) used to calculate the PAM50MET model. PAM50MET, final model.

Purpose:Predicting prognosis in HR+/HER2− metastatic breast cancer (MBC) might be clinically useful; however, no validated prognostic biomarkers exist in this setting to date.Patients and Methods:In phase III, EGF30008 trial, 484 patients with HER2− MBC who received letrozole and placebo or lapatinib were selected. PAM50 data, ECOG performance status, visceral disease, number of metastasis, biopsy type, and age were evaluated. A progression-free survival (PFS) Cox model was evaluated. The final model (PAM50MET) with a prespecified cutoff was validated in patients (n = 261) with HR+/HER2− advanced breast cancer (aBC) from BOLERO-2 (phase III trial that evaluated exemestane and placebo or everolimus).Results:In EGF30008, prognostic models with PAM50 plus clinical variables yielded higher C-index values versus models with only PAM50 or clinical variables. The PAM50MET model combined 21 variables: 2 PAM50 subtypes, basal signature, 14 genes, and 4 clinical variables. In EGF30008, the optimized cutoff was associated with PFS [HR = 0.37; 95% confidence interval (CI), 0.29–0.47; P < 0.0001] and overall survival (OS; HR = 0.37; 95% CI, 0.27–0.51; P < 0.0001). The median (months; 95% CI) PFS and OS were 22.24 (19.0–24.9) and not reached in PAM50MET-low versus 9.13 (8.15–11.0) and 33.0 (28.0–40.0) in PAM50MET-high groups, respectively. In BOLERO-2, the PAM50MET-low was associated with better PFS (HR = 0.72; 95% CI, 0.53–0.96; P = 0.028) and OS (HR = 0.51; 95% CI, 0.35–0.69; P < 0.0001). The median (months) (95% CI) PFS and OS were 6.93 (5.57–11.0) and 36.9 (33.4–NA) in PAM50MET-low versus 5.23 (4.2–6.8) and 23.5 (20.2–28.3) in PAM50MET-high groups, respectively.Conclusions:PAM50MET is prognostic in HR+/HER2− MBC, and further evaluation might help identify candidates for endocrine therapy only or novel therapies.

Supplementary Figure Legends

Supplementary Figure 5. Relative expression levels of lucitanib-specific RTK targets and ligands in PDX and patient tumors. A) mRNA expression levels of 2544 genes detected by HTG in Pt325 samples (Pre-tt versus On-tt). Highlighted are some differentially expressed genes between both samples, including FGFR1. Data is log-2 transformed. B) and C) Relative mRNA levels of lucitanib-specific RTK targets or cognate ligands in PDX and patient tumors from the MTKI/FGFRi-treated patient cohort from Figure 3D, respectively.

Supplementary Methods

The use of Cyclin Dependent Kinase 4/6 inhibitors (CDK4/6i) induce transcription in breast tumors. The control mechanism of such genomic changes and their physiological relevance are not elucidated yet. Now, we identified chromatin activation of tumors treated with CDK4/6i. These chromatin changes induce an increase of HER2 expression and signaling. Mechanically, we demonstrate that CDK4/6 binds to FOXA1 and phosphorylates its chromatin binding domain. The loss of FOXA1 phosphorylation allows the binding of this pioneer factor to novel chromatin regions enriched with genes involved in Receptor Tyrosine Kinase signaling. Our study demonstrates that FOXA1 is a new substrate of CDK4 and that its phosphorylation limits the binding of FOXA1 to conventional chromatin regions. Moreover, when patients are treated with CDK4/6i, a dephosphorylated FOXA1 binds to additional chromatin regions. By doing so, FOXA1 leads to an increase of HER2 expression and of the activation of HER2-MEK-ERK pathway in breast cancer patients.

Background: HER3 is overexpressed in 30-50% of breast cancers and has been associated with poor prognosis. Patritumab deruxtecan (HER3-DXd; U3-1402) is a HER3-directed ADC with a potent topoisomerase I (TOP1) inhibitor payload. A phase 1/2 study of HER3-DXd (NCT02980341) demonstrated promising antitumor activity in hormone receptor positive (HR+) metastatic BC patients with clinical activity observed across baseline levels of HER3 protein or mRNA expression. A window of opportunity clinical trial is currently ongoing to evaluate the biological activity of HER3-DXd in patients with treatment naïve BC according to HER3 mRNA/protein expression levels (NCT04610528). Here, we aimed to describe the activity of HER3-DXd in PDX models to identify robust biomarkers of response. Methods: The antitumor activity of HER3-DXd was assessed in 21 BC PDX models (14 HR+ and 7 triple negative). HER3-DXd sensitivity was established as a complete response that lasted longer than 120 days, following 4 weekly doses of 10 mg/kg. HER3-DXd antitumor activity was compared to the antitumor activity of irinotecan (50 mg/kg dosed once weekly), which was evaluated according to modified RECIST criteria in a subset of 14 BC PDX models. PDX models of acquired resistance to HER3-DXd were generated by repeated treatment cycles. Pharmacodynamic (PD) experiments were conducted by collecting tumor samples from PDXs after a single dose of HER3-DXd or irinotecan. Baseline HER3 expression was assessed by immunohistochemistry (IHC) and Western blot. mRNA expression of 72 genes including ERBB3 and genes from the PAM50 signature were measured using the nCounter platform. Two-class unpaired significance analysis of microarrays (SAM), using a false-discovery rate Citation Format: Andreu Òdena, Laia Monserrat, Fara Brasó-Maristany, Marta Guzmán, Judit Grueso, Olga Rodríguez, Maurizio Scaltriti, Sarat Chandarlapaty, Yang Qiu, Kumiko Koyama, Mafalda Oliveira, Aleix Prat, Violeta Serra. Antitumor activity of patritumab deruxtecan (HER3-DXd), a HER3-directed antibody drug conjugate (ADC) across a diverse panel of breast cancer (BC) patient-derived xenografts (PDXs) [abstract]. In: Proceedings of the 2021 San Antonio Breast Cancer Symposium; 2021 Dec 7-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2022;82(4 Suppl):Abstract nr P5-13-14.

Background: HoR+ breast cancer (BC) is an heterogeneous disease, comprising 4 molecular subtypes (i.e.Luminal A [LumA], Luminal B [LumB]), HER2-Enriched [HER2E] and Basal-like [BL]). In HoR+ MBC, non-LumA subtypes appear to be associated with poorer survival outcome compared to LumA. Here, we present a meta-analysis to validate the prognostic value of IS inHoR+ MBC. Methods: We performed a systematic review and trial-level meta-analysis of all available prospective phase II/III trials in HoR+ MBC where the prognostic role of the IS was assessed in terms of progression-free survival (PFS) or time-to-progression (TTP), if the former was not available. Hazard ratios (HR) and 95% confidence intervals (CI) had to be available or computable. Subgroup analyses according to treatment, menopausal and HER2 status were also performed. The random-effect model of Der Simonian and Laird was applied. Heterogeneity was assessed through Cochran’s Q and I2. Funnel plot with Egger’s and Begg’s test for publication bias, multiple sensitivity analyses and risk of bias analysis were also conducted. Revman 5.4 and R 3.6.1 for MacOS X were used for statistical analyses. Tests were two-sided and the threshold for significance was set at p Table 1.Included studies characteristicsStudy ReferenceTrial NameBreast CancerSubgroupMenopausal StatusTrial TypeTreatmentNon-Luminal ALuminal APrat A. et al. The Oncologist 2019BOLERO2HR+/HER2-negPostmenopausalRandomizedEverolimus+exemestane vs exemestane139122Jørgensen C.L.T. et al. Acta Oncologica 2014DBCGMainly HR+/HER2-negMixedRandomizedDocetaxel+gemcitabine vs docetaxel18684Prat A. et al. JAMA Oncol 2016EGF30008HR+/HER2+ and negPostmenopausalRandomizedLetrozole+lapatinib vs letrozole424377Prat A. et al. J Clin Oncol 2021MONALEESA 2HR+/HER2-negPostmenopausalRandomizedRibociclib+letrozole vs letrozole--Prat A. et al. J Clin Oncol 2021MONALEESA 3HR+/HER2-negPostmenopausalRandomizedRibociclib+fulvestrant vs fulvestrant618542Prat A. et al. J Clin Oncol 2021MONALEESA 7HR+/HER2-negPremenopausalRandomizedRibociclib+AI/TAM vs AI/TAM--Ciruelos E. et al. Clin Can Res 2020PATRICIAHR+/HER2+PostmenopausalNon-RandomizedPalbociclib+/-letrozole+trastuzumab3410 Citation Format: Francesco Schettini, Olga Martínez-Sáez, Nuria Chic, Fara Brasó-Maristany, Patricia Galván, Debora Martínez, Laia Paré, Maria Vidal, Barbara Adamo, Montserrat Muñoz, Tomás Pascual, Eva Ciruelos, Charles M Perou, Lisa A Carey, Aleix Prat. Prognostic value of intrinsic subtypes (IS) in hormone receptor-positive (HoR+) metastatic breast cancer (MBC): A systematic review and meta-analysis of prospective trials [abstract]. In: Proceedings of the 2021 San Antonio Breast Cancer Symposium; 2021 Dec 7-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2022;82(4 Suppl):Abstract nr P4-07-08.

Introduction. The incorporation of cyclin-dependent kinase inhibitors 4 and 6 (CDK4/6 inhibitors) with endocrine therapy in patients with hormone receptor-positive/HER2-negative (HR+/HER2-) advanced breast cancer has demonstrated its efficacy improving progression-free survival (PFS), overall response rate (ORR) and, more recently, overall survival (OS). However, patients eventually progress due to resistance to treatment. To date, no clinical or molecular markers defining the HR+/HER2- patient population that obtains the greatest benefit from these drugs have been found, apart from estrogen receptor positivity. However, data from multiple retrospective analysis suggest that within HR+/HER2- disease, the non-luminal intrinsic subtypes (20-30% of these patients) have a worse prognosis and may not benefit equally from every CDK4/6 inhibitor (Finn SABCS 2017, Prat. JCO 2021). Furthermore, the prognostic impact of tumor infiltrating lymphocytes (TILs) and gene expression related to the immune response in the context of HR+/HER2- advanced breast cancer have not been deeply investigated. Design. CDK-PREDICT is an observational, non-interventional, multicenter study that will include 114 patients with advanced breast cancer treated in first-line with CDK4/6 inhibitors + hormone therapy. The primary objective is to correlate the baseline intrinsic subtypes (defined by PAM50) with the efficacy (measured as PFS) of CDK4/6 inhibitors + hormone therapy. As secondary objectives, the correlation of the intrinsic subtypes with ORR and with the histopathological characteristics of the tumor will be analyzed. In addition, the expression of immune response and cell cycle genes, as well as the presence of TILs, will be correlated with the intrinsic subtypes and with PFS and ORR. Formalin-fixed paraffin-embedded (FFPE) tumor tissue samples will be hematoxylin and eosin (H&E) stained and percentage of TILs will be determined. RNA will be purified and analyzed at the nCounter (Nanostring Technologies) using a panel of 72 genes including the PAM50 genes and immune genes such as PD1, PDL1, CD8 or CD4. Overall, we aim to develop a predictive score combining clinical, genomic, and immune expression data integrating tumor biology and microenvironment. For inclusion in the study, a metastatic sample taken within 100 days prior to CDK4/6 inhibitors treatment is required. Once this sample has been collected, registered, and assessed for quality, patients will be followed up every 6 months until disease progression, death or withdrawal from the study. As of July 2021, 80 patients have been included at 5 sites in Spain. This study is included within the Biomarker program of SOLTI. Recruitment of this study started in June 2020. Acknowledgements.This project has received a research grant from “Instituto de Salud Carlos III (ISCIII), Ministerio de Economía y Competitividad” (Spain) awarded within the National Research Program with reference PI 18/01408, co-funded with European Union ERDF funds (European Regional Development Fund). Citation Format: Pablo Tolosa, Tomás Pascual, Cristina Hernando, Sonia Servitja, María Fernández Abad, Fara Brasó-Maristany, Javier Benítez, Laura Lema, Mario Martínez, Yolanda Ruano, Lucía Parrilla, Alejandra Bernardini, Ana María Roncero, Laia Paré, Jordi Canes, Patricia Villagrasa, Fernando Salvador, Aleix Prat, Eva Ciruelos. Solti-1801. Analysis of the efficacy of CDK4/6 inhibitors in combination with hormonal treatment in luminal breast cancer in relation to the intrinsic subtype and markers of immunity (CDK-predict) [abstract]. In: Proceedings of the 2021 San Antonio Breast Cancer Symposium; 2021 Dec 7-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2022;82(4 Suppl):Abstract nr OT2-19-03.

Background: Overexpression of human epidermal growth factor receptor 2 (HER2) occurs in 15-20% of breast cancers (BC). At the same time, HER2-low tumors (immunohistochemistry 1+ or 2+ with no gene amplification) comprise ~50% of all BC. Trastuzumab Deruxtecan (T-DXd) is an anti-HER2 antibody drug conjugate that has shown impressive and durable response rates not only in HER2+ and HER2low BC, but also in other cancer subtypes expressing the HER2 receptor. However, not all patients respond or benefit to the same extent. Thus, there is a need to identify predictive biomarkers. Here, we hypothesize that by performing several molecular studies in both tissue and plasma samples of those patients participating in the DESTINY studies receiving T-DXd, we will shed more light on the molecular features of HER2 expressing BC and will better characterize the patient population that benefits benefit from this promising new anti-HER2 agent. Methods: HER2-PREDICT is a multi-center, observational study within the biomarker program of SOLTI group, which will include patients who received T-DXd-only while participating in DESTINY trials. Both HER2-positive and HER2-low unresectable and/or metastatic breast cancer may be included in SOLTI-1804 HER2-PREDICT study (NCT04257162). All patients need to consent to obtain a fresh tumor biopsy or donate an archival metastatic biopsy. Primary tumors may be allowed under SOLTI acceptance. Additionally, for patients included before initiating T-DXd treatment blood samples for biomarker analyses on Cycle 1 Day 1 (C1D1), C2D1 and end of treatment will be obtained. Collection of tumor biopsies is an essential part of this study. Pathological analyses will include hematoxylin and eosin (H&E) staining of formalin-fixed paraffin-embedded (FFPE) tissue, identification of areas with the greater amount of tumor cells and determination of their tumor cell percentage. RNA will be isolated and analyzed using the nCounter platform (Nanostring Technologies). Molecular intrinsic subtypes will be identified by a research-based version of PAM50. Furthermore, we aim to evaluate 771 additional genes (+5 housekeeping genes) that encompass genomic signatures and individual genes of importance in breast cancer by means of the nCounter®Breast Cancer 360 Panel. Somatic mutations in PIK3CA, TP53, and additional genes (e.g., GATA3 and ERBB2) will be identified using next-generation sequencing (NGS) in FFPE tumor blocks and plasma samples. Objectives: The primary objectives are (1) to identify an optimal ERBB2 mRNA cut-off point predictive of T-DXd response and (2) to evaluate the correlation of baseline ERBB2 mRNA levels (as a continuous variable) with overall response rate (ORR). Secondary objectives include: to evaluate the association of ERBB2 mRNA levels, PAM50 intrinsic subtypes and immune-related genes with ORR, progression-free survival (PFS) and overall survival (OS); to design a new gene expression signature predictive of clinical benefit; to evaluate the correlation in early changes in circulating tumor DNA (ctDNA) with ORR, PFS and OS; and to identify acquired somatic mutations of resistance upon progression in plasma samples. The study is active in 13 sites in Spain, and 39 patients have been included in the trial since December 2019 until June 2021. Acknowledgments: We thank Daiichi Sankyo Inc. for their financial support for the study. Citation Format: Aleix Prat, Cristina Saura, Josefina Cruz, Esteban Nogales Fernández, Javier Salvador Bofill, Joaquin Gavilá, Maria José Bermejo-Perez, Vanesa Quiroga, Serafin Morales, Sonia Servitja, Ruben de Toro, Pilar Zamora, Patricia Galván, Nuria Chic, Débora Martínez, Fara Brasó-Maristany, Jordi Canes, Laia Paré, Juan M Ferrero-Cafiero, Tomás Pascual, Sonia Pernas. Solti-1804 HER2-PREDICT: Translational study of tumor samples from breast cancer patients treated with trastuzumab deruxtecan in the metastatic setting [abstract]. In: Proceedings of the 2021 San Antonio Breast Cancer Symposium; 2021 Dec 7-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2022;82(4 Suppl):Abstract nr OT1-12-01.

Purpose:FGFR1 amplification (FGFR1amp) is recurrent in metastatic breast cancer (MBC) and is associated with resistance to endocrine therapy and CDK4/6 inhibitors (CDK4/6is). Multi-tyrosine kinase inhibitors (MTKIs) and selective pan-FGFR inhibitors (FGFRis) are being developed for FGFR1amp breast cancer. High-level FGFR amplification and protein expression by IHC have identified breast cancer responders to FGFRis or MTKIs, respectively.Experimental Design:Here, we used preclinical models and patient samples to identify predictive biomarkers to these drugs. We evaluated the antitumor activity of an FGFRi and an MTKI in a collection of 17 breast cancer patient–derived xenografts (PDXs) harboring amplification in FGFR1/2/3/4 and in 10 patients receiving either an FGFRi/MTKI. mRNA levels were measured on FFPE tumor samples using two commercial strategies. Proliferation and angiogenesis were evaluated by detecting Ki-67 and CD31 in viable areas by immunofluorescence.Results:High FGFR1–4 mRNA levels but not copy-number alteration (CNA) is associated with FGFRi response. Treatment with MTKIs showed higher response rates than with FGFRis (86% vs. 53%), regardless of the FGFR1–4 mRNA levels. FGFR-addicted PDXs exhibited an antiproliferative response to either FGFRis or MTKIs, and PDXs exclusively sensitive to MTKI exhibited an additional antiangiogenic response. Consistently, the clinical benefit of MTKIs was not associated with high FGFR1–4 mRNA levels and was observed in patients previously treated with antiangiogenic drugs.Conclusions:Tailored therapy with FGFRis in molecularly selected MBC based on high FGFR1–4 mRNA levels warrants prospective validation in patients with CDK4/6i-resistant luminal breast cancer and in patients with TNBC without targeted therapeutic options.