Background

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).

Methods

Eighteen PDXs representative of HR+/HER2- (n=11, 61%), HER2+ (n=6, 33%) and triple-negative (n=1, 6%) BC were treated with ribociclib monotherapy (75 mg/kg/day). The % change in tumor volume from baseline was calculated at day 35. RNA was obtained from flash-frozen tumors at baseline and day 12. PAM50 GE was analyzed by nCounter and associated with tumor response (as a continuous variable) using quantitative Statistical Analysis Microarrays (SAM). Differential GE during ribociclib treatment was identified using two-class paired SAM. All SAM used a false-discovery rate<5%.

Results

Baseline PAM50 subtype distribution was Luminal B (44%), HER2-E (33%) and Basal-like (B-L) (22%). HER2-E and Luminal B PDXs showed a statistically significant higher response to ribociclib (mean change in volume >40% and >140%), than B-L (>660%). Baseline GE analysis identified 6 genes highly expressed in responders (FOXA1, ERBB2, GRB7, MLPH, GPR160 and CXXC5), and 7 lower expressed genes (SFRP1, KRT17, MYC, CDH3, KRT5, MIA and KRT14). Paired GE analyses across PDXs identified 12 upregulated genes during treatment, including estrogen activation-related genes (ESR1, PGR, FOXA1, MAPT or BLVRA); and 12 downregulated genes, including proliferation-related genes (MKI67 or KIF2C) and HER2-E-related genes (ERBB2 or TMEM45B). Similar results were obtained with HR+/HER2- PDXs when analyzed separately.

Conclusions

In BC PDXs, B-L biology associates with lower response to ribociclib monotherapy than Luminal or HER2-E. Ribociclib induces a luminal phenotype with high GE of estrogen-regulated genes and low GE of proliferation genes, a biological switch that could explain the better efficacy of ribociclib in the endocrine therapy (ET)-resistant HER2-E subtype observed in clinical trials when combined with ET.

Background

Obesity is associated with T-cell dysfunction and reduced antitumor immune response. In TNBC, high sTILs seem to predict pathologic complete response (pCR) and favorable prognosis only in normal weight patients (Desmedt et al. Cancer Res 2020).

Methods

TNBC patients, who received anthracycline-taxane-based chemotherapy in GeparDuo, GeparTrio, GeparQuinto, GeparSixto, GeparSepto, and GeparOcto, with available BMI and pretreatment sTILs (centrally assessed) were considered. Patients with BMI <18.5 kg/m² were excluded. Associations between BMI (normal weight, 18.5-<25 vs overweight/obese ≥25 kg/m²), sTILs (high ≥30% vs low <30) and pCR were assessed using Fisher`s exact test; between sTILs (continuous, dichtomized) and pCR (ypT0/is ypN0) according to BMI and interaction BMI*sTILs by logistic regression, between sTILs and disease-free survival (DFS) according to BMI and interaction BMI*sTILs by Cox regression.

Results

Of 1288 patients, 49.8% were normal weight, 50.2% overweight/obese; median age was 47 [21-78] vs 50 [21-76] years (p<0.001), cT3-4 12.0% vs 16.6% (p=0.021) and N+ 32.7% vs 37.0% (p=0.125). Normal weight patients had a higher pCR than overweight/obese patients (47.2% vs 39.9% p=0.009). Median sTILs was 30%, 50.4% of patients had high sTILs (normal weight 50.2% vs overweight/obese 50.6%, p=0.868). Higher level of sTILs was predictive for pCR both in normal weight (sTILs continuous OR 1.10, 95%CI 1.03-1.17, p=0.002) and in overweight/obese patients (OR 1.15, 95%CI 1.08-1.22, p<0.001). Interaction BMI*sTILs, p=0.302. Results were similar for dichotomized sTILs. Median follow-up was 54.7 months. Each 10% increase in sTILs was associated with an 11% reduction in the risk of a DFS event in normal weight (HR 0.89, 95%CI 0.82-0.95, p=0.001) and 8% in overweight/obese patients (HR 0.92, 95%CI 0.87-0.99, p=0.017). Interaction TILs*BMI, p=0.366.

Conclusions

Our results do not confirm differences in the predictive and prognostic role of sTILs according to BMI in TNBC as described previously. A joint analysis to explore the source of observed heterogeneity is planned.

Background

Breast cancer (BC) diagnosed at ages <40 years is known to present more aggressive tumor phenotypes and poorer clinical outcome. To gain a better understanding of the age-related expression of BC promoting genes, we aimed to identify transcriptional alterations supporting cancer progression in BC of young women.

Methods

We studied mRNA gene expression data from two METABRIC cohorts. Differentially expressed genes (DEGs) and gene sets (Gene Set Enrichment Analysis) differentially enriched between primary BC for patients <40/≥40 years were explored. Protein-protein interaction (PPI) networks were explored by the STRING database and visualized by Cytoscape software. Hub genes were identified by MCODE and CytoHubba plugins. Drug signatures were explored by Connectivity Map (CMAP).

Results

We identified 91 and 99 commonly up- and downregulated DEGs in women <40 in the two METABRIC cohorts. Gene sets reflecting proliferation were among the top results reported (REACTOME and gene ontology; FDR <10%). Six hub genes presenting highest PPI network connectivity were identified. High signature score (sum of hub genes expression values) was significantly associated with high tumor diameter, histologic grade, lymph node metastasis, ER negativity, basal-like and HER2 enriched subtypes (P <0.001). The signature score showed strong correlation with proliferation signatures OncotypeDx and PCNA (ρ=0.90-0.96, P <0.001), and associated with reduced cancer specific survival (P <0.001), also when adjusted for traditional clinico-pathologic variables in both cohorts (HR: 1.074-1.081, 95% CI: 1.046-1.109, P ≤0.003). The signature score associated with survival when adjusting for traditional clinico-pathologic variables in OncotypeDx-low tumors in both cohorts (HR: 1.54-1.077, 95% CI: 1.034-1.251, P <0.001). By CMAP, inhibitors of CDK, PI3K and AURKA are suggested with potential relevance for BC of the young.

Conclusions

The current study provides new insights into age-related gene expression alterations in BC. We found evidence of higher tumor cell proliferation in young BC patients, and identified a gene expression signature reflecting tumor proliferation, aggressive tumor features and reduced survival, also in subsets of low OncotypeDx score.

Background

Splicing is a central RNA-based process commonly altered in human cancers; however, how spliceosomal components are co-opted during tumorigenesis remains poorly defined. Previous work on MYC-driven triple negative breast cancer (TNBC) has revealed the spliceasome as an essential vulnerability that can be targeted to achieve disease control. Therefore, an important outstanding question is the contribution of individual components of the spliceosome machinery to the tumorigenic process downstream of MYC hyperactivation.

Methods

Our data is supported by extensive mechanistic analysis of spliceosome regulatory dynamics using both in vitro and in vivo models as well as patient material (RNA seq and tissue microarray of a cohort of TNBC patients as well as publicly available datasets).

Results

We unravel that specific spliceosomal components are translationally regulated during oncogenic stress. Indeed, we found that MYC enables SF3A3 translation through an eIF3D-dependent mechanism. This ensures accurate splicing of mRNAs enriched for mitochondrial regulators. Altered SF3A3 translation leads to metabolic reprogramming and stem-like properties that fuel MYC tumorigenic potential in vivo. Furthermore, SF3A3 protein analysis reveals a dichotomous role associated with MYC activity in triple negative breast cancers. Our analysis of human TNBC patient samples provides strong evidence that SF3A3 post-transcriptional regulation is associated with unique gene expression signatures that stratify the clinical outcomes in these patients.

Conclusions

These findings unveil a post-transcriptional interplay between splicing and translation that governs critical facets of MYC-driven oncogenesis.

Background

PIK3CA represents one of the most mutated genes, both in hormone receptor-positive BC and HER2-positive ones. Several clinical trials demonstrated that the detection of PIK3CA mutations in ctDNA could represent a predictive biomarker of PI3K-Inhibitors treatment response. BELLE-2, BELLE-3, and SOLAR-1 trials prospectively showed a significant progression-free survival benefit in ctDNA PIK3CA-mutant patients, suggesting PIK3CA mutation detection in ctDNA could represent a PI3K-Is predictive biomarker. Still, it would seem that liquid biopsy could be potentially more reliable than a tissue one, as emerged from BELLE-2 study. Yet, the diagnostic accuracy of liquid biopsy in detecting mutations on PIK3CA in BC is still being evaluated.

Methods

Our purpose was to provide a comparative analysis of diagnostic accuracy between tissue and liquid biopsies. Moreover, we deeply investigated if different diagnostic molecular techniques could influence the accuracy of liquid biopsy in detecting PIK3CA mutation. Variables which could potentially affect the results (technique, tumor burden, time elapsed between tissue and blood samples collection) were considered. The pooled analysis was carried out on 25 works for a total of 1966 patients, for which matched tumor tissue and plasma specimens have been collected.

Results

The results proved a better performance of NGS-based technologies in terms of sensitivity and specificity compared to ddPCR/BEAMing and Real Time-Polymerase Chain Reaction (RT-PCR). In particular, we reported AUC values for NGS-based assays of 0,98 (ddPCR/BEAMing 0,92; PCR 0,77), resulting in absence of significant heterogeneity for the pooled specificity (I² = 0 %, Cochran’s Q test p-value = 0,52)

Conclusions

We would suggest the use of NGS rather than conventional RT-PCR to screen the presence of a PIK3CA mutation in BC patients. Finally, given the not well-established applications of ctDNA analysis in the clinical management of BC, this meta-analysis could add intriguing insights supporting the introduction of liquid biopsy in clinical practice as a routine procedure.

Background

Hypoxia occurs in most solid tumors including BC and may negatively impact treatment response. We investigate the incidence of BACH1 (a key regulator of mitochondrial metabolism) and HIF1α (a hypoxia-inducible factor) expressions and their correlation with clinical outcomes in early HER2-negative BC.

Methods

We correlated tumor BACH1 and HIF1α mRNA expression from available RNA-Seq data with pathological complete response (pCR), disease-free survival (DFS) and overall survival (OS) in the GeparSepto trial (NCT01583426), which randomized pts with early-stage BC to either neoadjuvant solvent-based paclitaxel (P) or nP followed by EC. BACH1 and HIF1α values were analyzed as a continuous variable and categorized as low and high by median cut-off. Multivariate logistic (for pCR) and Cox (DFS and OS) regressions were used to adjust for age, cT, and cN.

Results

RNA-Seq expression data was available for 279 out of 810 HER2-negative BC pts, median age was 49 years (range 22-76) and median follow-up 49.8 months (range 2.3-62.4). There was a positive correlation between BACH1 and HIF1A expression levels (Pearson correlation 0.498). Overall, BACH1 and HIF1α continuous expressions were significantly associated with increased pCR (OR=4.21 [95%CI 1.44-12.30), p=0.009), OR=2.08 [95%CI 1.14-3.82], p=0.018, respectively) but did not impact survival in multivariate models. The table shows effects of BACH1 and HIF1α expression in nP vs P arm, both high BACH1 and HIF1α expressions were significant independent predictors of pCR and were associated with numerically better survival.

Conclusions

HER2-negative BC with elevated BACH1 and HIF1α expression benefits from nP-based treatment. There was a positive correlation between increased BACH1 and HIF1α levels for predicting pCR. We suggest that high BACH1 and HIF1α expressions enhance the efficacy of nP treatment, perhaps through triggering the hypoxic tumor adaptation.

Clinical trial identification

ClinicalTrials.gov identifier: NCT01583426

Background

HER2-low breast cancer (BC) is defined by an immunohistochemistry (IHC) assay score 1+ or 2+ with negative in situ hybridization (ISH). Genomic characterization of this subset of BC is still limited. We aimed to compare the genomic alterations found in a subgroup of HER2-low metastatic BC versus HER2-zero tumors (IHC score 0) in order to identify potential biomarkers.

Methods

A retrospective analysis was carried out in a sample of 121 metastatic BC patients from Hospital Universitario 12 de Octubre and HM CIOCC (Madrid, Spain) collected from 2017-2020. Next-generation sequencing (NGS) was performed in formalin-fixed paraffin embedded (FFPE) samples of BC. Biopsies included both primary or metastatic tumor, whichever most recent/accesible was available. Somatic mutations and copy number variations (gains and loss) were gathered from the NGS reports. Two-sided Chi-Square test was used for comparisons of proportions between groups and p values below 0.05 were deemed statistically significant.

Results

We identified a total of 67 HER2-low and 54 HER2-zero breast carcinomas. Both groups were similar in the median age of patients, menopausal status and metastatic pattern. The most frequent phenotype was luminal B for both groups, with a significantly higher prevalence in HER2-low carcinomas (65.7% and 37.0%, p<0.01). The most frequently mutated genes were (Table 1) PIK3CA (31.3% vs 35.2%, p=0.34) and TP53 (34.3% vs 48.2%, p=0.12) in both HER2-low and HER2-zero categories, respectively. We observed a higher prevalence of FGFR1 amplification (defined as ≥10 copy number gain) in the HER2-low group (12% vs 1.8%, p=0.03) compared to HER2-zero carcinomas.

Conclusions

In our sample, PIK3CA and TP53 were the most frequently mutated genes in both HER2-low and HER2-zero breast carcinomas. FGFR1 amplification was more prevalent in the HER2-low category compared to HER2-zero tumors. This finding needs to be further confirmed as a potential target in this subset of patients.

Background

The HER2-E subtype within HR+/HER2- ABC represents 10-25% and is characterized by poor prognosis. In the MONALEESA program, ribociclib + ET was found highly active in this subtype compared to ET. Here, we explored the prognostic and predictive value of HER2-E in patients (pts) treated with a CDK4/6 inhibitor (CDK4/6i) + ET in the real-world setting.

Methods

This is a retrospective study of 144 consecutive pts with HR+/HER2- ABC treated with a CDK4/6i + ET in the first line setting from 2014-2020 in Hospital 12 de Octubre (Madrid) and Clinic of Barcelona. Research-based PAM50 was performed in FFPE tumors collected before CDK4/6i (primary tumors or metastatic biopsies). Univariate and multivariable cox model progression-free survival (PFS) analyses were performed adjusting for the presence of visceral or “de novo” disease, menopausal status and performance status.

Results

114 pts (79%) had PAM50 data (50% primary/50% metastatic), and 47%/46%/7% received palbociclib/ribociclib/abemaciclib. Subtype distribution: Luminal A (33%), Luminal B (37%), HER2-E (19%), normal-like (8%) and Basal-like (5%). Median PFS for HER2-E disease was 7.4 months (mo) (95% confidence interval [CI] 5.0-22.0) and 21.1 mo (95% CI 16.6-31.3) for non-HER2-E disease (adjusted hazard ratio [aHRatio]=2.38, p=0.010). Median OS for HER2-E disease was 30.9 months (mo) (95% confidence interval [CI] 13.2-not reached [NR]) and NR (95% CI 47.2-NR) for non-HER2-E disease (aHRatio=4.39, p=0.021). Although exploratory and statistically non-significant, the PFS HRatio estimates of ribociclib vs palbociclib/abemaciclib in Luminal A, Luminal B and HER2-E subtype were 0.88, 0.90 and 0.44, respectively. Finally, the overall response rate of ribociclib in Luminal A/B and HER2-E disease was 40.5% and 42.9% vs 36.8% and 25.0% with palbociclib/abemaciclib.

Conclusions

In a real-world setting, we confirmed the poor prognosis of the HER2-E subtype in HR+/HER2- ABC. Despite the limitations, our results support the observation from the ML program where ribociclib in combination with ET was particularly active in the HER2-E subtype.

Background

Several genomic assays are available to profile early breast cancer (BC) that, according to current evidence, can provide reliable information including the risk of recurrence. However, little is known regarding their current use and the perception of utility across Europe. The PROCURE project aims to develop a consensus on the utility of breast cancer multigene signatures (BCMS) in treatment decision making for different eBC patient profiles based on the opinion of a panel of experts.

Methods

A Scientific Committee of 8 experts in BC from 8 European countries developed a Delphi questionnaire to be administered in two-waves to experienced clinicians across Europe, selected based on their experience in BC. The questionnaire includes 5 sections in order to characterize the participants and their expertise in BCMS, to understand the current clinical practice in eBC and the use of BCMS, to recall the participant’s opinion on the utility of the BCMS in eBC according to the patient profiles, to define recommendations on the use of BCMS in clinical practice and finally, to identify unmet needs and future applications of BCMS. 180 participants, including medical oncologists, surgeons, pathologists and gynaecologists, are expected to answer anonymously the online Delphi questionnaire. 70% agreement will be used to determine consensus on a topic.

Results

At the end of January 2021, 146 participants from 11 European countries (Austria, Denmark, France, Germany, Italy, Norway, Portugal, Spain, Sweden, Switzerland and the UK) registered to participate. 61 of them had already fully completed the 1^st wave Delphi questionnaire. Results from the 1^st wave will be presented to engage larger discussion with congress participants.

Conclusions

The PROCURE Project will provide useful information regarding how BCMS are currently used in clinical practice across Europe and will help to measure the utility attributed to the different BCMS by BC experts for their daily clinical practice, to establish recommendations on the use of BCMS to make treatment decision in different eBC patient profiles and to define current unmet needs and future applications of BCMS according to experts point of view.

Editorial acknowledgement

Adelphi Targis SL provided editorial assistance in the writing of the abstract.

Background

Methods

Results

Conclusions

Background

De-novo metastatic breast cancer (MBC) represents a distinct entity, characterized by different genomic and prognostic features compared to relapsed MBC. Although evidence suggests that half of all breast cancers show low HER2 expressions, and that HER2 expressions may increase between the early and metastatic stage, little is known on HER2-low expressions features of de-novo MBC.

Methods

We retrospectively reviewed clinical-pathological data of breast cancer patients consecutively referred to our New Drugs Division (from Jan2014 to Dec2019) with an available pathological analysis of the tumor (on either the primary lesion or a metastatic site). We divided HER2-negative cases by ASCO/CAP 2018 guidelines into an IHC 0 subgroup and a HER2-low subgroup (1+ and 2+/ISH-negative). χ2-test was used for comparisons between categorical parameters.

Results

442 breast cancer patients were eligible for the analysis, of whom 17% (N=75) had de-novo MBC. 65% of all de-novo MBC were luminal-like, 20% were HER2-positive and 15% were triple negative.
43% (n=32) of de-novo MBC showed low HER2-expressions, more commonly among luminal-like (49% HER2-low) than triple negative tumors (32% HER2-low), although this difference was not statistically significant (p=0.17).
Compared to de-novo MBC, low HER2-expressions were significantly more common in relapsed MBC (57% vs 43%, p< 0.03). By comparing HER2-expression in relapsed MBC based on the site of sampling, HER2-low expression rate did not differ if the biopsy was performed on breast/lymph nodes or in visceral sites (68% vs 58%, p=0.1). No significant difference was found in low HER2-expression between de-novo MBC and early stage breast cancers (43% vs 44%, p=0.98).

Conclusions

De-novo MBC shows a rate of low HER2-expression comparable with early breast cancer, and significantly lower compared with relapsed MBC. This difference did not appear to be related to the biopsy site. Confirmation of these observations on larger populations of patients is warranted.

Background

In recent years, studies have shown that GATA3 can promote the expression of ERα and inhibit tamoxifen (TAM) resistance, whereas miR-221/222 could promote epithelial-to-mesenchymal transition (EMT), which could result in TAM resistance. Moreover, GATA3 also inhibits EMT. However, the specific mechanism by which miR-221/222 interacts with GATA3 and induces TAM resistance remains unknown. Our study aims to investigate the regulatory mechanism of miR-221/222 on GATA3 expression and clarify its regulatory role in the process of TAM resistance.

Methods

After the establishment of TAM-resistant MCF7 cell lines, we detected and compared the endogenous expression levels of miR-221/222, GATA3, ERα, E-cadherin and vimentin in MCF7 and MCF-TAM^R strains, as well as in different breast cancer cell lines, by PCR and western blotting. After overexpressing miR-221/222 in MCF7 cell lines, we characterized the GATA3, ERα and E-cadherin protein levels, and evaluated the migration and invasion ability of MCF7 cells by comparing wound healing and transwell migration with or without a miR-221/222 inhibitor. In order to confirm the effect of miR221/222 and GATA 3 on TAM resistance, a Cell Counting Kit-8 (CCK8) assay was used to quantify TAM resistance in the MCF-TAM^R cell lines.

Results

In the TAM-resistant and ERα-negative breast cancer cell lines, endogenous expression of miR-221/222 and vimentin were higher, whereas the expression of GATA3, ERα and E-cadherin was relatively low. After overexpressing miR-221/222, the protein levels of GATA3, ERα and E-cadherin decreased, indicating an inverse correlation between miR-221/222 and GATA3, ERα and E-cadherin. Wound healing and transwell experiments showed that miR221/222 promoted the migration and invasion of breast cancer cells, and CCK8 assay demonstrated that miR-221/222 can promote TAM resistance and that GATA 3 can reverse it.

Conclusions

Overall, our results suggest that miR-221/222 may down-regulate GATA3 expression, thereby down-regulating ERα and promoting breast cancer EMT to ultimately promote tamoxifen resistance. This regulating network may be due to the targeting of miR-221/222 to the 3’ UTR of GATA3 mRNA.