HER2-enriched Research Articles

Abstract HER2-06: HER2-06 Outcome analysis of HER2-zero or HER2-low hormone receptor-positive (HR+) breast cancer patients - characterization of the molecular phenotype in combination with molecular subtyping

Abstract Background: Breast cancer with low HER2 expression (HER2-low) is of high clinical relevance because of new therapeutic options with antibody-drug conjugates. We have recently shown in a large cohort from neoadjuvant clinical trials that HER2-low breast cancer has different molecular characteristics as well as different clinical outcomes compared to HER2-zero. Considering the positive correlation between HER2-low expression and hormone receptor positivity observed consistently in many investigations, we have extended our analysis to HR+ tumors from the post-neoadjuvant PenelopeB trial. In PenelopeB, patients with HR+ breast cancer and residual disease after neoadjuvant chemotherapy (NACT) were randomized to post-neoadjuvant palbociclib versus placebo in addition to endocrine therapy. We evaluated the molecular phenotype and clinical outcomes of HER2-low compared to HER2-zero patients. Methods: A total of 1250 patients were randomized, HER2 status was available for 1151 tumors from pretherapeutic core biopsy, determined mainly by local pathology, and from 1213 tumors from the post-NACT sample, determined as part of central pathology. For 1119 patients a paired HER2-status was both available. HER2-zero was defined as IHC0 and HER2-low-positive was defined as IHC1+ or IHC2+/ISH-. Gene expression analysis of 2549 genes using the HTG oncology biomarker panel was performed in 620 pretherapeutic biopsies and 780 post-NACT residual tumor samples, with 539 paired gene expression samples. Breast cancer subtypes were determined using the AIMS approach. Results: In pretherapeutic biopsies, 695 tumors (60%) were HER2-low and 457 (40%) were HER2-zero. A HER2-low status in the biopsy was significantly linked to improved iDFS (HR 0.76 (0.60-0.96; p=0.02). In residual tumors, 632 tumors (60%) were HER2-low and 581 (40%) were HER2-zero, without any prognostic impact of HER2 low status. In addition, a shift of HER2-low-status comparing core biopsy and residual tumor was observed in 415 (37%) of 1119 tumors. 161 (14%) had a shift from HER2-zero to HER2-low and 254 (23%) shifted from HER2-low to HER2-zero. A shift from HER2-zero to HER2-low in the post-NACT samples was significantly linked to reduced iDFS (HR 1.43 [95%CI 1.01-2.01]), p=0.04), compared to HER2-low group, while a shift from HER2-low to HER2-zero was associated with better iDFS compared to HER2-zero group, although not statistically significant (p=0.17). We did not observe a significant correlation of HER2-low status and AIMS molecular subtypes. In particular, the HER2-enriched (HER2E) subtype was assigned to only 4.3% of HER2-zero and 3.1% of HER2-low tumors. Significant iDFS differences were observed for HER2-low-status in combination with AIMS subtypes (lumB/basal/HER2E vs. lumA/normL; overall p-value < 0.0001) for both pretherapeutic biopsies and residual tumor. Patients with post-NACT HER2-low tumors had an improved survival in the subgroups of aggressive AIMS subtypes (lumB/basal/HER2E), but not in the less aggressive AIMs subtypes (lumA/normL), with a positive test for interaction (p=0.02). For the pre-NACT samples a similar, but non-significant trend was observed. We evaluated a total of 620 core biopsies for differences in gene expression comparing HER2-low and HER2-zero tumors. A total of 417 genes were statistically significantly different, but in a hierarchical clustering there was no clear separation of HER2-low and HER2-zero tumors. Conclusions: In the PenelopeB cohort of HR+ tumors, a HER2-low status in pretherapeutic core biopsies is related to improved disease-free survival, especially for those tumors that have a more aggressive intrinsic subtype. A shift of HER2-low status was observed before and after chemotherapy, indicating an adaptation of the pathway activity to therapy-induced stress, which might become relevant for future diagnostic and therapeutic approaches. Citation Format: Carsten Denkert, Miguel Martín, Michael Untch, Hervé R. Bonnefoi, Erik S. Knudsen, Seock-Ah Im, Angela DeMichele, Agnieszka Witkiewicz, Laura Van ’t Veer, Sung-Bae Kim, Harry D. Bear, Nicole McCarthy, Karen Gelmon, Frederik Marmé, José Ángel García-Sáenz, Nicholas Turner, Federico Rojo, Martin Filipits, Lesley-Ann Martin, Peter A. Fasching, Christian Schem, Catherine M. Kelly, Toralf Reimer, Masakazu Toi, Hope Rugo, Michael Gnant, Andreas Makris, Yuan Liu, Karsten Weber, Sivaramakrishna Rachakonda, Sibylle Loibl. HER2-06 Outcome analysis of HER2-zero or HER2-low hormone receptor-positive (HR+) breast cancer patients - characterization of the molecular phenotype in combination with molecular subtyping [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 HER2-06.

Abstract PD7-08: Gene Expression Profiling of Breast Cancer Brain Metastasis shows enrichment for non-luminal subtypes with potential prognostic implications

Abstract Background: The incidence of breast cancer (BC) brain metastases (BM) is increasing as a result of both improved diagnostic techniques and longer survival due to better treatment approaches. However, the biological complexity of BCBMs is still poorly understood. We here evaluate the genomic profile of BCBMs and assess its prognostic implications. Methods: Clinical data and BM samples (FFPE) from BC patients undergoing neurosurgery (2003-2019) at three institutions were collected. Hormone receptor (HR) and HER2 status were evaluated on the BCBM. RNA extracted from BM samples was used to measure the expression of 758 BC–related genes and 18 housekeeping genes using the Breast Cancer 360 Panel on an nCounter platform (NanoString Technologies). Intrinsic molecular subtyping was determined using the previously reported PAM50 subtype predictor (Parker et al. JCO 2009). Median overall survival from neurosurgery (OS) was calculated using the Kaplan-Meier method. The correlations between expression of each gene/PAM50 signature, BC subtype and OS were studied using univariate and multivariate Cox-models. Results: Sixty-five BCBM samples were analyzed: 32% (N=21) were HR+/HER2-, 38% (N=25) HER2+ and 29% (N=19) HR-/HER2-. With a median follow-up of 33 months, no clinical variable was significantly associated with OS, despite a trend towards a shorter survival for patients with HR-/HER2- BMs, as compared to patients with HR+/HER2- and HER2+ subtypes (median OS 9.4 versus 22.1 and 20.0 months, respectively, log-rank p=NS). The intrinsic subtype distribution, as assessed by gene-expression profiling, was 37% Basal-like, 46% HER2-enriched (HER2-E), 15% Luminal B and 2% Normal-like. Non-luminal subtypes (basal-like and HER2-E) were extensively represented, both overall and in each BC subtype (52% in HR+/HER2- subgroup, 96% in HER2+ subgroup, see Table). The PAM50 basal-like signature was significantly associated with a worse OS (HR 2.7, 95% CI 1.0-7.2, p=0.045), even after correcting for BC subtype (HR 5.2, 95% CI 1.1-23.4, p=0.032). In fact, even within the subgroup of HR+/HER2- BCBMs, the PAM50 basal-like signature was strongly associated with a worse OS (HR 92.6, 95% CI 5.0-1860.1, p=0.003) and patients with basal-like HR+/HER2- BCBMs presented a median OS similar to patients with HR-/HER2- BCBMs (mOS 9.0 vs 9.4 months). We identified 36 genes whose high expression was significantly associated with a worse OS (p< 0.05) and one gene (LINC02381) whose high expression was significantly associated with better OS (p< 0.05); for 33 of these genes (BCL11A, BMP2, BNIP3, CAV1, CDH3, CDK6, CKB, CRYAB, CXCL12, EGFR, EYA4, FOXC1, FZD8, FZD9, GABRP, GAS1, GDF5, GPC4, IL6, KRT17, KRT5, KRT6B, KRT7, LAMB3, LINC02381, MYC, NOTCH1, PRKX, PSAT1, RUNX3, SNAI1, SPRY2, TTYH1), the association was confirmed even after correcting for BC subtype (p< 0.05). Conclusions: Non-luminal intrinsic subtypes are extensively represented in resected BCBMs, even if clinically classified as HR+/HER2-. Our data suggest that basal-like genomic features might be enriched in BCBMs and might be associated with worse survival. Distribution of PAM50 intrisic subtyping on the 65 brain metastases evaluated according to hormone receptor (HR) and HER2 status Citation Format: Gaia Griguolo, Maria Vittoria Dieci, Susan Fineberg, claudia Pinato, Michele Bottosso, Luc Bauchet, Federica Miglietta, Jack Jacob, Giovanni Zarrilli, Valérie Rigau, Maria Cristina Guarascio, Francesca Zanconato, Francesca Schiavi, Matteo Fassan, William Jacot, Stefano Piccolo, Amelie Darlix, Valentina Guarneri. Gene Expression Profiling of Breast Cancer Brain Metastasis shows enrichment for non-luminal subtypes with potential prognostic implications [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 PD7-08.

Abstract P2-13-03: KEYRICHED-1 - A prospective, multicenter, open label, neoadjuvant phase II single arm study with pembrolizumab in combination with dual anti-HER2 blockade with trastuzumab and pertuzumab in early breast cancer patients with molecular HER2-enriched intrinsic subtype

Abstract Background: De-escalating strategies seem promising inHER2-positive early breast cancer (EBC) and chemo-free regimens are thus of keyinterest. Recent data have underlined the role of tumor immunogenicity inresponse to de-escalated neoadjuvant anti-HER2 therapy. Therefore, theprospective single arm hypothesis-generating phase II KEYRICHED-1 trial (NCT03988036)investigates the pCR rate in patients with HER2-enriched EBC receiving fourcycles of the dual anti-HER2 blockade in combination with the checkpointinhibitor pembrolizumab. Initial studies with dual antibody-based HER2 blockadealone were able to achieve pCR-rates of 20-40%, which did not quite match the pCRrates obtained with concurrent chemotherapy. KEYRICHED-1 aims at achieving pCR-ratescomparable to standard chemotherapy-containing regimens by incorporating appropriatemolecular selection and immune oncology. Methods: A total of 48 pre- and postmenopausal patients with newly diagnosed HER22+ or 3+ EBC (stage I-III) and HER2-enriched (HER2-E) subtype by PAM50 wereenrolled in this single-arm study. All patients received four cycles of studytreatment with pembrolizumab (200mg), trastuzumab biosimilar (Trazimera®,loading dose 8mg/kg bodyweight (BW), maintenance dose 6mg/kg BW), and pertuzumab(loading dose 840mg/kg BW, maintenance dose 420mg/kg BW) q21d . Primaryendpoint was centrally confirmed pCR (ypT0/is, ypN0). The trial was planned asa Simon's two-Stage design (null and alternative pCR were 40% and 60%); interimanalysis after 16 patients had to show a pCR rate of at least 50% to continuerecruitment. Results: Between 05/2020 and 03/2021, 98 patients werescreened. N=52 (55%) had HER2-E subtype,of whom 48 patients entered thetreatment phase. Median patient age was 57 years (28-83). 65% had tumors > 2cm and 30% positive lymph node status. Centrally confirmed pCR rate in surgicalspecimens was 46% (95% CI 0.31-0.62) in the 43 patients of the per protocolpopulation, and 52% (95%CI 0.37-0.67) in all 46 evaluable patients (localassessment; two pCRs verified only by core biopsy) (p=0.22 and p=0.06 for nullhypothesis, respectively). Despite HER2-E subtype, no pCR was observed in the 4patients with immunohistochemical (IHC) HER2 2+/ISH-positive status in contrastto 20/39 (51.2%) pCRs in IHC HER2 3+ tumors. Centrally confirmed pCR rate in HR+/HER2+tumors was38.5% compared to 58.5% in HR-/HER2+ tumors. No new safety signals wereobserved. Conclusions: These are the first results of a neoadjuvant chemotherapy-free12-week de-escalation anti-HER2-regimen with trastuzumab and pertuzumab incombination with the PD-1 inhibitor pembrolizumab in patients with a HER2-E EBC.In the context of the WSG ADAPT HER2+ de-escalation trials the observed pCRrates compare favourably in HR+ as well as HR- HER2 EBC. Moreover, KEYRICHED-1demonstrates that with appropriate molecular patient selection clinicallymeaningful pCR rates in the range of those obtained with longer, more toxicchemotherapy-containing regimens can be achieved. Citation Format: Sherko Kuemmel, Oleg Gluz, Mattea Reinisch, Athina Kostara, Iris Scheffen, Monika Graeser, Kerstin Luedtke-Heckenkamp, Andreas Hartkopf, Felix Hilpert, Angela Kentsch, Carsten Ziske, Reinhard Depenbusch, Michael Braun, Jens Blohmer, Christine zu Eulenburg, Matthias Christgen, Stephan Bartels, Hans Kreipe, Enrico Pelz, Peter Schmid, Nadia Harbeck. KEYRICHED-1 - A prospective, multicenter, open label, neoadjuvant phase II single arm study with pembrolizumab in combination with dual anti-HER2 blockade with trastuzumab and pertuzumab in early breast cancer patients with molecular HER2-enriched intrinsic subtype [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 P2-13-03.

Abstract P3-05-02: A minimal DNA-methylation signature to estimate tumor content and molecular subtype in breast cancer tissue samples with potential application to liquid biopsy

Abstract Introduction: Breast cancer (BC) is a heterogeneous disease characterized by different molecular "intrinsic" subtypes (PAM50) with distinct prognostic and therapeutic implications. DNA methylation (DNAm) is one of the most studied epigenetic mechanisms involved in BC tumorigenesis and progression. Recently, many studies have demonstrated the capability of DNAm-based assays in detecting and monitoring the disease in liquid biopsy samples from patients with BC, thus confirming promising role of DNAm-based biomarkers in the context of precision oncology. Here we present a novel computational method that exploits a minimal set of informative, clonal DNAm sites to estimate tumor content and molecular subtype in BC tissue samples with potential application for liquid biopsy analyses. Methods: DNAm data of TCGA-BRCA comprising 737 tumor samples, including 135 Basal-like (Basal), 46 HER2-enriched (HER2e) and 556 Luminal A/B (LumA/B) samples, and 96 normal tissue samples were downloaded from GDC Data Portal. To estimate tumor purity, we selected DNAm sites based on the following characteristics: (1) AUC>0.8 or AUC<0.2 (hyper- and hypo-methylated sites, respectively) in tumor versus normal samples; (2) range of beta-values in tumor samples greater than 0.5; (3) max and min beta-values above 0.9 or below 0.1, for hyper- and hypo-methylated sites, respectively; (4) the 3rd and 1st quartiles of normal samples beta-values below 0.3 or above 0.7, for hyper- and hypo-methylated sites, respectively. Tumor purity was then estimated taking the median of beta-values for hyper and 1-beta for hypo of selected sites for each sample. This selection was carried out considering all TCGA-BRCA samples and by PAM50 subtype (Basal, HER2e and LumA/B). After correction for purity, the set of subtype-specific sites (n=89) was also used as input to a generalized multinomial regression model (GLMNET) to classify BC samples into molecular subtypes. The model was trained in the TCGA-BRCA dataset using a 5-fold cross-validation. The performance was then tested in two independent datasets retrieved through the recountmethylation R package: GSE72251 (n=117) and GSE84207 (n=254). Results: Our estimates of sample purities, exploiting only 20 DNAm sites, show high correlation with those from InfiniumPurify (R=0.90, p < 10-16), another DNAm-based tool that requires hundreds of sites for purity estimation. The correlation was further improved using subtype-specific sites (LumA/B: R=0.95, p < 10-16; HER2e: R=0.96, p < 10-16; Basal: R=0.93, p < 10-16). For classification of tumor samples into molecular subtypes, our model obtained high accuracy for all subtypes (99%, 84% and 99%, for Basal, HER2e and LumA/B, respectively) in the training dataset. In the test datasets the F1-scores for Basal, HER2e and LumA/B subtypes were 89%, 60% and 87% for GSE72251, 91%, 42% and 93% for GSE84207. Conclusions: Our computational framework exploiting a minimal signature of DNAm sites demonstrates high accuracy in quantifying tumor purity and predicting molecular subtypes in the training dataset. In the two independent test sets including >350 samples, high accuracy was obtained for LumA/B and Basal samples while HER2e samples showed lower F1-score due to a drop of recall, though obtaining precision >80% in both datasets. Based on these results, a DNAm-based targeted-seq assay has been designed and will be applied to a series of plasma samples from patients with metastatic BC collected within our center. First results of the assay will be presented during the meeting. Citation Format: Dario Romagnoli, Francesca Galardi, Francesca De Luca, Chiara Biagioni, Erica Moretti, Laura Biganzoli, Ilenia Migliaccio, Luca Malorni, Matteo Benelli. A minimal DNA-methylation signature to estimate tumor content and molecular subtype in breast cancer tissue samples with potential application to liquid biopsy [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 P3-05-02.

Abstract PD10-11: Keyriched-1- A prospective, multicenter, open label, neoadjuvant phase ii single arm study with pembrolizumab in combination with dual anti-HER2 blockade with trastuzumab and pertuzumab in early breast cancer patients with molecular HER2-enriched intrinsic subtype

Abstract Background: De-escalating strategies seem promising in HER2-positive early breast cancer (EBC) and chemo-free regimens are thus of key interest. Recent data have underlined the role of tumor immunogenicity in response to de-escalated neoadjuvant anti-HER2 therapy. Therefore, the prospective single arm hypothesis-generating phase II KEYRICHED-1 trial (NCT03988036) investigates the pCR-rate in patients with HER2-enriched EBC receiving four cycles of the dual anti-HER2 blockade in combination with the checkpoint inhibitor pembrolizumab. Initial studies with dual antibody-based HER2 blockade alone were able to achieve pCR-rates of 20-40%, which did not quite match the pCR-rates obtained with concurrent chemotherapy. KEYRICHED-1 aims at achieving pCR-rates comparable to standard chemotherapy-containing regimens by incorporating appropriate molecular selection and immune oncology.. Methods: A total of 48 pre- and postmenopausal patients with newly diagnosed HER2 2+ or 3+ EBC (stage I-III) and HER2-enriched (HER2-E) subtype by PAM50 were enrolled in this single-arm study. All patients received four cycles of study treatment with pembrolizumab (200mg), trastuzumab biosimilar (Trazimera®, loading dose 8mg/kg bodyweight (BW), maintenance dose 6mg/kg BW), and pertuzumab (loading dose 840mg/kg BW, maintenance dose 420mg/kg BW) q21d. Primary endpoint was centrally confirmed pCR (ypT0/is, ypN0). The trial was planned as a Simon's two-stage design (null and alternative pCR were 40% and 60%); interim analysis after 16 patients had to show a pCR rate of at least 50% to continue recruitment.. Results: Between 05/2020 and 03/2021, 98 patients were screened. N=52 (55%) had HER2-E subtype, of whom 48 patients entered the treatment phase. Median patient age was 57 years (28-83). 65% had tumors > 2 cm and 30% positive lymph node status. Centrally confirmed pCR-rate in surgical specimens was 46% (95% CI 0.31-0.62) in the 43 patients of the per-protocol-population, and 52% (95%CI 0.37-0.67) in all 46 evaluable patients (local assessment; two pCRs verified only by core biopsy) (p=0.22 and p=0.06 for null hypothesis, respectively). Despite HER2-E subtype, no pCR was observed in the four patients with immunohistochemical (IHC) HER2 2+/ISH-positive status in contrast to 20/39 (51.2%) pCR in IHC HER2 3+ tumors. Centrally confirmed pCR-rate in HR+/HER2+ tumors was 38.5% compared to 58.5% in HR-/HER2+ tumors. No new safety signals were observed.. Conclusions: These are the first results of a neoadjuvant chemotherapy-free 12-week de-escalation anti-HER2-regimen with trastuzumab and pertuzumab in combination with the PD-1 inhibitor pembrolizumab in patients with a HER2-E EBC. In the context of the WSG ADAPT HER2+ de-escalation trials the observed pCR-rates compare favorably in HR+ as well as HR- HER2+ EBC. Moreover, KEYRICHED-1 demonstrates that with appropriate molecular patient selection clinically meaningful pCR-rates in the range of those obtained with longer, more toxic chemotherapy-containing regimens can be achieved. Citation Format: Sherko Kuemmel, Oleg Gluz, Mattea Reinisch, Athina Kostara, Iris Scheffen, Monika Graeser, Rachel Wuerstlein, Ulrike Nitz, Kerstin Luedtke-Heckenkamp, Andreas Hartkopf, Felix Hilpert, Angela Kentsch, Carsten Ziske, Reinhard Depenbusch, Michael Braun, Jens Blohmer, Christine zu Eulenburg, Matthias Christgen, Stephan Bartels, Hans Kreipe, Enrico Pelz, Peter Schmid, Nadia Harbeck. Keyriched-1- A prospective, multicenter, open label, neoadjuvant phase ii single arm study with pembrolizumab in combination with dual anti-HER2 blockade with trastuzumab and pertuzumab in early breast cancer patients with molecular HER2-enriched intrinsic subtype [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 PD10-11.

Abstract PD15-02: HER2-enriched subtype and novel molecular subgroups drive aromatase inhibitor resistance and an increased risk of relapse in early ER+/HER2+ breast cancer

Abstract Background: Approximately 10% of estrogen receptor positive (ER+) early breast cancers (EBC) are human epidermal growth factor positive/overexpressed (HER2+). Resistance to aromatase inhibitors (AIs) is poorly understood among ER+HER2+ BC. We aimed to identify biomarkers of response and resistance using the HER2+ tumours in the POETIC trial. Methods: POETIC was a phase III trial of post-menopausal women with ER+ EBC randomized 2:1 to 2-weeks of peri-surgical AI vs control, followed by standard-of-care. Baseline formalin-fixed paraffin-embedded samples from 342 ER+HER2+ BC (237 treated/105 controls) were gene expression profiled using Breast cancer 360™ panel (NanoString) covering intrinsic subtypes and 40 key biological signatures. New molecular subgroups were identified using consensus clustering (CC).Proliferation rate was estimated as percentage of cancer-cells staining for Ki67. Ki67 response was defined by percentage-changes from baseline to surgery: poor (PR; reduction <50%), intermediate (IR; 50-75%) and good (GR; >75%). Residual Ki67 at 2 weeks timepoint (Ki672w) was defined as High (H) (≥ 10%) vs Low (L) (<10%). Association of molecular features with Ki67 response was assessed using logistic regression models and with time to recurrence (TTR) using multivariable Cox regression models adjusted for post-surgery clinicopathological variables: grade, tumour size, nodal status and age. Results: Ki672w was measured for all treated samples (n = 227, 48% H, 52% L) and a fraction of controls (n = 50, 96% H, 4% L). At baseline, 45% of tumours were HER2-enriched (HER2-E), 53% Luminal A/B and 2% Basal-Like. HER2-E associated with high Ki672W (84% H, 16% L) compared to non-HER2-E (29% H, 71% L, p<0.001). Amongst the 40 BC360 signatures, 9 were significantly associated with Ki67 response (Table). High expression of endocrine-related and apoptosis signatures associated with GR while HRD and TP53 with PR. These features were also related to Ki672W, with additional signatures involved in immune-checkpoint component and immune-enrichment (IM) (IDO1, PD-L1, IFN-gamma and T-reg) associated with high Ki672W.. Using CC, 4 molecular subgroups predicting differential response to AI were identified. Cluster (C) 1 was characterized by overexpression of IM features and low ER signaling (42%); C2 low IM but highest ERBB2 expression (15%); C3 ESR1 high and PgR low (4%) and C4 high endocrine signaling and lowest ERBB2 expression (39%). Distribution of Intrinsic subtypes varied across these molecular subgroups with the majority of C1 being HER2-E (62%) and Luminal B (27%); C2 HER2-E (74%); C3 Luminal B (64%) and C4 Luminal B (55%) and Luminal A (30%). HER2-E had significantly poorer TTR (HR 2.14; 95%CI1.11-4.17; p=0.0224) compared to Luminal tumours, remaining significant (HR 2.55, 95%CI1.14-5.69; p=0.0222) in the multivariable analysis. C2 (HR 4.67, 95%CI 1.90-11.51; p<0.001) and C4 (HR 2.42, 95%CI1.05-4.5.58; p=0.0385) were independent predictors of shorter TTR compared to C1, adding significant value beyond intrinsic subtypes (Likelihood ratio test, p=0.00429). Conclusions: Our results confirm that HER2-E, TP53 mutation, HRD and IM features are the main components driving poor early response to AI in ER+/HER2+ EBC. Novel molecular subgroups identify additional non-HER-E tumours not responding to AI with an increased risk of relapse. The appropriate additional treatment warrants further investigation. List of significant gene expression signatures associated with response as defined by reduction of Ki67. Odds ratio (OR) were determined by ordinal logistic regression analyses. OR: <1 indicating associated with good response and > 1.0 associated with worse response.Odds Ratiop-valuep-adjusted (False Discovery Rate)ESR10.65< 0.001< 0.001Estrogen Receptor Signaling0.44< 0.001< 0.001ERBB21.52< 0.001< 0.001TP53 mutant signature2.65< 0.001< 0.001FOXA10.42< 0.001< 0.001Apoptosis0.26< 0.0010.00228Homologous Recombination Deficiency (HRD)2.33< 0.0010.00349PgR0.820.001480.00737Hypoxia2.180.001660.00737 Citation Format: Milana A Bergamino Sirvén, Elena López-Knowles, Gabriele Morani, Holly Tovey, Lucy Kilburn, Chris Holcombe, Anthony Skene, Ian Smith, John Robertson, Gene Schuster, Judith M Bliss, Mitch Dowsett, Maggie CU Cheang, POETIC investigators. HER2-enriched subtype and novel molecular subgroups drive aromatase inhibitor resistance and an increased risk of relapse in early ER+/HER2+ breast cancer [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 PD15-02.

Abstract OT2-27-01: Solti-1718 NEREA Trial: Neratinib in hormone receptor (HR)-positive/HER2-negative HER2-enriched (HER2-E) advanced breast cancer (BC)

Abstract Background HR+/HER2-negative BC represents ∼70% of all newly diagnosed breast tumors. BC is a clinically and biologically heterogeneous disease where intrinsic subtypes play a role. Non-luminal subtypes within HR+/HER2-negative disease do not benefit to the same extent from the standard of care treatments as the luminal subtypes. Thus, other strategies are needed. HER2-E subtype represents approximately 6.6-11.0% of HR+/HER2-negative tumors and is enriched in twice as many cases in metastatic tumors. According to EGF30008 trial, HER2-E advanced BC patients despite presenting poor outcomes across treatments showed benefit from anti-HER2 therapy. SOLTI-1718 NEREA aims to evaluate whether EGFR/ERBB2 axis inhibition by neratinib improves efficacy in terms of progression-free survival (PFS) in patients with advanced HR+/HER2-negative disease resistant to endocrine treatment (ET). Methods SOLTI-1718 NEREA (NCT04460430) is an open-label, single-arm, multicenter, and multinational phase II clinical trial following a Simon’s 2-stage design with one interim and one final efficacy analysis. Locally advanced or metastatic HER2-E (determined at metastatic sample), HR+/HER2-negative BC patients who had recurrence or progression while receiving previous ET (with or without a CDK4/6 inhibitor) will be included. ≤ 1 prior line of chemotherapy will be allowed. The primary objective is to assess the efficacy of neratinib in combination with ET in HER2-E, HR+/HER2-negative patients in terms of PFS6 by local assessment by the investigator using RECIST v.1.1. Secondary endpoints include clinical benefit rate at 6 months, overall response rate, duration of response, time to response and incidence, duration, and severity of adverse events. The treatment schedule will consist of neratinib 240 mg daily in combination with ET, with either exemestane, fulvestrant, or tamoxifen (as per the investigator´s decision). All patients will take prophylactic loperamide with an established dosing scheme during the first cycle and on-demand in subsequent cycles. Tumor assessments will be performed at baseline and every 8 weeks during the first year, and every 12 weeks thereafter. An interim analysis will be performed after 33 patients are evaluable. If 15 to 27 patients achieve a PFS at 6 months (PFS6), the trial will continue to the second stage, otherwise, it will be stopped for futility (<15) or efficacy (≥28). A total of 56 evaluable patients will be included in stages I and II. The Spanish regulatory national competent authority approved the study on April 8th 2020. Recruitment started in July 2020. As of June 2021. Seven patients have been enrolled in 15 sites in Spain and 3 sites in Portugal. Acknowledgments: We thank PUMA BIOTECHNOLOGY, INC for their provision of Neratinib and financial contribution to the study. Citation Format: Eva Ciruelos, Cristina Saura, Xavier González-Farré, Javier Salvador Bofill, Maria Vidal, Isabel Blancas, Elena López-Miranda, Maria Iglesias, Miriam Arumi, Mireia Margelí, Catarina Pulido, Serafin Morales, Fernando Henao, Pilar Sánchez, Sara Alves, Diogo Branco, Joan Manel Gasent, Pedro Sánchez-Rovira, Ana Godoy, José L. Passos-Coelho, Santiago Escrivá-de-Romaní, Juan M Ferrero-Cafiero, Tomás Pascual, Aleix Prat. Solti-1718 NEREA Trial: Neratinib in hormone receptor (HR)-positive/HER2-negative HER2-enriched (HER2-E) advanced breast cancer (BC) [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 OT2-27-01.

Abstract GS2-00: Correlative analysis of overall survival by intrinsic subtype across the MONALEESA-2, -3, and -7 studies of ribociclib + endocrine therapy in patients with HR+/HER2− advanced breast cancer

Abstract Background: The MONALEESA (ML)-2, -3, and -7 trials have shown a significant benefit in overall survival (OS) with ribociclib (RIB) + endocrine therapy (ET) over placebo (PBO) + ET in HR+/HER2− advanced breast cancer. HR+ breast cancer is a clinically and biologically heterogeneous disease, with identified intrinsic subtypes that vary in incidence, survival rate, and response to treatment. In a pooled analysis of the ML studies, patients with both luminal and HER2-enriched (HER2E) subtypes exhibited a consistent progression-free survival benefit with RIB + ET. The HER2E subtype (RIB, 14%; PBO, 11%), which is associated with ET resistance and poor outcomes, exhibited the greatest relative reduction in risk of progression or death (61%) with RIB + ET. Here, we report a pooled analysis of the ML-2, -3, and -7 trials, presenting OS by intrinsic subtype.. Methods: Tumor samples from patients enrolled in the ML-2, -3, and -7 trials underwent PAM50-based subtyping (blinded from clinical data), and the correlation between intrinsic subtype and OS was analyzed. Gene expression profiling of formalin-fixed, paraffin-embedded tumor samples was performed using a customized NanoString nCounter GX 800-gene panel. The prognostic and/or predictive relationship between PAM50-based subtypes and OS was evaluated using univariable and multivariable Cox proportional hazards models. Multivariable models were adjusted for known clinical prognostic factors, including age, prior chemotherapy, prior ET, ECOG performance status, visceral disease (presence of liver/lung metastases), bone-only metastases, histological grade, number of metastatic sites, tumor type, and de novo metastatic disease.. Results: From the pooled patient population (N = 2066), 997 tumor (71% primary) samples from the RIB (n = 585) and PBO (n = 412) arms of the ML trials (ML-2, n = 318; ML-3, n = 414; ML-7, n = 265) were profiled. Subtype distribution was consistent across treatment arms (Table). A similar benefit with RIB vs PBO was observed in the ITT (hazard ratio [HR], 0.76; 95% CI, 0.67-0.86) and biomarker (HR, 0.75; 95% CI, 0.63-0.89) populations. In both univariable and multivariable analyses, intrinsic subtype was prognostic for OS in both the RIB and PBO arms (P < .0001 for both arms); patients with luminal A subtype had the best OS outcomes in both arms, whereas patients with basal-like subtype had the worst OS outcomes. Intrinsic subtype was also predictive of OS (subtype-treatment interaction: P = .016 [univariable], P = .007 [multivariable]) with a consistent OS benefit with RIB treatment in all subtypes except for basal-like (Table). Patients with HER2E (HR, 0.60; P = .018), luminal B (HR, 0.69; P = .023), and luminal A (HR, 0.75; P = .021) subtypes all derived benefit from RIB. In patients with the basal-like subtype (n = 30), the HR was 1.89 (P = .148); these results should be interpreted with caution due to the small sample size (3% in each arm) and exploratory nature of this analysis.Conclusions: This pooled analysis of the ML trials confirmed the prognostic and predictive value of intrinsic subtype (PAM50 based) for OS. The addition of RIB to ET resulted in consistent OS benefit across all subtypes except for basal-like. The. consistent survival benefit in the HER2E subtype, which is associated with endocrine resistance and a very poor prognosis compared with luminal disease, warrants further investigation. SubtypeTreatment ArmDistribution, n (%)OS, median (95% CI), monthsHR (95% CI)P ValueaLuminal ARIB320 (55)68.0 (61.5-NR)0.75 (0.58-0.96).021PBO222 (54)54.6 (48.3-66.2)Luminal BRIB154 (26)58.8 (48.3-79.2)0.69 (0.50-0.95).023PBO124 (30)44.9 (35.5-52.6)HER2-enrichedRIB95 (16)40.3 (33.4-49.0)0.60 (0.40-0.92).018PBO52 (13)29.4 (23.9-42.0)Basal-likeRIB16 (3)19.4 (10.7-33.2)1.89 (0.80-4.47).148PBO14 (3)21.2 (12.8-NR)aP values are all two-sided. NR, not reached. Citation Format: Lisa Carey, Nadia Solovieff, Fabrice André, Joyce O'Shaughnessy, David A Cameron, Wolfgang Janni, Gabe S Sonke, Yoon-Sim Yap, Denise A Yardley, Juan Pablo Zarate, Tetiana Taran, Faye Su, Agnes Lteif, Aleix Prat. Correlative analysis of overall survival by intrinsic subtype across the MONALEESA-2, -3, and -7 studies of ribociclib + endocrine therapy in patients with HR+/HER2− advanced breast cancer [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 GS2-00.

Abstract P1-05-02: Intratumor molecular tumor heterogeneity in low ER-expressing primary breast tumors

Abstract Background: Change in estrogen receptor (ER) status between primary breast cancer and distant recurrence are seen in the clinic in up to 15-20% of patients. This is difficult to reconcile with the current understanding of breast cancer molecular subtypes, the large-scale molecular differences between ER-positive (ER+) and ER-negative (ER-) cancers suggest different cellular origins, luminal versus basal breast epithelium, respectively. A potential explanation for ER status switch is the presence of mixed molecular subtypes at diagnosis. ER+ cancers with less than 100% positivity may be composed by both luminal and basal-like cancer cells, and a recurrence might arise from one subtype that survived adjuvant therapy. The goal of this study was to test if molecular subtype heterogeneity exists at diagnosis in less than 100% ER+ breast cancer using the NanoString GeoMx™ platform. Methods: GeoMx™ is a highly multiplexed assay that quantifies RNA expression from spatially discrete regions of interest (ROIs) within formalin fixed paraffin embedded (FFPE) tissue sections. We identified 4 cancers with 30-40% ER-positivity on routine immunohistochemistry (IHC) staining (clone SP1). Eight ROIs per case were selected to represent both ER-high and -low regions of the section using ER IHC (clone 1D5). Invasive tumor cells were defined as PanCK positive cells. 1,825 mRNA species were measured separately in each ROI. mRNA expression results (unique molecular index [UMI] counts) were quantile normalized and differential expression analyses for all genes, ESR1, ER-regulated gene set, and PAM50 gene set were performed between ROIs within the same cancer. We also assessed PAM50 subtypes of each cases on bulk RNA using NanoString BC360 panel. Results: Three of the 4 cases had heterogenous ER IHC (1D5) staining; the fourth case was uniformly ER-negative during ROI selection. For this last case, ROIs were selected based on PanCK staining from different locations in the cancer. Bulk PAM50 subtyping indicated 1 basal-like, 2 HER2-enriched (HER2-E), and 1 luminal (lum)A cancer. We also attempted ROI-level subtyping; however, Pearson correlations to the nearest centroid were low (<0.5) for all ROIs, possibly due to low expression of PAM50 genes or to the exploratory methodology used. There was substantial within cancer, between ROI, expression heterogeneity for many genes including ESR1 and sensitivity to endocrine therapy (SET) index genes. Conclusions: ER- and ER+ cells are intermixed which makes defining low and high ER regions challenging. We observed region to region variation in ESR1 and SET index gene expression. ROI-level PAM50 subtyping was unstable. The novel NanoString GeoMx™ DSP technology can be used to study intratumor molecular heterogeneity in regions of interest on FFPE breast cancer tissue sections. We found substantial region-to-region gene expression differences within tissue sections. Case ID1234ER IHC SP130%40%40%30%ER IHC 1D50%40%40%30%Bulk PAM50 SubtypeBasalHER2-ELumAHER2-EESR1 UMI average (range across ROIs)28.45 (22.4-34.6)64.9 (18.8-173)179.9 (114-245)34.8 (25.1-45.4)SET Index (genes positively correlated with ESR1) UMI average (range across ROIs)41.5 (39.1-44.8)120.1 (86.9-182.9)94.9 (72.9-118.4)143.5 (124.9-150.3)SET Index (genes negatively correlated with ESR1) UMI average (range across ROIs)57.6 (45-75.3)43.4 (38.8-52.6)33.7 (31.8-36.7)44.8 (41.4-50.6) Citation Format: Julia Foldi, Emily Reisenbichler, Liuliu Pan, Krsitina Sorg, Sarah E. Church, Lajos Pusztai. Intratumor molecular tumor heterogeneity in low ER-expressing primary breast tumors [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 P1-05-02.

Abstract PD2-07: Impact of using cross-platform gene expression profiling technologies and computational methods for intrinsic breast cancer subtyping in PALOMA-2 and PALLET

Abstract Background Intrinsic breast cancer molecular subtyping (IBCMS) provides significant prognostic information for patients (pts) with breast cancer (BC) treated with chemotherapy, human epidermal growth factor receptor 2 (HER2) targeted therapies, and endocrine therapies (ETs). Classifying tumors into intrinsic subtypes to determine optimal treatment is often applied using PAM50, commercially known as Prosigna. Meanwhile, Absolute Assignment of Breast Cancer Intrinsic Molecular Subtypes (AIMS) computational method was trained to predict PAM50-based IBCMS. As the PAM50 algorithm was developed to capture the major subtypes in a general pt population, clinicopathologic distribution of the study cohort and technology platform calibration should be considered in IBCMS analyses. This study compared different next-generation sequencing technologies and methodologic approaches of PAM50 on tumor samples from 2 randomized trials of postmenopausal women with estrogen receptor-positive (ER+)/HER2-negative (HER2-) BC. Methods PALOMA-2 is a double-blind, randomized study of first-line palbociclib (PAL) + letrozole (LET) for ER+/HER2- advanced BC (ABC). Tumor samples from consented pts were subtyped using the validated RUO PAM50 assay (ruoProsigna, NanoString); results were compared with published subtype results using AIMS on EdgeSeq Oncology Biomarker Panel (HTG Molecular Diagnostics). PALLET is a phase 2, randomized trial of PAL+LET as neoadjuvant therapy in pts with ER+ HER2- BC. Baseline frozen tumor biopsies underwent whole transcriptome mRNA-sequencing (mRNA-seq). IBCMS was performed using AIMS; PAM50 subtyping was performed on data normalised with subgroup-specific gene centering and microarray-RNA-sequencing calibration. Results In PALOMA-2, 222 pts had both ruoProsigna and AIMS data; an overall 54% agreement rate between methods was observed, with 46% (56/121) of Luminal B (LumB) subtype by ruoProsigna assigned as Luminal A (LumA) by AIMS and 67% (6/9) of basal-like by ruoProsigna as HER2-enriched (HER2-E) by AIMS (Table 1). In PALLET, 224 pts had mRNA-seq data; a 69% agreement between the two approaches (AIMS and PAM50) was observed, with only 4% (2/49) of LumB assigned as LumA by AIMS but 17% (26/156) and 16% (25/156) of LumA considered LumB or normal-like by AIMS, respectively. Progression-free survival (PFS) by ruoProsigna-derived subtype in PALOMA-2 showed that PAL+LET benefited all pts but those with a basal-like subtype (Table 2). With AIMS, PAL+LET provided a PFS benefit in pts with LumA and LumB subtypes, but was less effective in the HER2-E subtype. Conclusion Intrinsic subtyping has potential clinical utility. PAL+ET should be considered for ER+/HER2- ABC, except possibly in pts with a basal-like tumor, consistent with previous reports. A standardized clinical PAM50 assay and bioinformatics approach should be used as discrepancies in gene expression platforms and algorithms lead to different results and could misguide treatment decisions. Clinical trial identification: Pfizer (NCT01740427) Table 1.Intrinsic Subtyping by IBCMS MethodsPALOMA-2PALLETMethodruoProsignaPAM50 mRNAseqAIMS BasalHER2LumALumBGrand TotalBasalHER2LumALumBNormalGrand TotalBasal-like, n (%)1 (11)NANANA13 (75)0001 (8)4HER2-E, n (%)6 (67)6 (30)6 (8)13 (10)311 (25)3 (100)8 (5)6 (12)1 (8)19LumA, n (%)NA2 (10)60 (83)56 (46)1180097 (62)2 (4)099LumB, n (%)2 (22)12 (60)3 (4)52 (43)690026 (17)41 (84)067Normal-like, n (%)NANA3 (4)NA30025 (16)010 (83)35Grand Total9 (100)20 (100)72 (100)121 (100)2224 (100)3 (100)156 (100)49 (100)12 (100)224NA=Not available Table 2.Median PFS statistics by subtype in PALOMA-2PAL+LET PFS, monthsPBO+LET PFS, monthsHazard Ratio(95% CI)P ValueruoProsignaBasal-like8.2 (n=5)3.6 (n=4)0.39 (0.09-1.77)0.206HER2-E11.0 (n=12)5.1 (n=8)0.41 (0.15-1.11)0.071LumA37.2 (n=52)13.6 (n=20)0.42 (0.21-0.84)0.011LumB27.6 (n=79)13.8 (n=42)0.63 (0.40-1.00)0.049AIMSBasal-likeNANANANAHER2-E16.4 (n=21)8.4 (n=10)0.82 (0.32-2.1)0.684LumA30.6 (n=84)16.5 (n=34)0.56 (0.33-0.95)0.029LumB19.3 (n=41)8.8 (n=28)0.39 (0.23-0.67)<0.001NA=Not available; PBO=placebo Citation Format: Maggie Cheang, Mitch Dowsett, Mothaffar Rimawi, Stephen Johnston, Samuel Jacobs, Judith Bliss, Katherine Pogue-Geile, Lucy Kilburn, Zhou Zhu, Eugene F. Schuster, Hui Xiao, Lisa Swaim, Shibing Deng, Dongrui R. Lu, Eric Gauthier, Jennifer Tursi, Dennis J. Slamon, Hope S. Rugo, Richard S. Finn, Yuan Liu. Impact of using cross-platform gene expression profiling technologies and computational methods for intrinsic breast cancer subtyping in PALOMA-2 and PALLET [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 PD2-07.

The Effect of Breast Cancer Subtype on Symptom Improvement Following Palliative Radiotherapy for Bone Metastases

AimTo assess the relationship between breast cancer subtypes and patient-reported outcomes (PRO) following palliative radiotherapy for bone metastases. Materials and methodsProspectively collected PRO for all breast cancer patients treated with palliative, bone metastasis-directed radiotherapy from 2013 to 2016 in the province of British Columbia were analysed. The PRO questionnaire scored pain severity, level of function and symptom frustration at baseline and at 3–4 weeks following palliative radiotherapy using a 12-point scale. The primary outcome was the rate of overall response (any improvement in score); the secondary outcome was the rate of complete improvement in PRO (final PRO score of 0). Multivariate logistic analysis was used to compare response rates between molecular subgroup approximations of luminal A (LumA), luminal B (LumB), HER2-enriched (HER2) and triple negative (TN), as defined by grade and immunohistochemical staining. ResultsThere were 376 patients who underwent 464 courses of palliative radiation for bone metastases. Subtypes included: 243 LumA, 146 LumB, 46 HER2 and 29 TN. There were 216 multifraction radiotherapy courses (median dose 20 Gy) and 248 single-fraction radiotherapy courses (median dose 8 Gy). The overall response rate was 85% and the complete response rate was 25%. In comparison with LumA breast cancers, TN breast cancers were associated with a lower rate of overall response (69% versus 86%, P = 0.021) and a lower rate of complete response (10% versus 28.8%, P = 0.045) on multivariate analyses. ConclusionPatients with TN breast cancer have lower rates of pain, function and symptom frustration improvement following palliative radiation for bone metastases.

TGF\u03b21 pathway components in breast cancer tissue from aggressive subtypes correlate with better prognostic parameters in ER-positive and p53-negative cancers

BackgroundTGFβ signaling exerts context-specific effects in breast cancer (BC) pathogenesis and single nucleotide polymorphisms (SNPs) in TGFβ-signaling components play a role in the genetic control of their expression and in BC susceptibility and clinical presentation. However, studies investigating the association between the TGFβ-signaling molecules and BC prognosis rarely considered disease subtypes and SNPs. Therefore, the present study aimed to evaluate the expression of TGFβ-signaling components in BC tissue from patients with available data regarding TGFB1 and TGFBR2 SNPs and plasmatic TGFβ1 levels.MethodsImmunostaining for TGFβ1, TGFβRII and phosphorylated (p)-SMAD2/3 was investigated in primary tumor tissue from 34 patients with luminal-B-HER2+ (LB-HER2), HER2-enriched (HER2) and triple negative (TN) BC subtypes genotyped for TGFB1 (rs1800468, rs1800469, rs1800470 and rs1800471) and TGFBR2 (rs3087465) SNPs.ResultsStrong positive correlations were observed between TGFβ1, TGFβRII and p-SMAD2/3 in tumor tissue, and an inverse correlation was observed between intratumor and plasmatic TGFβ1 levels in TN BCs. In LB-HER2+ tumors, p-SMAD2/3 was associated with older age at diagnosis and inversely correlated with p53 staining and lymph-node metastasis, while tumor-size negatively correlated with TGFβ1 and TGFβRII in this BC subgroup. Also, in p53-negative BCs, tumor size and Ki67 negatively correlated with both TGFβ1, TGFβRII and p-SMAD2/3. No correlation was found between SNPs and TGFβ1-signaling components expression.ConclusionTGFβ1 canonical signaling is activated in approximately half of BCs, and correlation between TGFβ components indicate a paracrine activation, which may exert tumor suppressor effects in p53-negative or Luminal-B-HER2+ subgroups.

339TiP SOLTI-1502 ARIANNA: Targeting PAM50 HER2-enriched intrinsic subtype with enzalutamide in hormone receptor-positive/HER2-negative metastatic breast cancer

Pre-clinical evidence and retrospective studies suggest that PAM50 HER2-Enriched (HER2-E), hormone receptor-positive (HR+)/HER2-negative tumors have estrogen receptor (ER)-independency and poor prognosis but seem to have androgen receptor (AR)-addiction. Enzalutamide (EZM) is a potent inhibitor of androgen receptor signaling. AR expression has been shown to induce resistance to both tamoxifen and aromatase inhibitors in estrogen receptor HR–expressing cell lines. The trial hypothesizes that EZM induces a significant proliferative arrest in PAM50 HER2-E, HR+/HER2-negative advanced breast cancer (BC), leading to clinical benefit in this poor prognosis population.

16P Understanding the biologic determinants of ribociclib efficacy in breast cancer

Ribociclib improves survival in hormone receptor-positive (HR+)/HER2-negative (HER2-) advanced breast cancer (BC). Deeper understanding of the biology associated with ribociclib efficacy is needed, especially within the HER2-enriched (HER2-E) subtype given recent analysis of MONALEESA program. Here, we performed gene expression (GE) analysis with/without ribociclib monotherapy in BC patient-derived xenografts (PDX).

Abstract OT-28-05: Solti-1718 NEREA trial: Neratinib in hormone receptor (HR)-positive/HER2-negative HER2-Enriched (HER2-E) advanced breast cancer (BC)

Abstract Background: HR+/HER2-negative BC represent ∼70% of all newly diagnosed breast tumors. BC is a clinically and biologically heterogenous disease where intrinsic subtypes play a role(1-3). Non-luminal subtypes within HR+/HER2-negative disease do not benefit at the same extent from standard of care treatments as the luminal subtypes(1). Thus, other strategies are needed. HER2-E subtype represents approximately 6.6-11.0% of HR+/HER2-negative tumors and is enriched in twice as many cases in metastatic tumors. According to EGF30008 trial, HER2-E advance BC patients despite presenting poor outcomes across treatments, showed more benefit from anti-HER2 therapy. SOLTI-1718 NEREA aims to evaluate whether EGFR/ERBB2 axis inhibition by neratinib improves efficacy in terms of progression-free survival (PFS) in patients with advanced HR+/HER2-negative disease resistant to an endocrine treatment (ET). Methods: SOLTI-1718 NEREA is an open-label, single arm, multicenter and multinational phase II clinical trial following a Simon’s 2-stage design with one interim and one final efficacy analysis. Locally advanced or metastatic HER2-E, HR+/HER2-negative BC patients who had recurrence or progression while receiving previous ET will be included. Treatment schedule will consist on neratinib 240 mg daily in combination with ET, with either exemestane, fulvestrant or tamoxifen. All patients will take prophylactic loperamide with an established dosing scheme during the first cycle and on demand in subsequent cycles. Tumor assessments will be performed at baseline and every 8 weeks during the first year, and every 12 weeks thereafter. Interim analysis will be performed after 33 patients are evaluable. If 15 to 27 patients achieved a PFS at 6 months (PFS6), the trial will continue to second stage, otherwise it will be stopped for futility (<15) or efficacy (≥28). A total of 56 evaluable patients will be included in stage I and II. The primary objective is to assess the efficacy of neratinib in combination with ET in HER2-E, HR+/HER2-negative patients in terms of PFS6 by local assessment by the investigator using RECIST v.1.1. Secondary endpoints include clinical benefit rate at 6 months, overall response rate, duration of response, time to response and incidence, duration and severity of adverse events. The Spanish national competent authority approved the study on April 8th 2020. The study will enroll patients in 15 sites in Spain and 3 sites in Portugal. Recruitment will start on July 2020. We thank PUMA BIOTECHNOLOGY, INC for their provision of Neratinib and financial contribution to the study.

Abstract OT-09-08: Solti-1502 aRIANNA: Targeting PAM50 HER2-enriched intrinsic subtype with enzalutamide in hormone receptor-positive/HER2-negative metastatic breast cancer

Abstract Background Pre-clinical evidence and retrospective studies suggest that PAM50 HER2-Enriched (HER2-E), hormone receptor-positive (HR+)/HER2-negative tumors have estrogen receptor (ER)-independency and poor prognosis, but seem to have androgen receptor (AR)-addiction (1). Enzalutamide (EZM) is a potent inhibitor of androgen receptor signaling (3). AR expression has been shown to induce resistance to both tamoxifen and aromatase inhibitors in estrogen receptor HR-expressing cell lines (4,5) The hypothesis of the ARIANNA trial is that EZM induces a significant proliferative arrest in PAM50 HER2-E, HR+/HER2-negative advanced breast cancer (BC), leading to clinical benefit in this poor prognosis population. Methods ARIANNA is an exploratory, phase II clinical trial in two independent cohorts evaluating the effect of EZM on proliferation after 2 weeks (+7 days window) on treatment in pre- or post-menopausal female or male patients with endocrine-resistant, locally advanced or metastatic HR+/HER2-negative BC. Cohort A will include 22 patients with PAM50 HER2-E HR+/HER-negative tumors and Cohort B (control group) will include 22 patients with PAM50 Luminal A/B HR+/HER2-negative tumors. Fresh tumor biopsy will be obtained at screening and sent to central laboratory for PAM50 subtyping determination to confirm the molecular subtype status prior to study treatment initiation, and to determine PAM50 11-gene proliferation-related signature. Patients with PAM50 Basal-like or Normal-like tumors will be excluded. Patients will receive EZM 160 mg once a day (QD). After 2 weeks on treatment, a tumor biopsy from the same baseline lesion (or, if not feasible, a lesion in the same organ) will be obtained for the purpose of the primary endpoint analysis. After this on-treatment biopsy, exemestane 50mg QD can be added to EZM at physician’s discretion. Tumor assessment will be performed at screening and every 8 weeks thereafter. Treatment will be continued until disease progression, unacceptable toxicity, investigator’s decision or withdrawal of consent. An optional tumor biopsy will be collected at the end of treatment. The primary objective is to evaluate the anti-proliferative effect of EZM after 2 weeks of treatment in patients with HER2-E HR+/HER2-negative tumors, measured as relative changes in the PAM50 11-gene proliferation-related signature by the PAM50 nCounter-based assay between baseline and on-treatment tumor biopsies. Secondary objectives include: anti-proliferative effect of EZM after 2 weeks of treatment in patients included in Cohort B (control group), safety, overall response rate, progression-free survival, and further correlative molecular analyses both at the tumor tissue (IHC, RNA and DNA) and ctDNA level for both cohorts. The trial will enroll patients in 8 Spanish sites and recruitment period will be 18 months. Funding was granted by Breast Cancer Research Foundation and drug was supplied by Astellas Pharma Global Development, Inc./Pfizer, Inc. Trial identification: NCT04142060 1. Cochrane DR, Bernales S, Jacobsen BM, Cittelly DM, Howe EN, D’Amato NC, et al. Role of the androgen receptor in breast cancer and preclinical analysis of enzalutamide. Breast Cancer Res. 2014;16:R7. 3. Tran C, Ouk S, Clegg NJ, Chen Y, Watson PA, Arora V, et al. Development of a Second-Generation Antiandrogen for Treatment of Advanced Prostate Cancer. Science. 2009;324:787-90. 4. Rechoum Y, Rovito D, Iacopetta D, Barone I, Andò S, Weigel NL, et al. AR collaborates with ERα in aromatase inhibitor-resistant breast cancer. Breast Cancer Res Treat. 2014;147:473-85. 5. De Amicis F, Thirugnansampanthan J, Cui Y, Selever J, Beyer A, Parra I, et al. Androgen receptor overexpression induces tamoxifen resistance in human breast cancer cells. Breast Cancer Res Treat. 2010;121:1-11 Citation Format: Mafalda Oliveira, Sonia Pernas, Mireia Margelí, Salvador Blanch, Barbara Adamo, Javier Salvador Bofill, Diana Moreno, Xavier Gonzalez-Farré, José Rios, Charles M Perou, Aleix Prat, Tomás Pascual, Juan M Ferrero-Cafiero, Patricia Villagrasa, Luis Manso. Solti-1502 aRIANNA: Targeting PAM50 HER2-enriched intrinsic subtype with enzalutamide in hormone receptor-positive/HER2-negative metastatic breast cancer [abstract]. In: Proceedings of the 2020 San Antonio Breast Cancer Virtual Symposium; 2020 Dec 8-11; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2021;81(4 Suppl):Abstract nr OT-09-08.

Prolactin receptor-driven combined luminal and epithelial differentiation in breast cancer restricts plasticity, stemness, tumorigenesis and metastasis

Dedifferentiation increased cellular plasticity and stemness are established derivers of tumor heterogeneity, metastasis and therapeutic failure resulting in incurable cancers. Therefore, it is essential to decipher pro/forward-differentiation mechanisms in cancer that may serve as therapeutic targets. We found that interfering with expression of the receptor for the lactogenic hormone prolactin (PRLR) in breast cancer cells representative of the luminal and epithelial breast cancer subtypes (hormone receptor positive (HR+) and HER2-enriched (HER2-E) resulted in loss of their differentiation state, enriched for stem-like cell subpopulations, and increased their tumorigenic capacity in a subtype-specific manner. Loss of PRLR expression in HR+ breast cancer cells caused their dedifferentiation generating a mesenchymal-basal-like phenotype enriched in CD44+ breast cancer stem-like cells (BCSCs) showing high tumorigenic and metastatic capacities and resistance to anti-hormonal therapy. Whereas loss of PRLR expression in HER2-E breast cancer cells resulted in loss of their luminal differentiation yet enriched for epithelial ALDH+ BCSC population showing elevated HER2-driven tumorigenic, multi-organ metastatic spread, and resistance to anti-HER2 therapy. Collectively, this study defines PRLR as a driver of precise luminal and epithelial differentiation limiting cellular plasticity, stemness, and tumorigenesis and emphasizing the function of pro/forward-differentiation pathways as a foundation for the discovery of anti-cancer therapeutic targets.

FGFR4 regulates tumor subtype differentiation in luminal breast cancer and metastatic disease.

Mechanisms driving tumor progression from less aggressive subtypes to more aggressive states represent key targets for therapy. We identified a subset of luminal A primary breast tumors that give rise to HER2-enriched (HER2E) subtype metastases, but remain clinically HER2 negative (cHER2-). By testing the unique genetic and transcriptomic features of these cases, we developed the hypothesis that FGFR4 likely participates in this subtype switching. To evaluate this, we developed 2 FGFR4 genomic signatures using a patient-derived xenograft (PDX) model treated with an FGFR4 inhibitor, which inhibited PDX growth in vivo. Bulk tumor gene expression analysis and single-cell RNA sequencing demonstrated that the inhibition of FGFR4 signaling caused molecular switching. In the Molecular Taxonomy of Breast Cancer International Consortium (METABRIC) breast cancer cohort, FGFR4-induced and FGFR4-repressed signatures each predicted overall survival. Additionally, the FGFR4-induced signature was an independent prognostic factor beyond subtype and stage. Supervised analysis of 77 primary tumors with paired metastases revealed that the FGFR4-induced signature was significantly higher in luminal/ER+ tumor metastases compared with their primaries. Finally, multivariate analysis demonstrated that the FGFR4-induced signature also predicted site-specific metastasis for lung, liver, and brain, but not for bone or lymph nodes. These data identify a link between FGFR4-regulated genes and metastasis, suggesting treatment options for FGFR4-positive patients, whose high expression is not caused by mutation or amplification.

Association of T- and B-cell receptor repertoires with molecular subtypes and outcome in HER2+ breast cancer: An analysis of the NeoALTTO clinical trial.

511 Background: Clinicopathological and molecular features, including estrogen receptor (ER) status and PAM50 subtypes, have shown an association with immunogenicity and tumor-infiltrating lymphocyte (TIL) levels in breast cancer (BC). To investigate the complexity of the immune response in HER2+ BC, we explored the association of T- and B-cell receptor (TCR and BCR) repertoires with clinicopathological characteristics, PAM50 subtypes and outcome in the NeoALTTO phase III trial. Methods: RNA sequencing (RNAseq) data from baseline tumor biopsies were available for 254 out of the 455 patients enrolled. TCR and BCR repertoires were extracted from RNAseq data using the MiXCR software. Repertoire and diversity measures (read counts, number of clones, evenness, Gini index, Shannon entropy, length of the complementarity-determining region 3 [CDR3], top and second top clone proportions) were estimated. PAM50 subtypes were computed from RNAseq data. Univariate and multivariate (adjusted for clinicopathological characteristics, TIL levels dichotomized using the median value of 12.5% and treatment arm) Cox proportional hazard models were used for survival analysis, while logistic regressions were used for pathological complete response (pCR), defined as ypT0/is. All results reported had a false discovery rate (FDR) <0.05. Results: Higher BCR read counts, number of clones and Gini index were significantly associated with ER-negative as well as grade 3 tumors. Among the PAM50 subtypes, HER2-enriched (HER2-E) showed significantly higher BCR read counts, number of clones and Gini index along with lower evenness compared to luminal A and B, as well as higher length of CDR3 than luminal A. Of note, basal-like showed similar BCR diversity measures to HER2-E. No significant differences were noted for TCR diversity measures. In multivariate analyses, neither TCR nor BCR features were associated with pCR, while BCR evenness (HR 1.5; 95%CI 1.1-2.1) and Gini index (HR 0.66; 95%CI 0.5-0.88) were associated with event-free survival. Conclusions: BCR repertoire measures suggest a clonal expansion in HER2-E and basal-like PAM50 subtypes. Furthermore, the implementation of BCR-derived biomarkers can help to identify patients with an improved clinical outcome after neoadjuvant anti-HER2 treatment. Our findings highlight the heterogeneity of the immune response within HER2+ BC and provide support for biomarker-driven treatment strategies including immunotherapy in this BC subtype. Further validation is required. Clinical trial information: NCT00553358 .

170P 18F-FDG PET/CT features of the metastases from intrinsic NSIBC molecular types and its prognostic value

To analyze the 18F-FDG PET/CT imaging features of the metastases from nonspecific invasive breast cancer (NSIBC) according to intrinsic molecular subtypes, and its prognostic value in metastatic NSIBC. Female breast cancer patients has 18F-FDG PET/CT at our hospital 12/2013-10/2018 were collected retrospectively, including image features, clinicopathological, and disease status after PET/CT. All patients were divided as five groups: Luminal A (LA), Luminal B (LB), HER2 positive luminal (LHER), HER2 enriched (HER) and Basal like (TN). Nonparametric tests were used to analyze the difference of maximum standardized uptake value (SUVmax), metabolic tumor volume (MTV), total lesion glycolysis (TLG), heterogeneity index (HI) and coefficient of variation (COV) among metastases. And, ROC curve was used to evaluate the diagnostic performance of SUVmax for bone and lymph node metastases. Cox regression was used to test for relationship among PET/CT parameters, clinicopathological and progression-free survival (PFS). 54 metastatic NSIBC with 747 metastases were enrolled. There was significant in SUVmax and HI of the metastases among five groups (P=0.00). The metastases in lung had the highest HI (P=0.00). LA had the smallest SUVmax and HI (P=0.00); LHER had the highest SUVmax (P=0.00). Among different molecular subtypes, SUVmax for bone were: LA＜HER＜LB＜LHER. ROC curve analysis showed that among LHER and TN, the SUVmax cutoff values of 4.55 and 2.65 identified bone and lymph node metastases with high sensitivity (92.90% and 95.30%) and specificity (100.00% and 86.20%). More than 12 months follow-up after PET/CT, 34 metastatic NSIBC cases had progressive disease (PD), 17 cases had non PD. The whole body MTV (HR=1.01, P=0.00), molecular subtypes (LA (HR=0.309, P=0.028) and LHER (HR=0.312, P=0.031))and presence of recurrent (HR=2.15, P=0.035) were identified as independent prognostic factors of PFS. The metastases of NSIBC from different molecular subtypes presented with diverse 18F-FDG PET/CT imaging features. Based on individualized analysis of molecular subtypes and metastatic sites, can yield a better diagnostic performance. Moreover, wMTV is one of independent prognostic factors in metastatic NSIBC.