Angel Guerrero-Zotano

A polymorphism at the 3'-UTR region of the aromatase gene defines a subgroup of postmenopausal breast cancer patients with poor response to neoadjuvant letrozole

BackgroundAromatase (CYP19A1) regulates estrogen biosynthesis. Polymorphisms in CYP19A1 have been related to the pathogenesis of breast cancer (BC). Inhibition of aromatase with letrozole constitutes the best option for treating estrogen-dependent BC in postmenopausal women. We evaluate a series of polymorphisms of CYP19A1 and their effect on response to neoadjuvant letrozole in early BC.MethodsWe analyzed 95 consecutive postmenopausal women with stage II-III ER/PgR [+] BC treated with neoadjuvant letrozole. Response to treatment was measured by radiology at 4th month by World Health Organization (WHO) criteria. Three polymorphisms of CYP19A1, one in exon 7 (rs700519) and two in the 3-UTR region (rs10046 and rs4646) were evaluated on DNA obtained from peripheral blood.ResultsThirty-five women (36.8%) achieved a radiological response to letrozole. The histopathological and immunohistochemical parameters, including hormonal receptor status, were not associated with the response to letrozole. Only the genetic variants (AC/AA) of the rs4646 polymorphism were associated with poor response to letrozole (p = 0.03). Eighteen patients (18.9%) reported a progression of the disease. Those patients carrying the genetic variants (AC/AA) of rs4646 presented a lower progression-free survival than the patients homozygous for the reference variant (p = 0.0686). This effect was especially significant in the group of elderly patients not operated after letrozole induction (p = 0.009).ConclusionsOur study reveals that the rs4646 polymorphism identifies a subgroup of stage II-III ER/PgR [+] BC patients with poor response to neoadjuvant letrozole and poor prognosis. Testing for the rs4646 polymorphism could be a useful tool in order to orientate the treatment in elderly BC patients.

PI3K/AKT/mTOR: role in breast cancer progression, drug resistance, and treatment.

Anti-cancer cancer-targeted therapies are designed to exploit a particular vulnerability in the tumor, which in most cases results from its dependence on an oncogene and/or loss of a tumor suppressor. Mutations in the phosphoinositide 3-kinase (PI3K)/AKT/mTOR pathway are freqcuently found in breast cancers and associated with cellular transformation, tumorigenesis, cancer progression, and drug resistance. Several drugs targeting PI3K/ATK/mTOR are currently in clinical trials, mainly in combination with endocrine therapy and anti-HER2 therapy. These drugs are the focus of this review.

Intrinsic subtypes and gene expression profiles in primary and metastatic breast cancer

Biological changes that occur during metastatic progression of breast cancer are still incompletely characterized. In this study, we compared intrinsic molecular subtypes and gene expression in 123 paired primary and metastatic tissues from breast cancer patients. Intrinsic subtype was identified using a PAM50 classifier and χ2 tests determined the differences in variable distribution. The rate of subtype conversion was 0% in basal-like tumors, 23.1% in HER2-enriched (HER2-E) tumors, 30.0% in luminal B tumors, and 55.3% in luminal A tumors. In 40.2% of cases, luminal A tumors converted to luminal B tumors, whereas in 14.3% of cases luminal A and B tumors converted to HER2-E tumors. We identified 47 genes that were expressed differentially in metastatic versus primary disease. Metastatic tumors were enriched for proliferation-related and migration-related genes and diminished for luminal-related genes. Expression of proliferation-related genes were better at predicting overall survival in metastatic disease (OSmet) when analyzed in metastatic tissue rather than primary tissue. In contrast, a basal-like gene expression signature was better at predicting OSmet in primary disease compared with metastatic tissue. We observed correlations between time to tumor relapse and the magnitude of changes of proliferation, luminal B, or HER2-E signatures in metastatic versus primary disease. Although the intrinsic subtype was largely maintained during metastatic progression, luminal/HER2-negative tumors acquired a luminal B or HER2-E profile during metastatic progression, likely reflecting tumor evolution or acquisition of estrogen independence. Overall, our analysis revealed the value of stratifying gene expression by both cancer subtype and tissue type, providing clinicians more refined tools to evaluate prognosis and treatment. Cancer Res; 77(9); 2213-21. ©2017 AACR.

MYC and MCL1 Cooperatively Promote Chemotherapy-Resistant Breast Cancer Stem Cells via Regulation of Mitochondrial Oxidative Phosphorylation

Most patients with advanced triple-negative breast cancer (TNBC) develop drug resistance. MYC and MCL1 are frequently co-amplified in drug-resistant TNBC after neoadjuvant chemotherapy. Herein, we demonstrate that MYC and MCL1 cooperate in the maintenance of chemotherapy-resistant cancer stem cells (CSCs) in TNBC. MYC and MCL1 increased mitochondrial oxidative phosphorylation (mtOXPHOS) and the generation of reactive oxygen species (ROS), processes involved in maintenance of CSCs. A mutant of MCL1 that cannot localize in mitochondria reduced mtOXPHOS, ROS levels, and drug-resistant CSCs without affecting the anti-apoptotic function of MCL1. Increased levels of ROS, a by-product of activated mtOXPHOS, led to the accumulation of HIF-1α. Pharmacological inhibition of HIF-1α attenuated CSC enrichment and tumor initiation inxa0vivo. These data suggest that (1) MYC and MCL1 confer resistance to chemotherapy by expanding CSCs via mtOXPHOS and (2) targeting mitochondrial respiration and HIF-1α may reverse chemotherapy resistance in TNBC.

Association of FGFR1 with ERα Maintains Ligand-Independent ER Transcription and Mediates Resistance to Estrogen Deprivation in ER+ Breast Cancer

Purpose: FGFR1 amplification occurs in approximately 15% of estrogen receptor–positive (ER+) human breast cancers. We investigated mechanisms by which FGFR1 amplification confers antiestrogen resistance to ER+ breast cancer. Experimental Design: ER+ tumors from patients treated with letrozole before surgery were subjected to Ki67 IHC, FGFR1 FISH, and RNA sequencing (RNA-seq). ER+/FGFR1–amplified breast cancer cells, and patient-derived xenografts (PDX) were treated with FGFR1 siRNA or the FGFR tyrosine kinase inhibitor lucitanib. Endpoints were cell/xenograft growth, FGFR1/ERα association by coimmunoprecipitation and proximity ligation, ER genomic activity by ChIP sequencing, and gene expression by RT-PCR. Results: ER+/FGFR1–amplified tumors in patients treated with letrozole maintained cell proliferation (Ki67). Estrogen deprivation increased total and nuclear FGFR1 and FGF ligands expression in ER+/FGFR1–amplified primary tumors and breast cancer cells. In estrogen-free conditions, FGFR1 associated with ERα in tumor cell nuclei and regulated the transcription of ER-dependent genes. This association was inhibited by a kinase-dead FGFR1 mutant and by treatment with lucitanib. ChIP-seq analysis of estrogen-deprived ER+/FGFR1–amplified cells showed binding of FGFR1 and ERα to DNA. Treatment with fulvestrant and/or lucitanib reduced FGFR1 and ERα binding to DNA. RNA-seq data from FGFR1-amplified patients tumors treated with letrozole showed enrichment of estrogen response and E2F target genes. Finally, growth of ER+/FGFR1–amplified cells and PDXs was more potently inhibited by fulvestrant and lucitanib combined than each drug alone. Conclusions: These data suggest the ERα pathway remains active in estrogen-deprived ER+/FGFR1–amplified breast cancers. Therefore, these tumors are endocrine resistant and should be candidates for treatment with combinations of ER and FGFR antagonists. Clin Cancer Res; 23(20); 6138–50. ©2017 AACR.

An ERBB1-3 Neutralizing Antibody Mixture With High Activity Against Drug-Resistant HER2+ Breast Cancers With ERBB Ligand Overexpression

BackgroundnPlasticity of the ERBB receptor network has been suggested to cause acquired resistance to anti-human epidermal growth factor receptor 2 (HER2) therapies. Thus, we studied whether a novel approach using an ERBB1-3-neutralizing antibody mixture can block these compensatory mechanisms of resistance.nnnMethodsnHER2+ cell lines and xenografts (n ≥u20096 mice per group) were treated with the ERBB1-3 antibody mixture Pan-HER, trastuzumab/lapatinib (TL), trastuzumab/pertuzumab (TP), or T-DM1. Downregulation of ERBB receptors was assessed by immunoblot analysis and immunohistochemistry. Paired pre- and post-T-DM1 tumor biopsies from patients (nu2009=u200911) with HER2-amplified breast cancer were evaluated for HER2 and P-HER3 expression by immunohistochemistry and/or fluorescence in situ hybridization. ERBB ligands were measured by quantitative reverse transcription polymerase chain reaction. Drug-resistant cells were generated by chronic treatment with T-DM1. All statistical tests were two-sided.nnnResultsnTreatment with Pan-HER inhibited growth and promoted degradation of ERBB1-3 receptors in a panel of HER2+ breast cancer cells. Compared with TL, TP, and T-DM1, Pan-HER induced a similar antitumor effect against established BT474 and HCC1954 tumors, but was superior to TL against MDA-361 xenografts (TL mean = 2026u2009mm 3 , SDu2009=u2009924u2009mm 3 , vs Pan-HER mean = 565u2009mm 3 , SDu2009=u2009499u2009mm 3 , P = .04). Pan-HER-treated BT474 xenografts did not recur after treatment discontinuation, whereas tumors treated with TL, TP, and T-DM1 did. Post-TP and post-T-DM1 recurrent tumors expressed higher levels of neuregulin-1 (NRG1), HER3 and P-HER3 (all P < .05). Higher levels of P-HER3 protein and NRG1 mRNA were also observed in HER2+ breast cancers progressing after T-DM1 and trastuzumab (NRG1 transcript fold change ± SD; pretreatment = 2, SDu2009=u20091.9, vs post-treatment = 11.4, SDu2009=u200910.3, P = .04). The HER3-neutralizing antibody LJM716 resensitized the drug-resistant cells to T-DM1, suggesting a causal association between the NRG1-HER3 axis and drug resistance. Finally, Pan-HER treatment inhibited growth of HR6 trastuzumab- and T-DM1-resistant xenografts.nnnConclusionsnThese data suggest that upregulation of a NRG1-HER3 axis can mediate escape from anti-HER2 therapies. Further, multitargeted antibody mixtures, such as Pan-HER, can simultaneously remove and/or block targeted ERBB receptor and ligands, thus representing an effective approach against drug-sensitive and -resistant HER2+ cancers.

ER+ Breast Cancers Resistant to Prolonged Neoadjuvant Letrozole Exhibit an E2F4 Transcriptional Program Sensitive to CDK4/6 Inhibitors

Purpose: This study aimed to identify biomarkers of resistance to endocrine therapy in estrogen receptor–positive (ER+) breast cancers treated with prolonged neoadjuvant letrozole. Experimental Design: We performed targeted DNA and RNA sequencing in 68 ER+ breast cancers from patients treated with preoperative letrozole (median, 7 months). Results: Twenty-four tumors (35%) exhibited a PEPI score ≥4 and/or recurred after a median of 58 months and were considered endocrine resistant. Integration of the 47 most upregulated genes (log FC > 1, FDR < 0.03) in letrozole-resistant tumors with transcription-binding data showed significant overlap with 20 E2F4-regulated genes (P = 2.56E−15). In patients treated with the CDK4/6 inhibitor palbociclib before surgery, treatment significantly decreased expression of 24 of the 47 most upregulated genes in letrozole-resistant tumors, including 18 of the 20 E2F4 target genes. In long-term estrogen-deprived ER+ breast cancer cells, palbociclib also downregulated all 20 E2F4 target genes and P-RB levels, whereas the ER downregulator fulvestrant or paclitaxel only partially suppressed expression of this set of genes and had no effect on P-RB. Finally, an E2F4 activation signature was strongly associated with resistance to aromatase inhibitors in the ACOSOG Z1031B neoadjuvant trial and with an increased risk of relapse in adjuvant-treated ER+ tumors in METABRIC. Conclusions: In tumors resistant to prolonged neoadjuvant letrozole, we identified a gene expression signature of E2F4 target activation. CDK4/6 inhibition suppressed E2F4 target gene expression in estrogen-deprived ER+ breast cancer cells and in patients ER+ tumors, suggesting a potential benefit of adjuvant CDK4/6 inhibitors in patients with ER+ breast cancer who fail to respond to preoperative estrogen deprivation. Clin Cancer Res; 24(11); 2517–29. ©2018 AACR.

Abstract 4508: Genomic profiling of ER+ breast cancers treated with prolonged neoadjuvant letrozole reveal a high frequency of NOTCH2 mutations in clinically resistant tumors

Hypothesis: Approximately 20% of patients with early ER+ breast cancer (BC) treated with adjuvant antiestrogen therapy eventually relapse with endocrine-resistant metastatic disease. We hypothesized that profiling newly diagnosed ER+ breast tumors that persist following prolonged estradiol deprivation with letrozole would identify genomic alterations causally associated with endocrine resistance. Methods: We treated 57 postmenopausal women (median 77 years; range 60-86) with ER+/HER2- early BC with neoadjuvant letrozole (median 7.5 months; range 3-36) followed by surgery and adjuvant endocrine therapy. Patients were followed with serial ultrasounds and defined as non-responders if they developed recurrent locally or metastatic disease, or had a preoperative endocrine response index (PEPI) ≥4 (composite score of post-treatment ER, Ki67, T and N status). DNA extracted from post-treatment specimens was profiled using targeted exome sequencing of 300 cancer-related genes. We screened for variants with a high probability of disrupting protein function and excluded variants likely to be germline by filtering out every alteration not present in the Catalogue of Somatic Mutations in Cancer (COSMIC), if the variant had an allele frequency >0.2% as per the Exome Aggregation Consortium (ExAC) dataset. Results: Ten (10) patients had a PEPI 0 score, 32 patients were PEPI 1-3, and 15 were PEPI ≥4. After a median follow-up of 50 months (range 12-100), 9 patients have recurred with metastatic disease (4 with PEPI1-3, 5 with PEPI≥4). We identified 535 variants with a median coverage >200x (387 nonsynonymous, 23 stop-gain, 124 indels). The median number of mutations was similar in responder vs non responders (8.03 vs 8.6). Recurrently mutated genes (>5%) included PIK3CA (38%), NOTCH2 (31%), KMT2C (22%), VEZF1 (21%), TP53 (12%), NF1 (9%), MTOR (9%), ESR1 (7%) and ERBB2 (5%). Recurrent amplifications were detected in CCND1 (15%), ERBB2 (12%), FGF19 (12%) and FGFR1 (8%). The only mutations enriched in the non-responders and that correlated with long-term outcome were those in NOTCH2. The relapse-free survival rate at 40 months for patients with NOTCH2 mutations was 89% vs 41% for patients with wild-type NOTCH2 (p = 0.001). NOTCH2 mutations clustered as a frameshift deletion (P6fs) in exon 1, with a 20% average allele frequency. This mutation has been reported in TCGA and COSMIC at a mutation rate of 6%, across all tumor types and is predicted to produce an N-terminally truncated NOTCH2 protein (39 amino acids shorter) lacking the signal peptide. Conclusions: Genomic profiling of residual ER+ breast cancers treated with prolonged neoadjuvant letrozole revealed a high frequency of NOTCH2 mutations. These alterations in NOTCH2 may cause hormone independence in ER+ breast cancer and are worthy of further mechanistic and clinical investigation. Citation Format: Angel Guerrero-Zotano, Thomas Stricker, Katherine E. Hutchinson, Luigi Formisano, Jennifer Giltnane, Amparo Ruiz, Carlos L. Arteaga. Genomic profiling of ER+ breast cancers treated with prolonged neoadjuvant letrozole reveal a high frequency of NOTCH2 mutations in clinically resistant tumors. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 4508.

Monitoring cytokeratin19 (KRT19) levels as response of circulating tumor cells (CTC) to neoadjuvant chemotherapy (NAC) in breast cancer (BC).

CTRC-AACR San Antonio Breast Cancer Symposium: 2008 AbstractsnnAbstract #5022 nnBackground: KRT19 mRNA+ cells before the initiation of adjuvant chemotherapy has been shown as an independent prognostic factor for disease recurrence in patients with early BC. Its usefulness for monitoring response to NAC it is not clear. We have studied changes in KRT19 levels during NAC and correlated it with response. Material and Methods: We analyzed a prospective series of 70 BC patients treated with NAQ. Stage II (45%), Stage III (55%), HR negative (43%), HER-2 positive (42%). Treatment was based on sequential anthracyclines and taxanes. Trastuzumab was given concomitant in HER2 disease. KRT19 level expression was measured from lymphocytes of peripheral blood using a quantitative RT-PCR. The analysis was performed at diagnosis (baseline), before taxane administration, at the end of NAC, and after surgery. On the other hand, 27 healthy donors and 18 metastasic BC patients were also analyzed in order to determine a cut-off value for CTC positivity according to receiver operating characteristics [ROC] analysis. Association between variables was performed by Pearsons Chi-squared and Fishers tests. Results: We observed a differential rate in KRT19 expression, between the control and the metastasic series being higher in the last group (U-Mann-Withney test, p=0.010). According to the selected cut-off value (AUC 0.272; p=0.010 ), we detected CTC in 46/70 (65.7%) patients at baseline, 22/40 (55%) prior to taxane administration, 36/50 (72%) at the end of NAC and 13/17 (76.5%) after surgery.Forty out of 57 patients (70.2%) responded to NAC(15 CR and 25 PR). Decreased in CTCs levels during treatment wasnt associated with response, on the contrary, residual CTCs were detected after surgery in all those cases that responded (p=0.005). The presence of CTCs after completion of chemotherapy, previous to surgery, was inversely related to tumor stage (p=0.012). Negative tumors for HER2 and tumors with high levels of ER expression maintain the CTC levels detected at diagnosis (p=0.044 and p=0.057 respectively). DISCUSSION: We werent able to find a correlation between response and decrease in CTCs levels. The presence of residual CTCs in responding cases could suggest a release of tumor cells into blood by NAQ and surgery The prognostic factor of residual CTCs needs to be addressed with longer follow up, and taking into account the effect of others therapies. This study is financed by the grant GVA07/130. nnCitation Information: Cancer Res 2009;69(2 Suppl):Abstract nr 5022.