PAM50 Gene Research Articles

Ipatasertib plus Paclitaxel for Patients with PIK3CA/AKT1/PTEN-Altered Locally Advanced Unresectable or Metastatic Triple-Negative Breast Cancer in the IPATunity130 Phase III Trial.

In the randomized phase II LOTUS trial, combining ipatasertib with first-line paclitaxel for triple-negative breast cancer (TNBC) improved progression-free survival (PFS), particularly in patients with PIK3CA/AKT1/PTEN-altered tumors. We aimed to validate these findings in a biomarker-selected TNBC population. In Cohort A of the randomized double-blind placebo-controlled phase III IPATunity130 trial, taxane-eligible patients with PIK3CA/AKT1/PTEN-altered measurable advanced TNBC and no prior chemotherapy for advanced disease were randomized 2:1 to ipatasertib (400 mg, days 1-21) or placebo, both plus paclitaxel (80 mg/m2, days 1, 8, and 15), every 28 days until disease progression or unacceptable toxicity. The primary endpoint was investigator-assessed PFS. Between February 2018 and April 2020, 255 patients were randomized (168 to ipatasertib, 87 to placebo). At the primary analysis, there was no significant difference between treatment arms in PFS [hazard ratio 1.02, 95% confidence interval (CI), 0.71-1.45; median 7.4 months with ipatasertib vs. 6.1 months with placebo]. The final analysis showed no difference in overall survival between treatment arms (hazard ratio 1.08, 95% CI, 0.73-1.58; median 24.4 vs. 24.9 months, respectively). Ipatasertib was associated with more grade ≥3 diarrhea (9% vs. 2%) and adverse events leading to dose reduction (39% vs. 14%) but similar incidences of grade ≥3 adverse events (51% vs. 46%). Exploratory subgroup analyses by PAM50 and Burstein gene expression showed inconsistent results. Adding ipatasertib to paclitaxel did not improve efficacy in PIK3CA/AKT1/PTEN-altered advanced TNBC. Biomarkers for benefit from PI3K/AKT pathway inhibition in TNBC remain poorly understood.

Comparative biological activity of palbociclib and ribociclib in hormone receptor-positive breast cancer

This study examines the biological effects of palbociclib and ribociclib in hormone receptor-positive breast cancer, pivotal to the HARMONIA prospective phase III clinical trial. We explore the downstream impacts of these CDK4/6 inhibitors, focusing on cell lines and patient-derived tumor samples. We treated HR+ breast cancer cell lines (T47D, MCF7, and BT474) with palbociclib or ribociclib (100 nM or 500 nM), alone or combined with fulvestrant (1 nM), over periods of 24, 72, or 144 h. Our assessments included PAM50 gene expression, RB1 phosphorylation, Lamin-B1 protein levels, and senescence-associated β-galactosidase activity. We further analyzed PAM50 gene signatures from the CORALLEEN and NeoPalAna phase II trials. Both CDK4/6 inhibitors similarly inhibited proliferation across the cell lines. At 100 nM, both drugs partially reduced p-RB1, with further decreases at 500 nM over 144 h. Treatment led to reduced Lamin-B1 expression and increased senescence-associated β-galactosidase activity. Both drugs enhanced Luminal A and reduced Luminal B and proliferation signatures at both doses. However, the HER2-enriched signature significantly diminished only at the higher dose of 500 nM. Corresponding changes were observed in tumor samples from the CORALLEEN and NeoPalAna studies. At 2 weeks of treatment, both drugs significantly reduced the HER2-enriched signature, but at surgery, this reduction was consistent only with ribociclib. Our findings suggest that while both CDK4/6 inhibitors effectively modulate key biological pathways in HR+/HER2- breast cancer, nuances in their impact, particularly on the HER2-enriched signature, are dose-dependent, influenced by the addition of fulvestrant and warrant further investigation.

Abstract PO2-13-05: Deciphering pregnancy-associated breast cancer: distinctive molecular profile and clinical implications from GEICAM/2017-07 EMBARCAM study

Abstract Background: Pregnancy-Associated Breast Cancer (PABC) is defined as BC diagnosed during pregnancy, breastfeeding, or within the first year postpartum. This challenging entity has a distinct biology and worst prognosis and presents an increased risk for metastasis and death compared to non-PABC patients. Identification of molecular pathways that define this malignancy is crucial to explain its biological characteristics and potential association with pregnancy as well as serve to identify new biomarkers with preventive and clinical implications. This study assesses the gene expression profile of a PABC cohort from the Registry Study of Pregnancy and Breast Cancer (GEICAM/2017-07 EMBARCAM study, NCT04603820) to identify distinct molecular signatures and altered pathways for this entity. Methods: This was a multicenter, observational and ambispective age-matched cohort of PABC patients (n=46) and non-PABC patients (n=49). We analyzed the expression of 776 genes involved in 23 key breast cancer pathways, using the nCounter® Breast Cancer 360™ Panel (NanoString Technologies) on BC tumor samples, including molecular subtypes by PAM50 gene signature. Associations between individual differential genes and epidemiological and clinical features of these patients were explored. Normalized differential expression values were log2-transformed for statistical analysis in nSolver Software (NanoString Technologies). P-values were adjusted within each gene/signature and on the grouping variable level difference t-test using the Benjamini and Yekutieli False Discovery Rate (FDR) adjustment. Results: PABC patients’ clinical subtypes were distributed as 43,5% HR-positive/HER2-negative, 17,4% HER2-positive and 39,1% triple negative BC. These patients reported higher family history of BC/ovarian cancer compared to non-PABC (52.17% vs 26.53%; p=0.0124). Differential gene expression showed distinct molecular profiles in PABC and non-PABC patients: significantly genes enrichment involved in cell proliferation and p53 signature for PABC patients and higher expression of apoptosis, TGF-β, and PTEN signatures for non-PABC (adj p< 0.05). Furthermore, CDK4 expression signature (adj p=0.124) and DNA damage repair signatures Homologous Recombination Deficiency (HDR) (adj p=0.083) and BRCAness (adj p=0.220) were also upregulated in PABC tumors. The most significantly upregulated genes in PABC patients (CCNA2, DEPDC1, FAM83D, CDC20, CDKN3, TRIP13, TTK, MAD2L1, KIF2C, and UBE2C) (adj p< 0.05) were involved in cell cycle regulation and DNA damage repair. PABC basal-like tumors were found to be significantly more prevalent than non-PABC, as per the PAM50 classification (45.7% vs 20.4%; p=0.020). Interestingly, basal-like PABC showed higher expression of ESR1 than basal-like non-PABC (logFC=1.486; p=0.022). In contrast, luminal A PABC tumors exhibited lower ESR1 expression vs non-PABC (logFC=-1.5; p=0.021). Additionally, HER2-enriched PABC tumors displayed lower B7-H3/CD276 expression compared to HER2-enriched non-PABC (adj p=0.031). Conclusions: Our study shows that PABC is potentially a clinical and molecular different entity with predominance of the basal-like subtype. Moreover, our results suggest the activation of oncogenic pathways related to cell proliferation, DNA damage repair and p53 mutations which may lead to a clinically more aggressive phenotype for PABC patients. Likewise, the enrichment of BRCAness and HRD signatures found in PABC patients in our study may suggest an increased genetic instability due to a breakdown in the DNA damage repair. These findings may be clinically relevant and translate to new treatment options in PABC patients, who may benefit from therapies targeting DNA repair or cell-cycle checkpoints, such as PARP inhibitors. Citation Format: Juan de la Haba-Rodríguez, Regina Peña, Marina Pollan, Yolanda Jerez Gilarranz, Jose Ponce, Antonio Fernández Aramburu, Blanca Cantos, Ana Santaballa Bertrán, Elena Galve, María Eva Pérez, Susana de la Cruz, María Helena López-Ceballos, Yolanda Fernández, Fernando Moreno, Sonia González, Manuel Ruíz - Borrego, Isabel Blancas, José L Alonso-Romero, Álvaro Jiménez-Arranz, Raúl Rincón, Silvia Guil, Alejandra Díaz-Chacón, Rosalía Caballero, Begoña Bermejo, Angel Guerrero. Deciphering pregnancy-associated breast cancer: distinctive molecular profile and clinical implications from GEICAM/2017-07 EMBARCAM study [abstract]. In: Proceedings of the 2023 San Antonio Breast Cancer Symposium; 2023 Dec 5-9; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2024;84(9 Suppl):Abstract nr PO2-13-05.

Few-shot genes selection: subset of PAM50 genes for breast cancer subtypes classification

BackgroundIn recent years, researchers have made significant strides in understanding the heterogeneity of breast cancer and its various subtypes. However, the wealth of genomic and proteomic data available today necessitates efficient frameworks, instruments, and computational tools for meaningful analysis. Despite its success as a prognostic tool, the PAM50 gene signature’s reliance on many genes presents challenges in terms of cost and complexity. Consequently, there is a need for more efficient methods to classify breast cancer subtypes using a reduced gene set accurately.ResultsThis study explores the potential of achieving precise breast cancer subtype categorization using a reduced gene set derived from the PAM50 gene signature. By employing a “Few-Shot Genes Selection” method, we randomly select smaller subsets from PAM50 and evaluate their performance using metrics and a linear model, specifically the Support Vector Machine (SVM) classifier. In addition, we aim to assess whether a more compact gene set can maintain performance while simplifying the classification process. Our findings demonstrate that certain reduced gene subsets can perform comparable or superior to the full PAM50 gene signature.ConclusionsThe identified gene subsets, with 36 genes, have the potential to contribute to the development of more cost-effective and streamlined diagnostic tools in breast cancer research and clinical settings.

CDK4/6-Inhibitors Versus Chemotherapy in Advanced HR+/HER2-Negative Breast Cancer: Results and Correlative Biomarker Analyses of the KENDO Randomized Phase II Trial.

The optimal treatment approach for hormone receptor-positive/HER2-negative metastatic breast cancer (HR+/HER2-negative MBC) with aggressive characteristics remains controversial, with lack of randomized trials comparing cyclin-dependent kinase (CDK)4/6-inhibitors (CDK4/6i) + endocrine therapy (ET) with chemotherapy + ET. We conducted an open-label randomized phase II trial (NCT03227328) to investigate whether chemotherapy + ET is superior to CDK4/6i + ET for HR+/HER2-negative MBC with aggressive features. PAM50 intrinsic subtypes (IS), immunological features, and gene expression were assessed on baseline samples. Among 49 randomized patients (median follow-up: 35.2 months), median progression-free survival (mPFS) with chemotherapy + ET (11.2 months, 95% confidence interval [CI]: 7.7-15.4) was numerically shorter than mPFS (19.9 months, 95% CI: 9.0-30.6) with CDK4/6i + ET (hazard ratio: 1.41, 95% CI: 0.75-2.64). Basal-like tumors under CDK4/6i + ET exhibited worse PFS (mPFS: 11.4 months, 95% CI: 3.00-not reached [NR]) and overall survival (OS; mOS: 18.8 months, 95% CI: 18.8-NR) compared to other subtypes (mPFS: 20.7 months, 95% CI: 9.00-33.4; mOS: NR, 95% CI: 24.4-NR). In the chemotherapy arm, luminal A tumors showed poorer PFS (mPFS: 5.1 months, 95% CI: 2.7-NR) than other IS (mPFS: 13.2 months, 95% CI: 10.6-28.1). Genes/pathways involved in BC cell survival and proliferation were associated with worse outcomes, as opposite to most immune-related genes/signatures, especially in the CDK4/6i arm. CD24 was the only gene significantly associated with worse PFS in both arms. Tertiary lymphoid structures and higher tumor-infiltrating lymphocytes also showed favorable survival trends in the CDK4/6i arm. The KENDO trial, although closed prematurely, adds further evidence supporting CDK4/6i + ET use in aggressive HR+/HER2-negative MBC instead of chemotherapy. PAM50 IS, genomic, and immunological features are promising biomarkers to personalize therapeutic choices.

Perturbation and stability of PAM50 subtyping in population-based primary invasive breast cancer

PAM50 gene expression subtypes represent a cornerstone in the molecular classification of breast cancer and are included in risk prediction models to guide therapy. We aimed to illustrate the impact of included genes and biological processes on subtyping while considering a tumor’s underlying clinical subgroup defined by ER, PR, and HER2 status. To do this we used a population-representative and clinically annotated early-stage breast tumor cohort of 6233 samples profiled by RNA sequencing and applied a perturbation strategy of excluding co-expressed genes (gene sets). We demonstrate how PAM50 nearest-centroid classification depends on biological processes present across, but also within, ER/PR/HER2 subgroups and PAM50 subtypes themselves. Our analysis highlights several key aspects of PAM50 classification. Firstly, we demonstrate the tight connection between a tumor’s nearest and second-nearest PAM50 centroid. Additionally, we show that the second-best subtype is associated with overall survival in ER-positive, HER2-negative, and node-negative disease. We also note that ERBB2 expression has little impact on PAM50 classification in HER2-positive disease regardless of ER status and that the Basal subtype is highly stable in contrast to the Normal subtype. Improved consciousness of the commonly used PAM50 subtyping scheme will aid in our understanding and interpretation of breast tumors that have seemingly conflicting PAM50 classification when compared to clinical biomarkers. Finally, our study adds further support in challenging the common misconception that PAM50 subtypes are distinct classes by illustrating that PAM50 subtypes in tumors represent a continuum with prognostic implications.

Human basal-like breast cancer is represented by one of the two mammary tumor subtypes in dogs

BackgroundAbout 20% of breast cancers in humans are basal-like, a subtype that is often triple-negative and difficult to treat. An effective translational model for basal-like breast cancer is currently lacking and urgently needed. To determine whether spontaneous mammary tumors in pet dogs could meet this need, we subtyped canine mammary tumors and evaluated the dog–human molecular homology at the subtype level.MethodsWe subtyped 236 canine mammary tumors from 3 studies by applying various subtyping strategies on their RNA-seq data. We then performed PAM50 classification with canine tumors alone, as well as with canine tumors combined with human breast tumors. We identified feature genes for human BLBC and luminal A subtypes via machine learning and used these genes to repeat canine-alone and cross-species tumor classifications. We investigated differential gene expression, signature gene set enrichment, expression association, mutational landscape, and other features for dog–human subtype comparison.ResultsOur independent genome-wide subtyping consistently identified two molecularly distinct subtypes among the canine tumors. One subtype is mostly basal-like and clusters with human BLBC in cross-species PAM50 and feature gene classifications, while the other subtype does not cluster with any human breast cancer subtype. Furthermore, the canine basal-like subtype recaptures key molecular features (e.g., cell cycle gene upregulation, TP53 mutation) and gene expression patterns that characterize human BLBC. It is enriched in histological subtypes that match human breast cancer, unlike the other canine subtype. However, about 33% of canine basal-like tumors are estrogen receptor negative (ER−) and progesterone receptor positive (PR+), which is rare in human breast cancer. Further analysis reveals that these ER−PR+ canine tumors harbor additional basal-like features, including upregulation of genes of interferon-γ response and of the Wnt-pluripotency pathway. Interestingly, we observed an association of PGR expression with gene silencing in all canine tumors and with the expression of T cell exhaustion markers (e.g., PDCD1) in ER−PR+ canine tumors.ConclusionsWe identify a canine mammary tumor subtype that molecularly resembles human BLBC overall and thus could serve as a vital translational model of this devastating breast cancer subtype. Our study also sheds light on the dog–human difference in the mammary tumor histology and the hormonal cycle.

Retrospective study of transcriptomic profiling identifies Thai triple-negative breast cancer patients who may benefit from immune checkpoint and PARP inhibitors.

Triple-negative breast cancer (TNBC) is a rare and aggressive breast cancer subtype. Unlike the estrogen receptor-positive subtype, whose recurrence risk can be predicted by gene expression-based signature, TNBC is more heterogeneous, with diverse drug sensitivity levels to standard regimens. This study explored the benefit of gene expression-based profiling for classifying the molecular subtypes of Thai TNBC patients. The nCounter-based Breast 360 gene expression was used to classify Thai TNBC retrospective cohort subgroups. Their expression profiles were then compared against the previously established TNBC classification system. The differential characteristics of the tumor microenvironment and DNA damage repair signatures across subgroups were also explored. Thai TNBC cohort could be classified into four main subgroups, corresponding to the LAR, BL-2, and M subtypes based on Lehmann's TNBC classification. The PAM50 gene set classified most samples as basal-like subtypes except for Group 1. Group 1 exhibited similar enrichment of the metabolic and hormone response pathways to the LAR subtype. Group 2 shared pathway activation with the BL-2 subtype. Group 3 showed an increase in the EMT pathway, similar to the M subtype. Group 4 showed no correlation with Lehmann's TNBC. The tumor microenvironment (TME) analysis showed high TME cell abundance with increased expression of immune blockade genes in Group 2. Group 4 exhibited low TME cell abundance and reduced immune blockade gene expressions. We also observed distinct signatures of the DNA double-strand break repair genes in Group 1. Our study reported unique characteristics between the four TNBC subgroups and showed the potential use of immune checkpoint and PARP inhibitors in subsets of Thai TNBC patients. Our findings warrant further clinical investigation to validate TNBC's sensitivity to these regimens.

PAM50 intrinsic subtypes, risk of recurrence score and breast cancer survival in HIV-positive and HIV-negative patients—a South African cohort study

PurposeTreatment decision making for patients with breast cancer increasingly depends on analysis of markers or systems for estimating risk of breast cancer recurrence. Breast cancer intrinsic subtypes and risk of recurrence (ROR) scores have been found to be valuable in predicting survival and determining optimal treatment for individual patients. We studied the association of breast cancer survival with the PAM50 gene expression assay in HIV-positive and HIV-negative patients.MethodRNA was extracted from formalin-fixed paraffin-embedded specimens of histologically confirmed invasive carcinoma and was purified using the AllPrep® DNA/RNA FFPE kit, Qiagen (Hilden, Germany). The NanoString RUO PAM50 algorithm was used to determine the molecular subtype and the risk of recurrence score of each sample. The overall and disease-free survival were determined with comparison made among HIV-positive and -negative patients. We then generated Kaplan–Meier survival curves, calculated p-values and estimated hazard ratios and their 95% confidence intervals using Cox regression models.ResultsOf the 384 RNA samples analysed, 98.4% met the required RNA quality standard and the specified QC threshold for the test. Luminal B was the most common PAM50 intrinsic subtype and 82.1% of patients were at high risk for disease recurrence based on ROR score. HIV infection, PAM50-based HER2-enriched and basal-like intrinsic subtypes, and high ROR were associated with poor overall and disease-free survival. HIV-positive patients with luminal A & B subtypes had significantly worse survival outcomes than HIV-negative luminal patents.ConclusionAggressive tumour biology was common in our cohort. HIV infection, PAM50 HER2-enriched,basal-like intrinsic subtypes and high ROR score were associated with poor overall and disease-free survival. HIV infection impacted survival in patients with luminal subtypes only.

Trastuzumab deruxtecan (T-DXd) vs treatment of physician’s choice (TPC) in patients (pts) with HER2-low, hormone receptor-positive (HR+) unresectable and/or metastatic breast cancer (mBC): Exploratory biomarker analysis of DESTINY-Breast04.

1020 Background: DESTINY-Breast04 (NCT03734029) showed improved progression-free survival (PFS) and overall survival for T-DXd vs TPC in pts with HER2-low (IHC 1+ or 2+/ISH-negative) mBC. We present exploratory biomarker analysis in pts with HER2-low, HR+ mBC. Methods: Biopsy specimens collected from 326 pts after prior treatment were analyzed using RNA-sequencing and intrinsic subtypes estimated by PAM50 gene expression. ESR1 and PIK3CA mutations and known gene alterations associated with resistance to CDK4/6 inhibitors (CDK4/6i) were assessed in baseline (BL) circulating tumor DNA (ctDNA) samples from 414 pts by Guardant OMNI. Association with objective response rate (ORR) and PFS was evaluated. Results: Frequencies of BL intrinsic subtypes in the T-DXd and TPC arms were 41.3% and 46.6% for Luminal A, 48.0% and 37.9% for Luminal B, and 9.0% and 11.7% for HER2 enriched, respectively. According to ctDNA results in the T-DXd and TPC arms, respectively, ESR1 mutations were observed in 51.3% and 54.0% of pts, PIK3CA mutations in 36.1% and 41.6% of pts, and at least one CDK4/6i resistance marker (pts with prior CDK4/6i) was detected in 71.5% and 70.2% of pts. Improved T-DXd efficacy was seen regardless of intrinsic subtype (Luminal A, Luminal B, HER2-enriched), ESR1 mutation, PIK3CA mutation, or CDK4/6i resistance markers (Table). Conclusions: Greater clinical benefit was consistently observed with T-DXd vs TPC independent of intrinsic subtype, ESR1 mutation, PIK3CA mutation, or known CDK4/6i resistance marker status. Clinical trial information: NCT03734029 . [Table: see text]

Abstract 2050: Molecular subclassification of NCI PDMR breast cancer models using PAM50 gene expression signature

Abstract Background: Breast cancer is the second most common cancer in women. In 2022, it accounted for 15% of total new cancer cases and is the number four cause of death among all cancer types. To benefit from precision medicine, distinguishing molecular subtypes for prognosis and treatment in a clinical setting is essential. While intrinsic subtype classification from NGS results of patients is well established, the approach has not been comprehensively described for patient-derived xenograft (PDX) models, which have been shown to be powerful in translational research. The National Cancer Institute's Patient-Derived Models Repository (NCI PDMR; https://pdmr.cancer.gov) provides rich information in developing the method. Materials and Methods: Normalized gene expression data of breast cancer PDX and patient specimens (originators) were extracted using tximport and DESeq2 based on RNA-seq analysis. The immunohistochemistry (IHC) was used to determine the status of ER, PR and HER2 receptor expression in these tumor specimens. The PAM50 classification was performed by the R package Genefu. For further analysis, the PAM50 centroids for all 5 subtypes were also obtained from Genefu. Results: Using the RNA-seq data from 43 PDX models (180 PDX samples, 4~6 samples/model), we were able to predict subtypes at the model level based on the PAM50 method: There are 1 Luminal A subtypes, 5 Luminal B; 6 Her2; 30 Basal and 1 Normal, which encompasses the whole spectrum of PAM50. Thirty originators were also included and there are 8 Luminal A, 9 Luminal B, 2 Her2 and 11 Basal. With the matched 11 originators and the PDX models, 91% of their predicted subtypes are identical; 0.80 Cohen’s kappa was obtained, indicating high inter-rater agreement. We also described subsequent analysis with IHC data-based subtypes. For the 10 originators having IHC-based subtypes, 90% agreement was observed; for 24 PDX models with IHC data, 88% was observed. Of all the 180 PDX samples, 33 of the 43 PDX models (77%) have consistent predicted PAM50 molecular subtypes across different passages and lineages. Within the discordant samples, we observed cases such as a mixture of luminal B and Basal, which can be reasonably interpreted by AR positive signal from IHC. The discrepancy encourages further PDX subclassification from the Basal subtype. Conclusions: Using our high-throughput gene expression profiles from many patients and samples from patient derived models, we have demonstrated the feasibility of applying classic PAM50 classification algorithm, which was originally developed with microarray data, to be able to recognize the expression signals from our RNA-seq data. Overall, this study should set a primer for the identification of PDX-based subtypes, starting from breast cancer. Citation Format: Peter I. Wu, Lindsay Dutko, Shahanawaz Jiwani, Li Chen, Biswajit Das, Ting-Chia Chang, Yvonne A. Evrard, Chris A. Karlovich, Alyssa Chapman, Brandie Fullmer, Ashley Hayes, Ruth Thornton, Nikitha Nair, Kelly Benauer, Gloryvee Rivera, Thomas Forbes, John Carter, Suzanne Borgel, Tiffanie Miner, Chelsea McGlynn, Justine Mills, Shannon Uzelac, Tia Shearer, Lauren Hicks, Michelle Norris, Carley Border, Sergio Alcoser, Thomas Walsh, Michael Mullendore, Michelle Eugeni, Dianne Newton, Melinda G. Hollingshead, P. M. Williams, James H. Doroshow. Molecular subclassification of NCI PDMR breast cancer models using PAM50 gene expression signature [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 2050.

Discordance between PAM50 intrinsic subtyping and immunohistochemistry in South African women with breast cancer

PurposeBreast cancer is a heterogeneous disease with different gene expression profiles, treatment options and outcomes. In South Africa, tumors are classified using immunohistochemistry. In high-income countries multiparameter genomic assays are being utilized with implications for tumor classification and treatment.MethodsIn a cohort of 378 breast cancer patients from the SABCHO study, we investigated the concordance between tumor samples classified by IHC and the PAM50 gene assay.ResultsIHC classified patients as ER-positive (77.5%), PR-positive (70.6%), and HER2-positive (32.3%). These results, together with Ki67, were used as surrogates for intrinsic subtyping, and showed 6.9% IHC-A-clinical, 72.7% IHC-B-clinical, 5.3% IHC-HER2-clinical and 15.1% triple negative cancer (TNC). Typing using the PAM50 gave 19.3% luminal-A, 32.5% luminal-B, 23.5% HER2-enriched and 24.6% basal-like. The basal-like and TNC had the highest concordance, while the luminal-A and IHC-A group had the lowest concordance. By altering the cutoff for Ki67, and realigning the HER2/ER/PR-positive patients to IHC-HER2, we improved concordance with the intrinsic subtypes.ConclusionWe suggest that the Ki67 be changed to a cutoff of 20–25% in our population to better reflect the luminal subtype classifications. This change would inform treatment options for breast cancer patients in settings where genomic assays are unaffordable.

Abstract P1-13-16: Dissecting the biological activity of different CDK4/6 inhibitors (CDK4/6i) in hormone receptor-positive/HER2-negative (HR+/HER2-) breast cancer (BC)

Abstract 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.

Abstract PD13-01: PD13-01 Elacestrant in postmenopausal women with estrogen receptor positive and HER2-negative early breast cancer: primary efficacy and safety analysis of the preoperative, window of opportunity SOLTI-1905-ELIPSE trial

Abstract Introduction Elacestrant is the first oral, non-steroidal, selective estrogen receptor degrader (SERD) to demonstrate improved efficacy compared to standard of care endocrine treatment, with greater relative benefit in ESR1-mutated tumors, and manageable safety profile in pretreated patients with metastatic breast cancer (BC) (Bidard F.C., JCO 2022). SOLTI ELIPSE trial (NCT04797728) is a prospective, multicenter, window of opportunity trial designed to assess whether a short-course of preoperative elacestrant may suppress tumor proliferation in postmenopausal women with estrogen receptor positive (ER+)/HER2-negative early BC (Vidal M., SABCS 2021). Here, we present the results of the primary efficacy and safety study analysis. Methods Eligible patients with operable, untreated ER+/HER2-negative BC that were T1c (≥1.5 cm)-T3 by ultrasound, clinically or radiologically N0 and had a locally assessed Ki67 ≥10%, received elacestrant 400 mg once a day continuously for a total of 4 weeks. At the study treatment completion, patients were treated according to local practice. Centralized assessment of post-treatment (D28) Ki67 from surgical specimen or tumor biopsy was required for the primary endpoint evaluation. Primary efficacy endpoint was complete cell cycle arrest (CCCA), defined as Ki67≤2.7%, at D28. Ki67 geometric relative change, variation in tumor infiltrating lymphocytes (TILs), switch in PAM50 subtypes and differential expression of 192 genes from baseline (D1) to D28 was also explored. Adverse events (AEs) were graded according to CTCAE v5.0. Results Between April 2021 and February 2022, 24 patients were enrolled and 22 were evaluable for the primary endpoint. Baseline characteristics were: mean age 69 years (range 50-81); ductal histology 74%; T1c 61%; T2 39%; grade 1-2 83%; median local Ki67 20% (10-70). Baseline PAM50 subtypes distribution was: Luminal A (n=12), Luminal B (n=8), Basal-like (n=1), Normal-like (n=1). At D28, CCCA was achieved in the 27% (n=6) of the patients. Fourteen patients (64%) had D28 Ki67 ≤10%. Paired centralized Ki67 was available in 19 patients. A statistically significant 41% (95% CI, -24 to -58) Ki67 relative reduction (rr) from D1 was observed (p=0.007). CCCA rate was 31% and 17% in patients with D1 Ki67 < 20% (n=13) and D1 Ki67 ≥20% (n=6), respectively. Ki67 varied consistently in both Ki67 < 20% (rr=-38%; 95% CI, -16 to -60) and Ki67 ≥20% (rr=-46%; 95% CI, -20 to -72) groups. Overall, elacestrant was associated with a shift towards a more endocrine sensitive and less proliferative phenotype based on PAM50 gene signatures. CCCA occurred in 45% of Luminal A tumors, whereas no CCCA was observed among Luminal B tumors. Levels of TILs were significantly higher at D28 (mean difference, +3.73; p=0.004). Elacestrant induced high expression of immune-response genes including IGJ, GZMB, CD4, CD8a and suppressed proliferation (e.g., UBE2T, MYBL2, BIRC5, MK67) and estrogen-signaling (e.g., ESR1, PGR, CCND1, BRCA2) genes (false discovery rate 5%). These changes in gene expression were observed both in tumors with D28 Ki67≤2.7% and in those with D28 Ki67 >10%. Overall, 87% of the patients reported any grade AEs. Treatment-related AE occurred in 1 patient (grade 3 cutaneous rash) and led to treatment discontinuation. Most frequently reported AEs (all grade 1) were hot flush (n=6), dyspepsia (n=2), anemia (n=2) and constipation (n=2). No serious AEs were reported. Conclusions In untreated ER+/HER2-negative early BC a short-course preoperative treatment with elacestrant was associated with relevant biological and molecular response and with manageable safety profile. Globally, these findings support further exploration of this highly potent, novel oral SERD in early BC. Citation Format: Maria Vidal, Tomás Pascual, Claudette Falato, Rodrigo Sanchez-Bayona, Montserrat Muñoz, Isaac Cerbrecos, Xavier Gonzalez-Farré, Tomás Cortadellas, Mireia Margelí, Miguel Angel Luna, Christian Siso, Kepa Amillano, Patricia Galván, Fernando Salvador, Alejandra Espinosa, Laia Paré, Esther Sanfeliu, Aleix Prat, Meritxell Bellet Ezquerra. PD13-01 Elacestrant in postmenopausal women with estrogen receptor positive and HER2-negative early breast cancer: primary efficacy and safety analysis of the preoperative, window of opportunity SOLTI-1905-ELIPSE 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 PD13-01.

Abstract P6-10-04: Homologous recombination deficiency across subtypes of primary breast cancer

Abstract Background: Homologous recombination deficiency (HRD) is highly prevalent in triple-negative breast cancer (TNBC) and predictive of response to PARP inhibition in the primary setting (Eikesdal et al, Ann Oncol, 2021). However, the prevalence of HRD across breast cancer subtypes has not been established. Methods: Pretreatment tumor biopsies from 201 patients (32 TNBC and 169 non-TNBC) with primary breast cancer in the phase II PETREMAC trial (ClinicalTrials #NCT02624973) were examined. These samples underwent targeted cancer gene panel sequencing and BRCA1 promoter methylation analysis to assess HRD status defined by homologous recombination repair (HRR) gene mutations and/or BRCA1 promoter methylation. HRR genes included BRCA1, BRCA2, BRIP1, BARD1, and PALB2 by strict definition (HRR-S), and additionally ABL1, ATM, ATR, ATRX, BLM, CDK12, CHEK1, EMSY, ERCC4, FANCA, FANCC, FANCD2, FANCE, FANCF, FANCG, MEN1, MRE11, NBN, PTEN, and SETD2 by wider definition (HRR-W). HRD strict (HRD-S) was defined as biallelic gene inactivation by HRR-S mutations or BRCA1 methylation. Finally, tumors underwent PAM50 gene expression subtyping and evaluation of functional HRD by RAD51 nuclear foci analysis, for which a low score has been associated with HRD. Results: HRD-S was present in 13% of the breast cancers (total: n= 27/201; TNBC: 15/32; 47%; non-TNBC: 12/169; 7%), whereas HRD-W (HRR-W or BRCA1 methylation) was observed in 29% (total: n=58/201; TNBC: 19/32; 59%; non-TNBC: 39/169; 23%). Among 190 tumors analyzed for PAM50 intrinsic subtype, HRD-S was detected in 3/60 and 4/48 (5% and 8%) of tumors classified as luminal A and B, respectively, 1/35 (3%) of HER2-enriched, 4/21 (19%) of normal-like, and 12/26 (46%) of basal-like tumors. Out of 58 non-TNBC biopsies examined by RAD51 staining, four (7%) were classified as HRD-S and all these were scored as RAD51 low. The remaining 54 non-TNBC samples were homologous recombination proficient, and none of these exhibited functional HRD by RAD51 low scores. All four HRD-S/RAD51 low tumors were hormone receptor-positive, HER2 negative, and belonged to the luminal A (n=1), luminal B (n=2), and basal-like (n=1) subtypes, with HRD caused by germline BRCA1 (gBRCA1), gBRCA2, somatic BRCA1 mutations and BRCA1 methylation, respectively. Conclusion: The prevalence of HRD across all breast cancer subtypes suggests that HRD analysis and therapy targeting such DNA repair defects should be tested in future clinical trials. Citation Format: Christina Engebrethsen, Synnøve Yndestad, Andrea Herencia-Ropero, Oleksii Nikolaienko, Olav Karsten Vintermyr, Reidun K. Lillestøl, Laura Minsaas, Beryl Leirvaag, Gjertrud Iversen, Bjørnar Gilje, Egil Blix, Helge Espelid, Steinar Lundgren, Jürgen Geisler, Liv Jorunn Vassbotn, Hildegunn S. Aase, Turid Aas, Alba Llop-Guevara, Violeta Serra, Per Eystein Lønning, Stian Knappskog, Hans Petter Eikesdal. Homologous recombination deficiency across subtypes of primary breast cancer [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 P6-10-04.

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.

Sample Preparation Approach Influences PAM50 Risk of Recurrence Score in Early Breast Cancer.

Simple SummaryThe PAM50 risk of recurrence (ROR) score is predictive of the risk of distant recurrence and the benefits of adjuvant therapy in early breast cancer. The Prosigna assay utilizes RNA from tumor cells selected via macrodissection of formalin-fixed, paraffin-embedded (FFPE) tissue sections to measure the activity of the PAM50 genes. An alternative and widely used extraction method is RNA purification from fresh-frozen (FF) bulk tissue without enriching the tumor cellularity. However, the impact of the RNA preparation approach on ROR scores and subsequent treatment selection has not been systematically evaluated. We compared the different approaches and found high correlation between risk of recurrence scores estimated from macrodissected FFPE tissue and scores estimated from bulk FF tumor tissue. However, important discrepancies were revealed for luminal tumors, which may have consequences for treatment recommendations for these patients.The PAM50 gene expression subtypes and the associated risk of recurrence (ROR) score are used to predict the risk of recurrence and the benefits of adjuvant therapy in early-stage breast cancer. The Prosigna assay includes the PAM50 subtypes along with their clinicopathological features, and is approved for treatment recommendations for adjuvant hormonal therapy and chemotherapy in hormone-receptor-positive early breast cancer. The Prosigna test utilizes RNA extracted from macrodissected tumor cells obtained from formalin-fixed, paraffin-embedded (FFPE) tissue sections. However, RNA extracted from fresh-frozen (FF) bulk tissue without macrodissection is widely used for research purposes, and yields high-quality RNA for downstream analyses. To investigate the impact of the sample preparation approach on ROR scores, we analyzed 94 breast carcinomas included in an observational study that had available gene expression data from macrodissected FFPE tissue and FF bulk tumor tissue, along with the clinically approved Prosigna scores for the node-negative, hormone-receptor-positive, HER2-negative cases (n = 54). ROR scores were calculated in R; the resulting two sets of scores from FFPE and FF samples were compared, and treatment recommendations were evaluated. Overall, ROR scores calculated based on the macrodissected FFPE tissue were consistent with the Prosigna scores. However, analyses from bulk tissue yielded a higher proportion of cases classified as normal-like; these were samples with relatively low tumor cellularity, leading to lower ROR scores. When comparing ROR scores (low, intermediate, and high), discordant cases between the two preparation approaches were revealed among the luminal tumors; the recommended treatment would have changed in a minority of cases.

129P Integration of gene expression and tumor-infiltrating lymphocytes (TILs) to predict pCR after neoadjuvant chemotherapy and nivolumab for patients with luminal B-like breast cancer in the phase II GIADA trial

We evaluated the impact of PAM50 molecular subtypes and gene expression signatures on the pathological response to neoadjuvant chemotherapy followed by Nivolumab for patients with Luminal B-like breast cancer.

Clinical, pathological, and PAM50 gene expression features of HER2-low breast cancer

Novel antibody-drug conjugates against HER2 are showing high activity in HER2-negative breast cancer (BC) with low HER2 expression (i.e., 1+ or 2+ and lack of ERBB2 amplification). However, the clinical and molecular features of HER2-low BC are yet to be elucidated. Here, we collected retrospective clinicopathological and PAM50 data from 3,689 patients with HER2-negative disease and made the following observations. First, the proportion of HER2-low was higher in HR-positive disease (65.4%) than triple-negative BC (TNBC, 36.6%). Second, within HR-positive disease, ERBB2 and luminal-related genes were more expressed in HER2-low than HER2 0. In contrast, no gene was found differentially expressed in TNBC according to HER2 expression. Third, within HER2-low, ERBB2 levels were higher in HR-positive disease than TNBC. Fourth, HER2-low was not associated with overall survival in HR-positive disease and TNBC. Finally, the reproducibility of HER2-low among pathologists was suboptimal. This study emphasizes the large biological heterogeneity of HER2-low BC, and the need to implement reproducible and sensitive assays to measure low HER2 expression.

Olaparib monotherapy as primary treatment in unselected triple negative breast cancer

The antitumor efficacy of PARP inhibitors (PARPi) for breast cancer patients harboring germline BRCA1/2 (gBRCA1/2) mutations is well established. While PARPi monotherapy was ineffective in patients with metastatic triple negative breast cancer (TNBC) wild type for BRCA1/2, we hypothesized that PARPi may be effective in primary TNBCs without previous chemotherapy exposure. In the phase II PETREMAC trial, patients with primary TNBC >2 cm received olaparib for up to 10 weeks before chemotherapy. Tumor biopsies collected before and after olaparib underwent targeted DNA sequencing (360 genes) and BRCA1 methylation analyses. In addition, BRCAness (multiplex ligation-dependent probe amplification), PAM50 gene expression, RAD51 foci, tumor-infiltrating lymphocytes (TILs) and PD-L1 analyses were performed on pretreatment samples. The median pretreatment tumor diameter was 60 mm (range 25-112 mm). Eighteen out of 32 patients obtained an objective response (OR) to olaparib (56.3%). Somatic or germline mutations affecting homologous recombination (HR) were observed in 10/18 responders [OR 55.6%, 95% confidence interval (CI) 33.7-75.4] contrasting 1/14 non-responders (OR 7.1%; CI 1.3-31.5, P= 0.008). Among tumors without HR mutations, 6/8 responders versus 3/13 non-responders revealed BRCA1 hypermethylation (P= 0.03). Thus, 16/18 responders (88.9%, CI 67.2-96.9), in contrast to 4/14 non-responders (28.6%, CI 11.7-54.7, P= 0.0008), carried HR mutations and/or BRCA1 methylation. Excluding one gPALB2 and four gBRCA1/2 mutation carriers, 12/14 responders (85.7%, CI 60.1-96.0) versus 3/13 non-responders (23.1%, CI 8.2-50.3, P= 0.002) carried somatic HR mutations and/or BRCA1 methylation. In contrast to BRCAness signature or basal-like subtype, low RAD51 scores, high TIL or high PD-L1 expression all correlated to olaparib response. Olaparib yielded a high clinical response rate in treatment-naïve TNBCs revealing HR deficiency, beyond germline HR mutations. ClinicalTrials.gov identifier: NCT02624973.