Abstract
Resistance to therapy in metastatic breast cancer patients results from acquired mutations and selection of resistant clones. Understanding the mechanisms of resistance in the clinical ...setting is complicated by the lack of genomic characterization of tumor specimens.
This case report describes a patient with ER+ and dynamic HER2+ status with exposure to a range of different therapies, suggesting that inference of tumor subclonal composition and evolution can be leveraged to study treatment resistance.
A female patient was diagnosed with estrogen receptor positive (ER+) and human epidermal growth factor receptor positive (HER2+) stage I (T1N0) breast cancer. After receiving standard of care adjuvant chemotherapy (AC-TH) and Tamoxifen for two years, the patient developed metastatic disease to the liver. Upon metastatic disease, she received first line treatment with the trastuzumab emtansine antibody-drug conjugate (T-DM1). After fifteen months, enlargement of supraclavicular and retroperitoneal lymph nodes was observed, Letrozole was added to the treatment regimen until the patient progressed after eight months. Subsequent therapy with Fulvestrant and the CDK4/6 inhibitor Palbocicilb was administered for three months until the patient developed brain metastases.
We analyzed biopsies taken at different time points of metastatic disease and, using whole exome sequencing (WES), reconstructed the subclonal architecture of each of the tumor specimens and inferred the evolutionary history of these clones. These results suggest potential mechanisms of resistance to the different therapies administered during disease progression.
Biopsies obtained originated from a retroperitoneal lymph node at 3 years after diagnosis, a liver metastasis at 4 years after diagnosis and a brain metastasis after 4 years and 3 months after diagnosis. WES was performed on these tumor specimens and characterization of mutational events and copy number alterations was made using standard analytical pipelines.
Inference of subclonal populations in the three tumor specimens analyzed revealed presence of a founding clone with mutations common to the three metastatic biopsies with alterations in TP53 p.K305* and PIK3CA p.E545K. A clone unique to the ER+/HER2- retroperitoneal lymph node metastasis shows no known driver mutations. In the liver metastasis (ER+/HER2-), we observed the presence of a dominant clone (cellular prevalence 93.5%) with mutation in ESR1 p.G442R and another subclone detected at low cellular prevalence (~5%) with an amplification in the ERBB2 gene. In the brain metastasis (ER+/HER2-), the latest tumor specimen obtained, we observed clonal selection of the ERBB2-amplified subpopulation (clonal prevalence 60%).
The subclonal population dynamics and the different alterations in the clones present at different time points of tumor evolution provide a rationale to understand the therapeutic sensitivity and resistance observed in this patient. The ER+/HER2- retroperitoneal lymph node metastasis which appeared under T-DM1 treatment, suggests that this subclone is resistant to T-DM1, explained by the HER2 negative status, and was selected while the patient was on this regime. At the time of appearance of this clone, Letrozole was added to the treatment and a clone with an ESR1 p.G442R mutation appeared and dominated the tumor population, suggesting that this mutation provides resistance to the aromatase inhibitor. When treatment was switched to Fulvestrant and Palbociclib, a potentially dormant clone with ER+ and HER2+ features re-appeared in the tumor.
Evolutionary analysis of this patient's tumor specimens underscores the importance of multiple therapies directed at all tumor vulnerabilities present, since targeting a single driver feature of the tumor will result in selection of other resistant subclones.
Citation Format: Jorge E. Buendia-Buendia, Sonia Pernas, Ofir Cohen, Ping Ping Mao, Kailey Kowalski, Nancy Lin, Eric Winer, Nikhil Wagle. Clonal heterogeneity and tumor evolution inform treatment resistance in a patient with HER2+ breast cancer abstract. In: Proceedings of the 2019 San Antonio Breast Cancer Symposium; 2019 Dec 10-14; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2020;80(4 Suppl):Abstract nr P3-01-09.
Remarkable progress in sequencing technology over the past 20 years has made it possible to comprehensively profile tumors and identify clinically relevant genomic alterations. In breast cancer, the ...most common malignancy affecting women, we are now increasingly able to use this technology to help specify the use of therapies that target key molecular and genetic dependencies. Large sequencing studies have confirmed the role of well-known cancer-related genes and have also revealed numerous other genes that are recurrently mutated in breast cancer. This growing understanding of patient-to-patient variability at the genomic level in breast cancer is advancing our ability to direct the appropriate treatment to the appropriate patient at the appropriate time—a hallmark of “precision cancer medicine.” This review focuses on the technological advances that have catalyzed these developments, the landscape of mutations in breast cancer, the clinical impact of genomic profiling, and the incorporation of genomic information into clinical care and clinical trials.
Young age at breast cancer diagnosis correlates with unfavorable clinicopathologic features and worse outcomes compared with older women. Understanding biological differences between breast tumors in ...young versus older women may lead to better therapeutic approaches for younger patients.
We identified 100 patients ≤35 years old at nonmetastatic breast cancer diagnosis who participated in the prospective Young Women's Breast Cancer Study cohort. Tumors were assigned a surrogate intrinsic subtype based on receptor status and grade. Whole-exome sequencing of tumor and germline samples was performed. Genomic alterations were compared with older women (≥45 years old) in The Cancer Genome Atlas, according to intrinsic subtype.
Ninety-three tumors from 92 patients were successfully sequenced. Median age was 32.5 years; 52.7% of tumors were hormone receptor-positive/HER2-negative, 28.0% HER2-positive, and 16.1% triple-negative. Comparison of young to older women (median age 61 years) with luminal A tumors (N = 28 young women) revealed three significant differences: PIK3CA alterations were more common in older patients, whereas GATA3 and ARID1A alterations were more common in young patients. No significant genomic differences were found comparing age groups in other intrinsic subtypes. Twenty-two patients (23.9%) in the Young Women's Study cohort carried a pathogenic germline variant, most commonly (13 patients, 14.1%) in BRCA1/2.
Somatic alterations in three genes (PIK3CA, GATA3, and ARID1A) occur at different frequencies in young versus older women with luminal A breast cancer. Additional investigation of these genes and associated pathways could delineate biological susceptibilities and improve treatment options for young patients with breast cancer. See related commentary by Yehia and Eng, p. 2209.
Abstract
Background: Deciphering the molecular landscape of resistance to the CDK4/6 inhibitors represents a critically important question for patients with hormone-receptor positive (HR+) metastatic ...breast cancer (MBC). Emerging insights from sequencing efforts suggest that inactivating alterations in the RB1 tumor suppressor occur in a small minority of patients and that a variety of heterogeneous mediators provoke resistance in patient samples. Proteins implicated in CDK4/6i resistance include cell cycle regulators such as cyclin E1/2, CDK6, and aurora kinase as well as known oncogenic signal transduction mediators involved in activation of the RAS-MEK and AKT-mTOR pathways. The insulin-like growth factor 1 receptor (IGF1R) has been implicated in modulating anti-estrogen resistance, and IGF1R inhibitors are currently in various stages of pre-clinical and clinical development.
Methods: We identified patients with amplification events in IGF1R from a database containing targeted sequencing of solid tumor samples obtained from patients with HR+ MBC enrolled on a research biopsy protocol. Tumor biopsies may have been obtained at various points during each patient’s clinical treatment course. HR+ T47D cells were modified to over-express IGF1R via lentiviral infection and selection. Derivative cell lines were treated with IGF-1 ligand and downstream activation of the PI3K/AKT and RAS/MEK pathways were assessed via western blotting. Control cells (expressing GFP) were mixed with IGF1R-expressing cells 1:1 and cultured in the presence of IGF-1 ligand and palbociclib or other drugs, for 1-3 weeks. At the timepoint of interest, cells were harvested and the relative proportion of GFP or IGF1R-expressing cells were interrogated via flow cytometry.
Results: We identified seven patients with HR+ MBC and IGF1R amplifications via targeted sequencing of tumor biopsies. Five of these patients had exposure to CDK4/6i-based therapy in the metastatic setting. Three patients demonstrated intrinsic resistance to CDK4/6i treatment (with duration <6 months) and biopsies were obtained prior to CDK4/6i exposure or, in one case, while on treatment. In an additional patient, after nine months of CDK4/6i-based therapy, an IGF1R amplification was present at the time of progression. In one counter-example, a baseline biopsy revealed IGF1R amplification and subsequent clinical benefit with CDK4/6i, exceeding 10 months, was noted. T47D cells over-expressing IGF1R demonstrated increased pERK and pAKT activation following introduction of IGF-1 ligand. Control GFP and IGF1R-expressing cells were plated 1:1 and cultured in the presence of IGF-1 ligand and palbociclib. In a flow cytometry-based competition assay, an IGF-1 dose-dependent increase in the relative proportion of IGF1R-expressing cells was noted after one, two, and three weeks of palbociclib treatment. The extent of IGF1R-expressing cell enrichment was attenuated in the presence of either a MEK inhibitor or an IGF1R inhibitor.
Conclusions: IGF1R amplification events were identified in tumor biopsy samples that reflect either intrinsic or acquired resistance to CDK4/6i-based therapy. HR+ breast cancer cells which over-express IGF1R demonstrate enrichment under palbociclib drug selection in a flow cytometry-based competition assay, which was abrogated by concurrent use of a MEK or IGF1R inhibitor. These results suggest that IGF1R may join the increasingly heterogeneous landscape of CDK4/6i resistance mediators. Further exploration of this possibility is warranted. A subset of patients with IGF1R-mediated CDK4/6i resistance could benefit from therapeutic strategies designed to downregulate MEK or IGF1R activity.
Citation Format: Seth A. Wander, Pingping Mao, Maxwell R. Lloyd, Gabriela N. Johnson, Kailey Kowalski, Utthara Nayar, Lillian M. Guenther, Kimberly Stegmaier, Eric P. Winer, Nancy U. Lin, Nikhil Wagle. Igf1r mediates cdk4/6 inhibitor (cdk4/6i) resistance in tumor samples and in cellular models abstract. In: Proceedings of the 2020 San Antonio Breast Cancer Virtual Symposium; 2020 Dec 8-11; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2021;81(4 Suppl):Abstract nr PD7-08.
Abstract
Background: Forkhead box A1 (FOXA1) is an essential pioneer transcription factor (TF) evoking other key TFs-mediated lineage-specific transcriptional programs in several endoderm-derived ...organs. Aberrant FOXA1 augmentation, via genetic alterations, occurs in 10-15% of ER+ primary and metastatic breast cancer (BC). We have recently shown that high levels of FOXA1 (H-FOXA1) induces enhancer and transcriptional reprogramming to promote endocrine-resistant (EndoR) and pro-metastatic phenotypes. Using the core transcriptional regulatory circuitry (CRC) mapping method, we identified the AP-1 TF JUNB as a key CRC component in BC cells expressing H-FOXA1. In this study, we aimed to further characterize key AP-1 components that play a role in mediating H-FOXA1-induced transcriptional reprogramming in EndoR and metastatic BC. Methods: The ROSE and HOMER tools were used to identify super-enhancers (SEs) and the predicted SE-harboring TFs in MCF7-parental (P) cells with ectopic FOXA1 overexpression (OE), and in MCF7 tamoxifen-resistant (TamR) cells with endogenous FOXA1 amplification and OE. Cell growth, migration, and co-immunoprecipitation assays, were performed using ER+ BC P cells with ectopic FRA1 OE. A FOXA1 core gene signature (GS) was deduced using the BETA.plus algorithm to analyze our previously reported RNA-seq and ChIP-seq data derived from MCF7-P cells with ectopic FOXA1 OE. Additional RNA-seq analyses include MCF7-P cells with ectopic FRA1 OE, FOXA1 OE and simultaneous FRA1 siRNA knockdown (KD), and MCF7-TamR cells with FRA1 KD. We identified a FOXA1/FRA1-centered GS and its clinical relevance was examined using expression profiles of TCGA, METABRIC, and a metastatic biopsy study from cohort of patients with ER+ metastatic BC from Dana-Farber Cancer Institute.Results: We identified FRA1 as one of the top TFs selectively harboring SEs at their gene loci in MCF7-TamR vs. P cells. Both FRA1 and JUNB expression was elevated in TamR vs. P cells and altered concordantly with FOXA1 in P and TamR cells upon FOXA1 OE or KD, respectively. As we identified JUNB as a CRC component with binding sites enriched at the SEs in BC cells expressing H-FOXA1, we hypothesized that FRA1 and JUNB form a feed-forward transcriptional axis amplifying H-FOXA1-induced enhancer reprogramming. We found that JUNB co-immunoprecipitated with FRA1 in MCF7-TamR and MCF7-P cells with ectopic FRA1 OE, suggesting that FRA1 forms a heterodimer with JUNB to exert AP-1 activity. Ectopic FRA1 OE reduced P cell endocrine sensitivity, increased cell migration, and elicited a transcriptome enriched for the FOXA1-induced core GS. In P cells with ectopic FOXA1 OE, we identified a FRA1-dependent GS (n = 27) that is highly enriched for interferon signaling. This FOXA1/FRA1 GS was highly expressed in luminal B vs. A subtype of primary tumors, further elevated in ER+ metastases, where its expression was positively correlated with FRA1 mRNA levels. Notably, this FOXA1/FRA1 GS was not dependent on FRA1 in P cells without FOXA1 OE, suggesting its relevance in the context of H-FOXA1.Conclusions: Here we show that a FOXA1/FRA1-centered transcriptional axis induces an interferon signaling-enriched GS associated with poor outcome of ER+ BC and metastasis. A FRA1/JUNB AP-1 complex may form a feed-forward transcriptional axis to amplify H-FOXA1 signaling. The FOXA1/FRA1-centered GS could be used to stratify patients with ER+ BC who may need additional targeted therapies. Further studies are warranted to elucidate the interplay between FOXA1 and FRA1/JUNB in regulating interferon signaling, which may guide approaches to improve patient outcomes, possibly with immunotherapy using immune checkpoint inhibitors.
Citation Format: Xiaoyong Fu, Resel Pereira, Lanfang Qin, Carmine De Angelis, Sarmistha Nanda, Martin Shea, Agostina Nardone, Rinath Jeselsohn, Ofir Cohen, Nikhil Wagle, Mothaffar Rimawi, C Kent Osborne, Rachel Schiff. A FOXA1/FRA1-centered transcriptional axis regulates interferon signaling in high FOXA1-associated endocrine-resistant and metastatic breast cancer abstract. In: Proceedings of the 2020 San Antonio Breast Cancer Virtual Symposium; 2020 Dec 8-11; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2021;81(4 Suppl):Abstract nr PD8-03.
To evaluate the success rate and long-term outcome of cyclocryotherapy for refractory pediatric glaucoma.
Retrospective interventional case series.
A total of 64 eyes of 49 patients from 2 ...institutions with pediatric glaucomas resistant to conventional medical and surgical therapies treated with cyclocryotherapy from 1975 to 1996 were included in this review.
Cyclocryotherapy was performed on eyes with pediatric glaucoma resistant to maximal medical and surgical interventions. Each cyclocryotherapy session was evaluated in terms of area treated, temperature, and number of applications placed.
Criteria for success included intraocular pressure (IOP) of 21 mmHg or less without devastating complications or need for further glaucoma surgery.
The mean baseline pretreatment IOP of all eyes was 30.0 ± 8.1 mmHg. Six months after their last treatment, 42 eyes (66%) were successes. Longer term follow-up (mean, 4.8 ± 3.3 years) yielded a lower final success rate in 28 eyes (44%). For these 28 eyes, mean IOP was reduced from 30.3 ± 7.8 mmHg pretreatment to 16.8 ± 4.0 mmHg after their last cyclocryotherapy treatment session (P < 0.001). The average number of cyclocryotherapy sessions for successful eyes was 4.1 ± 4.0 (range, 1–17). The mean follow-up time for these successful eyes was 4.9 ± 3.4 years. Devastating complications attributable to cyclocryotherapy included phthisis (5 eyes) and retinal detachment (5 eyes). Devastating complications occurred more frequently among eyes with aniridia than among all other eyes (nonaniridics) (50% vs. 11%, respectively;
P < 0.05).
Cyclocryotherapy is an effective means of lowering IOP and is a reasonable treatment option in selected pediatric patients with refractory glaucoma. Eyes with aniridia experienced a very high rate of phthisis after cyclocryotherapy and may be poor candidates for this treatment.
Abstract
Background
Clinical predictors of local recurrence following radiation among patients with brain metastases (BrM) provide limited explanatory power. We developed a DNA-based signature of ...radiotherapeutic efficacy among patients with BrM to better characterize recurrence risk.
Methods
We identified 570 patients with 1487 BrM managed with whole-brain (WBRT) or stereotactic radiation therapy at Brigham and Women’s Hospital/Dana-Farber Cancer Institute (2013–2020) for whom next-generation sequencing panel data (OncoPanel) were available. Fine/Gray’s competing risks regression was utilized to compare local recurrence on a per-metastasis level among patients with versus without somatic alterations of likely biological significance across 84 genes. Genes with a q-value ≤ 0.10 were utilized to develop a “Brain-Radiation Prediction Score” (“Brain-RPS”).
Results
Genomic alterations in 11 (ATM, MYCL, PALB2, FAS, PRDM1, PAX5, CDKN1B, EZH2, NBN, DIS3, and MDM4) and 2 genes (FBXW7 and AURKA) were associated with decreased or increased risk of local recurrence, respectively (q-value ≤ 0.10). Weighted scores corresponding to the strength of association with local failure for each gene were summed to calculate a patient-level RPS. On multivariable Fine/Gray’s competing risks regression, RPS 1.66 (1.44–1.91, P < .001), metastasis-associated edema 1.60 (1.16–2.21), P = .004, baseline size 1.02 (1.01–1.03), P < .001 and receipt of WBRT without local therapy 4.04 (2.49–6.58), P < .001 were independent predictors of local failure.
Conclusions
We developed a genomic score to quantify local recurrence risk following brain-directed radiation. To the best of our knowledge, this represents the first study to systematically correlate DNA-based alterations with radiotherapeutic outcomes in BrM. If validated, Brain-RPS has potential to facilitate clinical trials aimed at genome-based personalization of radiation in BrM.
Inhibition of proliferation in estrogen receptor-positive (ER
) breast cancers after short-term antiestrogen therapy correlates with long-term patient outcome. We profiled 155 ER
/human epidermal ...growth factor receptor 2-negative (HER2
) early breast cancers from 143 patients treated with the aromatase inhibitor letrozole for 10 to 21 days before surgery. Twenty-one percent of tumors remained highly proliferative, suggesting that these tumors harbor alterations associated with intrinsic endocrine therapy resistance. Whole-exome sequencing revealed a correlation between 8p11-12 and 11q13 gene amplifications, including
and
, respectively, and high Ki67. We corroborated these findings in a separate cohort of serial pretreatment, postneoadjuvant chemotherapy, and recurrent ER
tumors. Combined inhibition of FGFR1 and CDK4/6 reversed antiestrogen resistance in ER
/
coamplified CAMA1 breast cancer cells. RNA sequencing of letrozole-treated tumors revealed the existence of intrachromosomal
fusion transcripts and increased expression of gene signatures indicative of enhanced E2F-mediated transcription and cell cycle processes in cancers with high Ki67. These data suggest that short-term preoperative estrogen deprivation followed by genomic profiling can be used to identify druggable alterations that may cause intrinsic endocrine therapy resistance.
Constitutively active estrogen receptor α (ER/ESR1) mutations have been identified in approximately one-third of ER+ metastatic breast cancers. Although these mutations are known as mediators of ...endocrine resistance, their potential role in promoting metastatic disease has not yet been mechanistically addressed. In this study, we show the presence of ESR1 mutations exclusively in distant but not local recurrences in five independent breast cancer cohorts. In concordance with transcriptomic profiling of ESR1-mutant tumors, genome-edited ESR1 Y537S and D538G-mutant cell models exhibited a reprogrammed cell adhesive gene network via alterations in desmosome/gap junction genes and the TIMP3/MMP axis, which functionally conferred enhanced cell-cell contacts while decreasing cell-extracellular matrix adhesion. In vivo studies showed ESR1-mutant cells were associated with larger multicellular circulating tumor cell (CTC) clusters with increased compactness compared with ESR1 wild-type CTCs. These preclinical findings translated to clinical observations, where CTC clusters were enriched in patients with ESR1-mutated metastatic breast cancer. Conversely, context-dependent migratory phenotypes revealed cotargeting of Wnt and ER as a vulnerability in a D538G cell model. Mechanistically, mutant ESR1 exhibited noncanonical regulation of several metastatic pathways, including secondary transcriptional regulation and de novo FOXA1-driven chromatin remodeling. Collectively, these data provide evidence for ESR1 mutation-modulated metastasis and suggest future therapeutic strategies for targeting ESR1-mutant breast cancer.
Context- and allele-dependent transcriptome and cistrome reprogramming in mutant ESR1 cell models elicit diverse metastatic phenotypes related to cell adhesion and migration, which can be pharmacologically targeted in metastatic breast cancer.
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