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The absence of effective therapeutic targets and aggressive nature of triple-negative breast cancer (TNBC) renders this disease subset difficult to treat. Although estrogen receptor beta (ERβ) is expressed in TNBC, studies on its functional role have yielded inconsistent results. However, recently, our preclinical studies, along with other observations, have shown the potential therapeutic utility of ERβ in the context of mutant p53 expression. The current case study examines the efficacy of the selective estrogen receptor modulator tamoxifen in p53-mutant TNBC with brain metastases. Significant increase in ERβ protein expression and anti-proliferative interaction between mutant p53 and ERβ were observed after cessation of tamoxifen therapy, with significant regression of brain metastases. This case study provides supporting evidence for the use of tamoxifen in p53-mutant, ERβ+TNBC, especially in the setting of brain metastasis.

Supplemental tables

MAF of mutations detected

Survival curves

Purpose:Plasma genotyping may identify mutations in potentially “actionable” cancer genes, such as BRCA1/2, but their clinical significance is not well-defined. We evaluated the characteristics of somatically acquired BRCA1/2 mutations in patients with metastatic breast cancer (MBC).Experimental Design:Patients with MBC undergoing routine cell-free DNA (cfDNA) next-generation sequencing (73-gene panel) before starting a new therapy were included. Somatic BRCA1/2 mutations were classified as known germline pathogenic mutations or novel variants, and linked to clinicopathologic characteristics. The effect of the PARP inhibitor, olaparib, was assessed in vitro, using cultured circulating tumor cells (CTCs) from a patient with a somatically acquired BRCA1 mutation and a second patient with an acquired BRCA2 mutation.Results:Among 215 patients with MBC, 29 (13.5%) had somatic cfDNA BRCA1/2 mutations [nine (4%) known germline pathogenic and rest (9%) novel variants]. Known germline pathogenic BRCA1/2 mutations were common in younger patients (P = 0.008), those with triple-negative disease (P = 0.022), and they were more likely to be protein-truncating alterations and be associated with TP53 mutations. Functional analysis of a CTC culture harboring a somatic BRCA1 mutation demonstrated high sensitivity to PARP inhibition, while another CTC culture harboring a somatic BRCA2 mutation showed no differential sensitivity. Across the entire cohort, APOBEC mutational signatures (COSMIC Signatures 2 and 13) and the “BRCA” mutational signature (COSMIC Signature 3) were present in BRCA1/2-mutant and wild-type cases, demonstrating the high mutational burden associated with advanced MBC.Conclusions:Somatic BRCA1/2 mutations are readily detectable in MBC by cfDNA analysis, and may be present as both known germline pathogenic and novel variants.

Supplemental Table S3. Actionable mutations found in cfDNA and tumor tissue genotyping in 12 patients who went on to receive matched therapy after having both forms of testing.

Purpose:Actionable mutations can guide genotype-directed matched therapy. We evaluated the utility of tissue-based and plasma-based genotyping for the identification of actionable mutations and selection of matched therapy in patients with metastatic breast cancer (MBC).Experimental Design:Patients with MBC who underwent tissue genotyping (institutional platform, 91-gene assay) or plasma-based cell-free DNA (cfDNA, Guardant360, 73-gene assay) between January 2016 and December 2017 were included. A chart review of records to identify subtype, demographics, treatment, outcomes, and tissue genotyping or cfDNA results was performed. The incidence of actionable mutations and the selection of matched therapy in tissue genotyping or cfDNA cohorts was determined. The impact of matched therapy status on overall survival (OS) in tissue genotyping or cfDNA subgroups was determined with Cox regression analysis.Results:Of 252 patients who underwent cfDNA testing, 232 (92%) had detectable mutations, 196 (78%) had actionable mutations, and 86 (34%) received matched therapy. Of 118 patients who underwent tissue genotyping, 90 (76%) had detectable mutations, 59 (50%) had actionable mutations, and 13 (11%) received matched therapy. For cfDNA patients with actionable mutations, matched versus nonmatched therapy was associated with better OS [HR 0.41, 95% confidence interval (CI): 0.23–0.73, P = 0.002], and this remained significant in a multivariable analysis correcting for age, subtype, visceral metastases, and brain metastases (HR = 0.46, 95% CI: 0.26–0.83, P = 0.010).Conclusions:Plasma-based genotyping identified high rates of actionable mutations, which was associated with significant application of matched therapy and better OS in patients with MBC.See related commentary by Rugo and Huppert, p. 3275

Purpose:While FGFR1 amplification has been described in breast cancer, the optimal treatment approach for FGFR1-amplified (FGFR1+) metastatic breast cancer (MBC) remains undefined.Experimental Design: We evaluated clinical response to endocrine and targeted therapies in a cohort of patients with hormone receptor–positive (HR+)/HER2− MBC and validated the functional role of FGFR1-amplification in mediating response/resistance to hormone therapy in vitro.Results:In the clinical cohort (N = 110), we identified that patients with FGFR1+ tumors were more likely to have progesterone receptor (PR)-negative disease (47% vs. 20%; P = 0.005), coexisting TP53 mutations (41% vs. 21%; P = 0.05), and exhibited shorter time to progression with endocrine therapy alone and in combination with CDK4/6 inhibitor, but not with a mTOR inhibitor (everolimus), adjusting for key prognostic variables in multivariate analysis. Furthermore, mTOR-based therapy resulted in a sustained radiological and molecular response in an index case of FGFR1+ HR+/HER2− MBC. In preclinical models, estrogen receptor–positive (ER+)/FGFR1-amplified CAMA1 human breast cancer cells were only partially sensitive to fulvestrant, palbociclib, and alpelisib, but highly sensitive to everolimus. In addition, transduction of an FGFR1 expression vector into ER+ T47D cells induced resistance to fulvestrant that could be overcome by added TORC1 inhibition, but not PI3K or CDK4/6 inhibition.Conclusions:Collectively, these findings suggest that while FGFR1 amplification confers broad resistance to ER, PI3K, and CDK4/6 inhibitors, mTOR inhibitors might have a unique therapeutic role in the treatment of patients with ER+/FGFR1+ MBC.

Supplementary figures

Supplemental Table S2. Characteristics of patients who underwent both cfDNA and tumor tissue genotyping testing.

Supplemental Figure S1. Consort diagram of patients included in this study.

Supplemental Table S1a. Genes included in Guardant360® panel and related genotypedirected therapy.

Purpose: Plasma genotyping may identify mutations in potentially “actionable” cancer genes, such as BRCA1/2, but their clinical significance is not well-defined. We evaluated the characteristics of somatically acquired BRCA1/2 mutations in patients with metastatic breast cancer (MBC). Experimental Design: Patients with MBC undergoing routine cell-free DNA (cfDNA) next-generation sequencing (73-gene panel) before starting a new therapy were included. Somatic BRCA1/2 mutations were classified as known germline pathogenic mutations or novel variants, and linked to clinicopathologic characteristics. The effect of the PARP inhibitor, olaparib, was assessed in vitro, using cultured circulating tumor cells (CTCs) from a patient with a somatically acquired BRCA1 mutation and a second patient with an acquired BRCA2 mutation. Results: Among 215 patients with MBC, 29 (13.5%) had somatic cfDNA BRCA1/2 mutations [nine (4%) known germline pathogenic and rest (9%) novel variants]. Known germline pathogenic BRCA1/2 mutations were common in younger patients (P = 0.008), those with triple-negative disease (P = 0.022), and they were more likely to be protein-truncating alterations and be associated with TP53 mutations. Functional analysis of a CTC culture harboring a somatic BRCA1 mutation demonstrated high sensitivity to PARP inhibition, while another CTC culture harboring a somatic BRCA2 mutation showed no differential sensitivity. Across the entire cohort, APOBEC mutational signatures (COSMIC Signatures 2 and 13) and the “BRCA” mutational signature (COSMIC Signature 3) were present in BRCA1/2-mutant and wild-type cases, demonstrating the high mutational burden associated with advanced MBC. Conclusions: Somatic BRCA1/2 mutations are readily detectable in MBC by cfDNA analysis, and may be present as both known germline pathogenic and novel variants.

Background: BM in MBC patients cause significant morbidity and mortality. BRCA1 germline mutations have previously been shown to be associated with an increased risk of developing BM (Lee et al, 2011), with an incidence as high as 15% (Zavitsanos et al, 2016). We previously reported that a subset of MBC patients may have somatic BRCA mutations in the absence of germline BRCA mutations (Vidula N, SABCS, 2017). In this study, we evaluated the incidence and clinical characteristics of BM in MBC patients with somatic BRCA mutations detected by cfDNA. Methods: MBC patients with somatic BRCA1 or 2 mutations detected by cfDNA (Guardant360TM, next generation sequencing, 73 gene panel; mutations classified as somatic by Guardant360TM) with at least 4 months of follow-up post-testing at an academic institution were identified. From this cohort, we identified patients who developed BM post cfDNA testing. A retrospective review of medical records and Guardant360TM reports was conducted to identify demographics, tumor subtype, type of cfDNA BRCA mutation, whether the BRCA mutation was known to be pathogenic, germline BRCA mutation status, mutant allele fraction (MAF), clonality (MAF ratio of BRCA mutation/gene mutation with highest MAF ≥ 0.25 for clonal, and Results: Of 36 MBC patients with somatic BRCA mutations, 9 (25%) developed BM and 27 (75%) did not have BM (non-BM). The median time to development of BM was 6.7 months after cfDNA testing. Of the BM patients, 5 (56%) had triple-negative (TN) and 4 (44%) had hormone receptor positive (HR+)/HER2- MBC in comparison with the non-BM cases where 5 (19%) had TN, 19 (70%) had HR+/HER2-, and 3 (11%) had HER2+ MBC. Very few patients (1 BM and 2 non-BM) had known co-existing separate germline BRCA mutations (rest not known BRCA carriers confirmed by negative germline testing and/or absence of family history suggestive of a germline BRCA phenotype). The median age at MBC diagnosis was similar for BM and non-BM patients (57 years). PIK3CA and TP53 mutations were commonly seen in both BM and non-BM cases. Additionally, MYC, EGFR, and CCNE1 mutations were commonly seen in BM cases. As outlined in Table 1, among patients with BM, the somatic BRCA mutations were commonly BRCA1, clonal, known to be pathogenic (56%), and present at a higher MAF, but these findings did not reach statistical significance possibly due to the small sample size. Brain tumor tissue mutation status in BM patients and correlation with cfDNA results will be presented at the meeting. Conclusions: We observed a relatively high incidence (25%) of BM in MBC patients with somatic BRCA mutations detectable by cfDNA, which were often known to be pathogenic mutations (56%), and often associated with co-existing MYC, EGFR, and CCNE1 mutations. Further research using a larger cohort with adequate statistical power is needed to validate these findings, and may help identify MBC patients at risk for BM using a liquid biopsy. Table 1.Characteristic BMNon-BMPrior anthracycline and/or platinum6 (67%)16 (59%)Type of somatic BRCA mutationBRCA16 (67%)11 (41%)BRCA22 (22%)15 (56%)BRCA1 and 21 (11%)1 (4%)Median BRCA MAF0.40.17ClonalityClonal6 (67%)13 (48%)Subclonal3 (33%)14 (52%)Mutation known to be pathogenic5 (56%)7 (26%)Common co-existing mutationsPIK3CA5 (56%)12 (44%)TP535 (56%)15 (56%)MYC5 (56%)8 (30%)EGFR5 (56%)8 (30%)CCNE15 (56%)6 (22%)KIT3 (33%)5 (19%) Citation Format: Neelima Vidula, Andrzej Niemierko, Giuliana Malvarosa, Priscilla Brastianos, Erica Blouch, Kristen Shannon, Steven Isakoff, Seth Wander, Laura Spring, Jerry Younger, Kristin Price, Beverly Moy, Dejan Juric, Leif Ellisen, Aditya Bardia. Brain metastases (BM) in patients with metastatic breast cancer (MBC) and circulating cell-free DNA (cfDNA) somatic BRCA mutations [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 P4-09-06.

Background: The AKT1 oncogene represents a central node in the well-characterized PI3K/AKT/mTOR signal transduction pathway involved in the regulation of cellular proliferation as well as therapeutic resistance. With the emergence of multiple targeted therapies for HR+ MBC, we sought to explore the prevalence of AKT1 alterations, the spectrum of co-occurring mutations, and any potential impact on response to endocrine-based therapy, including CDK 4/6 and PIK3CA inhibitors. Methods: Presence or absence of AKT1 mutation was interrogated via cell-free DNA (cfDNA) analysis (Guardant assay) among patients with HR+/HER2- MBC receiving treatment at the Massachusetts General Hospital who underwent routine clinical cfDNA testing. Genomic sequencing results were then compared to detailed clinical annotation based upon retrospective chart review. In the laboratory, HR+/HER2- T47D breast cancer cells were transfected with AKT1, under the control of a doxycycline-inducible promoter, to induce AKT1 overexpression. Sensitivity to escalating doses of fulvestrant and palbociclib were interrogated via cell-titer-glo viability assay in vitro. Results: Among 272 patients with HR+/HER2- MBC, we identified 13 patients with AKT1 mutation (4.7%). 12/13 (92%) patients had a canonical AKT1E17K alteration, one patient (8%) had an AKT1 E49K alteration. AKT1 mutations occurred concurrently with alterations in ESR1 (31%), PIK3CA (23%), TP53 (23%), ERBB2 (15%), and RB1 (8%). All patients had been exposed to some form of anti-estrogen prior to sequencing, either via (neo)adjuvant therapy and/or in the metastatic setting; 5/13 (38%) had exposure to a CDK4/6 inhibitor prior to cfDNA sequencing. Following the identification of an AKT1 alteration, examples of rapid clinical progression on fulvestrant, CDK4/6i, and/or everolimus were identified, however the presence of an AKT1 alteration was not sufficient to predict pan-resistance to these agents, as counter-examples of clinical benefit were also identified. In an index patient with an AKT1 mutation and prior progression on letrozole and palbociclib, subsequent treatment with fulvestrant and abemaciclib did not yield clinical benefit, highlighting the potential role of AKT1 in mediating clinical resistance to endocrine therapy and CDK4/6i. In the pre-clinical model, HR+/HER2- breast cancer cells demonstrated resistance to both fulvestrant and palbociclib following upregulation of exogenous AKT1 expression. Updated clinical data with additional patients and response to regimens incorporating an anti-estrogen, a CDK4/6 inhibitor, an mTOR inhibitor, and/or a PIK3CA inhibitor will be presented at the meeting. Conclusions: Activating AKT1 mutations can be identified via targeted sequencing of cfDNA in patients with HR+/HER2- MBC, and may play an important role in mediating resistance to anti-estrogens, CDK4/6 inhibitors, and other emerging targeted therapies. Heterogeneity in response to endocrine-based therapy following the identification of an AKT1 alteration may be related to tumor-extrinsic factors, the AKT1 allelic fraction, or cooperativity between multiple resistance drivers. Further research is needed to confirm these findings and guide optimal therapeutic sequencing strategies for patients with HR+/HER2- AKT1 mutant MBC. Citation Format: Seth Wander, Pingping Mao, Maxwell Lloyd, Kailey Kowalski, Gabriela N. Johnson, Giuliana Malvarosa, Beverly Moy, Leif W. Ellisen, John Iafrate, Nikhil Wagle, Aditya Bardia. AKT1 alterations following exposure to endocrine-based therapy in patients with hormone-receptor positive (HR+) metastatic breast cancer (MBC): Clinical and functional implications [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 P6-10-08.

PURPOSE Accurate monitoring of therapeutic response remains an important unmet need for patients with metastatic breast cancer (MBC). Analysis of tumor genomics obtained via circulating tumor DNA (ctDNA) can provide a comprehensive overview of tumor evolution. Here, we evaluated ctDNA change as an early prognostic biomarker of subsequent radiologic progression and survival in MBC. PATIENTS AND METHODS Paired blood samples from patients with MBC were analyzed for levels of ctDNA, carcinoembryonic antigen, and cancer antigen 15-3 at baseline and during treatment. A Clinical Laboratory Improvement Amendments–certified sequencing panel of 73 genes was used to quantify tumor-specific point mutations in ctDNA. Multivariable logistic regression analysis was conducted to evaluate the association between ctDNA rise from baseline to during-treatment (genomic progression) and subsequent radiologic progression and progression-free survival (PFS). RESULTS Somatic mutations were detected in 76 baseline samples (90.5%) and 71 during-treatment samples (84.5%). Patients with genomic progression were more than twice as likely to have subsequent radiologic progression (odds ratio, 2.04; 95% CI, 1.74 to 2.41; P < .0001), with a mean lead time of 5.8 weeks. Genomic assessment provided a high positive predictive value of 81.8% and a negative predictive value of 89.7%. The subset of patients with genomic progression also had shorter PFS (median, 4.2 v 8.3 months; hazard ratio, 2.97; 95% CI, 1.75 to 5.04; log-rank P < .0001) compared with those without genomic progression. CONCLUSION Genomic progression, as assessed by early rise in ctDNA, is an independent biomarker of disease progression before overt radiologic or clinical progression becomes evident in patients with MBC.

Plasma genotyping identifies potentially actionable mutations at variable mutant allele frequencies, often admixed with multiple subclonal variants, highlighting the need for their clinical and functional validation. We prospectively monitored plasma genotypes in 143 women with endocrine-resistant metastatic breast cancer (MBC), identifying multiple novel mutations including HER2 mutations (8.4%), albeit at different frequencies highlighting clinical heterogeneity. To evaluate functional significance, we established ex vivo culture from circulating tumor cells (CTCs) from a patient with HER2-mutant MBC, which revealed resistance to multiple targeted therapies including endocrine and CDK 4/6 inhibitors, but high sensitivity to neratinib (IC50: 0.018 μM). Immunoblotting analysis of the HER2-mutant CTC culture line revealed high levels of HER2 expression at baseline were suppressed by neratinib, which also abrogated downstream signaling, highlighting oncogenic dependency with HER2 mutation. Furthermore, treatment of an index patient with HER2-mutant MBC with the irreversible HER2 inhibitor neratinib resulted in significant clinical response, with complete molecular resolution of two distinct clonal HER2 mutations, with persistence of other passenger subclones, confirming HER2 alteration as a driver mutation. Thus, driver HER2 mutant alleles that emerge during blood-based monitoring of endocrine-resistant MBC confer novel therapeutic vulnerability, and ex vivo expansion of viable CTCs from the blood circulation may broadly complement plasma-based mutational analysis in MBC.

Background: The availability of multiple therapies has transformed the landscape of MBC, but also brought the challenge of selecting the right therapy for an individual patient. Furthermore, in patients with Hormone Receptor positive (HR+) breast cancer who have bone metastases only it may be difficult to assess effectiveness of therapy via imaging. Peripheral ctDNA detection and analysis by next-generation sequencing (NGS) has gained popularity in cancer diagnosis and therapeutics due to its relative noninvasiveness, ease of use, and high sensitivity. Here, we explore the utility of ctDNA change as a predictor for disease progression in MBC. We hypothesized that genomic progression is a harbinger of subsequent radiologic progression in patients with MBC. Methods: We analyzed change from pre-treatment (baseline) to on-treatment ctDNA mutant allele fraction (MAF) among patients with MBC. Patients receiving standard-of-care therapies or investigational agents on clinical trials at our institution were included. All patients were followed from the date of baseline test until death or data cutoff (6/20/2018). All peripheral blood specimens were collected and analyzed between 1/7/2016 and 3/1/2018 via NGS (Guardant360®). Peripheral blood specimens were sequenced prior to initiation of a new therapeutic regimen (baseline) and subsequently at least once while on-treatment, on average 4-12 weeks later. All patients had a follow-up CT scan of chest, abdomen and pelvis 2-4 weeks after the on-treatment NGS. A priori, we defined genomic progression as increase in ctDNA total MAF of at least 20% from baseline. We utilized Cox regression analysis to identify whether genomic progression was a predictor of radiologic progression, adjusting for common prognostic variables. Results: All patients (N= 77) were female, predominantly White (83.1%), and median age was 57 (range 32 to 77). Fifty one out of 77 patients (66.2%) were ER+, 5 HER2+, and 9 had triple negative breast cancer. The median MAF at baseline was 2.2% (range 0% - 61.7%). Common genomic alterations in ctDNA included PIK3CA, TP53, ESR1, AKT1, NF1. 27 out of 77 (35%) patients showed disease progression on the first subsequent CT scan, while 59 out of 77 (76.6%) progressed during the follow up time. We found that an increase in ctDNA MAF of at least 20% was a strong predictor of disease progression (HR =2.46, CI [1.14-5.32], p=0.02), compared to those who had a MAF increase of less than 20% or a decrease in total MAF. In multi-variable analysis, adjusting for age, number of prior therapies, type of therapy, and visceral metastases, increase in ctDNA remained a significant predictor for subsequent disease progression (HR =3.84, CI [1.63-9.07], p=0.002). Subset results in patients with bone metastases only, and relative comparison of ctDNA with standard tumor markers will be presented at the meeting. Conclusions: Genomic progression, identified by an increase in ctDNA MAF, is potentially an early predictor of subsequent disease progression in patients with MBC. Further research is needed to prospectively evaluate the clinical utility of ctDNA change as a surrogate marker in guiding treatment decision-making for patients with MBC. Citation Format: Velimirovic M, Juric D, Niemierko A, Spring LM, Vidula N, Malvarosa G, Yuen M, Moy B, Isakoff SJ, Ellisen LW, Bardia A. Genomic progression, detected by circulating tumor DNA (ctDNA) sequencing, as an early predictor of disease progression in metastatic breast cancer (MBC) [abstract]. In: Proceedings of the 2018 San Antonio Breast Cancer Symposium; 2018 Dec 4-8; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2019;79(4 Suppl):Abstract nr P4-08-08.

Purpose: Actionable mutations can guide genotype-directed matched therapy. We evaluated the utility of tissue-based and plasma-based genotyping for the identification of actionable mutations and selection of matched therapy in patients with metastatic breast cancer (MBC). Experimental Design: Patients with MBC who underwent tissue genotyping (institutional platform, 91-gene assay) or plasma-based cell-free DNA (cfDNA, Guardant360, 73-gene assay) between January 2016 and December 2017 were included. A chart review of records to identify subtype, demographics, treatment, outcomes, and tissue genotyping or cfDNA results was performed. The incidence of actionable mutations and the selection of matched therapy in tissue genotyping or cfDNA cohorts was determined. The impact of matched therapy status on overall survival (OS) in tissue genotyping or cfDNA subgroups was determined with Cox regression analysis. Results: Of 252 patients who underwent cfDNA testing, 232 (92%) had detectable mutations, 196 (78%) had actionable mutations, and 86 (34%) received matched therapy. Of 118 patients who underwent tissue genotyping, 90 (76%) had detectable mutations, 59 (50%) had actionable mutations, and 13 (11%) received matched therapy. For cfDNA patients with actionable mutations, matched versus nonmatched therapy was associated with better OS [HR 0.41, 95% confidence interval (CI): 0.23–0.73, P = 0.002], and this remained significant in a multivariable analysis correcting for age, subtype, visceral metastases, and brain metastases (HR = 0.46, 95% CI: 0.26–0.83, P = 0.010). Conclusions: Plasma-based genotyping identified high rates of actionable mutations, which was associated with significant application of matched therapy and better OS in patients with MBC. See related commentary by Rugo and Huppert, p. 3275

PURPOSE: Plasma genotyping may identify mutations in potentially “actionable” cancer genes, such as BRCA1/2, but their clinical significance is not well defined. We evaluated the characteristics of somatically acquired BRCA1/2 mutations in patients with MBC. METHODS: Patients with MBC undergoing routine cell-free DNA (cfDNA) next generation sequencing (73 gene panel) before starting a new therapy were included. Somatic BRCA1/2 mutations were classified as known germline-pathogenic mutations or novel variants, and linked to clinicopathological characteristics. The effect of the PARP inhibitor olaparib was assessed in vitro, using cultured circulating tumor cells (CTCs) from a patient with a somatically acquired BRCA1 mutation and a second patient with an acquired BRCA2 mutation. RESULTS: Among 215 MBC patients, 29 (13.5%) had somatic cfDNA BRCA1/2 mutations (nine (4%) known germline-pathogenic and rest (9%) novel variants). Known germline-pathogenic BRCA1/2 mutations were common in younger patients (p=0.008), those with triple-negative disease (p=0.022), and they were more likely to be protein-truncating alterations and be associated with TP53 mutations. Functional analysis of a CTC culture harboring a somatic BRCA1-mutation demonstrated high sensitivity to PARP inhibition, while another CTC culture harboring a somatic BRCA2 mutation showed no differential sensitivity. Across the entire cohort, APOBEC mutational signatures (COSMIC Signatures 2 and 13) and the “BRCA” mutational signature (COSMIC Signature 3) were present in BRCA1/2 mutant and wild-type cases, demonstrating the high mutational burden associated with advanced MBC. CONCLUSION: Somatic BRCA1/2 mutations are readily detectable in MBC by cfDNA analysis, and may be present as both known germline-pathogenic and novel variants.

Background Research biopsies (RBs) are essential to understanding tumor biology and mechanisms of resistance and to advancing precision medicine. However, RBs have associated risks and may not benefit the patient. Objectives The purpose of this integrative review is to summarize and synthesize the current literature on the experience, attitudes, and understanding of patients with cancer related to RBs. Methods Articles from January 2010 to February 2017 were retrieved via a search of MEDLINE®. Articles included reported on the willingness, perceptions, understanding, attitudes, and/or experience of patients with cancer related to RBs. Findings Nine of 216 identified studies were selected. Studies exploring patient willingness to undergo RBs (n = 6) identified RBs as a potential barrier to clinical trial participation. Studies exploring patient understanding and informed consent (n = 3) revealed variable patient knowledge of the risks and benefits of RBs.

Introduction: BRCA mutations may impact patient outcomes, as well as chemotherapy response in patients with breast cancer (BC). While germline BRCA mutations have been well-studied, the incidence and clinical impact of somatic BRCA mutations have not been well-described. We evaluated the presence of BRCA mutations, and the association between somatic BRCA mutations with clinical outcomes in patients with metastatic breast cancer (MBC). Methods: We identified patients with MBC who underwent ctDNA testing by Guardant360 at our institution before the start of a new therapy. From this subset of patients, we subsequently identified those patients with circulating tumor DNA (ctDNA) BRCA 1 or 2 mutations. We conducted a retrospective review of medical and pathology records to identify tumor subtype, germline BRCA testing results, and tissue genotyping results based on institutional Snapshot-NGS genotyping assay. In addition, we conducted a multivariate analysis to evaluate the hazard ratio (HR) for the association between ctDNA BRCA mutation and progression free survival (PFS) adjusting for age, number of prior therapies, and type of therapy. Results Among patients with MBC (N = 178), 27 (15.2%) had BRCA alterations detected by ctDNA analysis. Among patients with ctDNA BRCA alterations, the median age at metastatic diagnosis was 53; 16/24 (66.6%) had hormone receptor (HR)+/HER2- BC, 5/24 (20.8%) had triple negative (TN) BC, 2/24 (8.3%) had HR-/HER2+ BC, and 1/24 (4.2%) had HR+/HER2+ BC. Of patients with ctDNA BRCA mutations, only a minority (16.7%) had BRCA alterations detected by genotyping of archival tumor, and only 1 (3.7%) had a germline BRCA mutation (BRCA 1). In multivariate analysis, patients with BRCA mutant tumors, had similar median PFS as compared to non-BRCA mutant breast cancer (HR: 1.17; p = 0.58). Overall survival analysis and impact of BRCA mutations on response to therapy, particularly DNA damaging agents, will be presented at the meeting. Conclusions: BRCA mutations by ctDNA are detectable in a significant proportion of MBC patients. Most BRCA mutations detected by ctDNA were not identified by genotyping of archival tissue, and were not associated with germline BRCA mutations, suggesting that somatic BRCA mutations may be detected by sensitive blood-based genotyping assays in patients who are not known BRCA carriers. The therapeutic impact of DNA damaging agents and PARP inhibitors in MBC patients with somatic BRCA alterations is not known and warrants additional research. Citation Format: Vidula N, Isakoff SJ, Niemierko A, Malvarosa G, Park H, Abraham E, Spring L, Peppercorn J, Moy B, Ellisen LW, Juric D, Bardia A. Somatic BRCA mutation detection by circulating tumor DNA analysis in patients with metastatic breast cancer: Incidence and association with tumor genotyping results, germline BRCA mutation status, and clinical outcomes [abstract]. In: Proceedings of the 2017 San Antonio Breast Cancer Symposium; 2017 Dec 5-9; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2018;78(4 Suppl):Abstract nr PD1-13.

Background: While the human epidermal growth factor receptor 2 (HER2) gene has long been linked with the pathogenesis and prognosis of breast cancer, its significance has been recognized only when the receptor has been amplified. However, rare, but actionable, somatic mutations in HER2, without HER2 gene amplification, have been described in breast cancer based on molecular analysis of metastatic specimens (frequency ˜ 1%). Little is known about the incidence based on blood-based genotyping assays, as well as impact of HER2 mutations on clinical outcomes in patients with ER+/HER2- metastatic breast cancer (MBC). Methods: We evaluated the presence of HER2 mutations based on routine tissue and blood-based genotyping assays, ordered by treating oncologists at our institution, for patients with estrogen receptor positive (ER+)/HER2 negative MBC. The tissue analysis was based on SNAPSHOT-NGS, an anchored multiplex polymerase chain reaction assay that detects single nucleotide variants (SNV) and insertions/deletions (indel), and the blood-based genotyping analysis was based on circulating tumor DNA (ctDNA) detection using the Guardant 360 panel, a next-generation sequencing (NGS) assay capable of detecting mutations with comparable sensitivity to SNAPSHOT. Patients with acquired HER2 mutations were identified, and multivariate analysis was performed to evaluate the hazard ratio (HR) for the association between HER2 mutations and progression free survival (PFS), adjusting for age and number of prior therapies. Results: Among the ER+/HER2- MBC patients (N=118), 11% (N= 13) were found to have acquired HER2 mutation by ctDNA analysis, but no HER2 mutations were identified in any of patients based on tissue-based molecular analysis of archival specimens. Among patients with HER2 mutant, ER+/HER2- MBC, the median age at metastatic diagnosis was 57.34 (range 51.5-67.1) years, 7.7% had de-novo metastatic disease, and 30.8% had prior CDK 4/6 inhibitor therapy. In terms of outcomes, in the multivariate model, patients with HER2 mutant breast cancer had similar PFS when treated with endocrine and targeted therapy combination (HR = 0.24; p = 0.21), and trended towards worse PFS with chemotherapy (HR = 2.69; p = 0.06), as compared to non-HER2 mutant group, albeit duration of follow up was limited (median duration = 6.7 months). Of note, most of the detected HER2 mutations were activating or deleterious, but not all were clonal. Updated outcome data, including overall survival, will be presented at the meeting. Conclusions: A much larger subset of patients with ER+/HER2- MBC have HER2 mutations detectable by ctDNA, but not by tissue, which highlights the need for blood-based biomarker monitoring for identification of actionable mutations, as well as the potential clinical utility in development of genotype driven trials for patients with HER2 mutant, ER+/HER2- MBC. Citation Format: Medford A, Juric D, Niemierko A, Malvarosa G, Park H, Shellock M, Spring L, Moy B, Isakoff S, Ellisen L, Bardia A. HER2 mutations detected by ctDNA in ER+/HER2- metastatic breast cancer patients: Incidence and impact on clinical outcomes [abstract]. In: Proceedings of the 2017 San Antonio Breast Cancer Symposium; 2017 Dec 5-9; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2018;78(4 Suppl):Abstract nr P5-20-02.

This abstract was withdrawn by the authors.

Introduction: Robust clinical genotyping assays are crucial to accelerate development of targeted therapies toward tumor-specific oncogene pathways. Indeed, there has been an explosion in the development of genotyping assays and stratified enrollment in clinical trials based on molecular characteristics of the tumor. However, availability of tissue can be a significant barrier for genotyping, particularly patients with estrogen receptor positive (ER+) breast cancer, who frequently have bone only metastasis. Detection of mutations in circulating tumor DNA (ctDNA) represents an attractive alternate strategy. However, there is a paucity of data comparing genotyping results from tissue and ctDNA, and potential clinical utility for patients with metastatic breast cancer. Methods: Analysis of ctDNA was based on Guardant 360 panel (2015), a next-generation sequencing (NGS) based assay that covers point mutations in 70 genes (complete or critical exon coverage) with select amplifications, fusions, and indels, using NGS Illumina HiSeq platform, validated to detect alterations in samples with at least 2% ctDNA. Molecular profiling of tissue was based on the institutional lab-developed test “Snapshot-NGS assay”, utilizing a multiplex polymerase chain reaction (PCR) technology called Anchored Multiplex PCR (AMP) for single nucleotide variant (SNV) and insertion/deletion (indel) detection in genomic DNA targeting hotspots and exons in 39 genes using NGS Illumina MiSeq platform, validated to detect SNV and indel variants at 5% allelic frequency or higher in target regions. Results: In the analytical dataset, a total of 42 patients with metastatic breast cancer (median age 62) at our institution had ctDNA and tissue Snapshot-NGS results available. Out of these, 95% had at least one genetic alteration detected in ctDNA, before start of a new therapy. The common genetic alterations in ER+ disease (n = 33) included PIK3CA (39.4%), ESR1 (27.3%), and AKT1 (15.2%); HER2+ disease (n = 2) included PIK3CA and CCND1; TNBC (n = 7) included TP53 (71.4%). Gene amplifications in FGFR, PIK3CA, CCND1, MYC, and KRAS were also noted. In general, the average number of molecular alterations was higher in ctDNA than the tissue specimen (mean = 5.1 vs 1.1; p < .001). Of 34 patients with actionable alterations detected in ctDNA, 76.5% of these patients did not have actionable alterations detected in tissue. Of 29 patients with actionable alterations detected in tissue (primary or metastatic), 13.8% of these patients did not have any actionable alterations detected in ctDNA. Conclusion: A significantly higher number of genomic alterations were detected in ctDNA compared to tissue, including actionable alterations linked to potential targeted therapies. Given these findings and the difficulties with tissue-based genotyping assays, blood based biomarker assays might provide an alternative and effective way to obtain a comprehensive molecular profile, and potentially guide management decisions for patients with metastatic breast cancer. Citation Format: Malvarosa G, Spring L, Juric D, Moy B, Bardia A. Comparison of genotyping results from tissue and circulating DNA (ctDNA) in patients with metastatic breast cancer [abstract]. In: Proceedings of the 2016 San Antonio Breast Cancer Symposium; 2016 Dec 6-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2017;77(4 Suppl):Abstract nr P1-05-05.

Research Biopsies in Oncology – Patient Willingness, Perceptions, Understanding, and Experience: An Integrative Review. Background Tumors are classified into subsets based on molecular alterations to select personalized therapies, and research biopsies are essential to advance personalized medicine. However, research biopsies can be associated with complications, results might not directly benefit the patient particularly on-treatment biopsies obtained for evaluation of pharmacodynamics effect, and mandatory requirement can impact patient enrollment in clinical trials. Understanding patient perceptions and experiences surrounding research biopsies may highlight areas for improvement and could expand participation in clinical trials. Objectives The purpose of this integrative review explores and summarizes the current literature on oncology patient perceptions, willingness and experience of research biopsies evaluates data to determine potential implications for practice improvement, patient experience, and clinical trial participation. Methods Articles from January 2010-February 2016 were retrieved via a systematic search restricted to English of MEDLINE/Ovid. Search terms included breast cancer, cancer, research, biopsy, perceptions and attitudes. Inclusion criteria were studies that included patients with cancer and had information on patient willingness, perceptions, understanding, attitudes, and/or experience around research biopsies. Abstracts were independently reviewed for inclusion by two authors. For each selected manuscript, the following information was abstracted: primary author, sample and setting, results, conclusion, and assessment of strength of evidence level and quality using John Hopkins Nursing Evidence Based Practice (JHNEBP) Rating Scales. Results The search resulted in 175 unique studies. 17 abstracts were selected for full manuscript review. From these, 8 were excluded for not meeting inclusion criteria. The 9 selected studies ranged in sample size from 10-362 and all articles were rated IIIA-B evidence level. The majority of studies (66.7%) focused on breast cancer patients. Studies exploring patient willingness to undergo research biopsies (N=8) revealed variability in patient willingness to undergo research biopsies and identified research biopsies identified as a potential barrier to clinical trial participation. Studies exploring patient understanding of the risks and benefits of research biopsies (N=3) revealed overall poor understanding among patients. One study explored anxiety surrounding the research biopsy procedure, suggesting significant anticipatory anxiety is a significant concern. Issues surrounding informed consent were raised in several studies. Conclusions Research biopsies can be associated with significant anxiety, potentially impact enrollment in clinical trials, and consequently there is a need for development of strategies to improve patient experience and education. While a separate consent for research biopsies may be onerous, tailored educational materials, focus groups, as well as development of alternate strategies such as liquid biopsies, are essential to ensuring advancement in cancer care and outcomes. Key Words: cancer, research, biopsy, perceptions, attitudes. Citation Format: Abraham E, Spring L, D'Alleva J, Malvarosa G, Tripp E, Post K, Bardia A, Habin K. Research biopsies in oncology-patient willingness, perceptions, understanding, and experience: An integrative review [abstract]. In: Proceedings of the 2016 San Antonio Breast Cancer Symposium; 2016 Dec 6-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2017;77(4 Suppl):Abstract nr P4-20-07.

Purpose: While FGFR1 amplification has been described in breast cancer, the optimal treatment approach for FGFR1-amplified (FGFR1+) metastatic breast cancer (MBC) remains undefined. Experimental Design: We evaluated clinical response to endocrine and targeted therapies in a cohort of patients with hormone receptor–positive (HR+)/HER2− MBC and validated the functional role of FGFR1-amplification in mediating response/resistance to hormone therapy in vitro. Results: In the clinical cohort (N = 110), we identified that patients with FGFR1+ tumors were more likely to have progesterone receptor (PR)-negative disease (47% vs. 20%; P = 0.005), coexisting TP53 mutations (41% vs. 21%; P = 0.05), and exhibited shorter time to progression with endocrine therapy alone and in combination with CDK4/6 inhibitor, but not with a mTOR inhibitor (everolimus), adjusting for key prognostic variables in multivariate analysis. Furthermore, mTOR-based therapy resulted in a sustained radiological and molecular response in an index case of FGFR1+ HR+/HER2− MBC. In preclinical models, estrogen receptor–positive (ER+)/FGFR1-amplified CAMA1 human breast cancer cells were only partially sensitive to fulvestrant, palbociclib, and alpelisib, but highly sensitive to everolimus. In addition, transduction of an FGFR1 expression vector into ER+ T47D cells induced resistance to fulvestrant that could be overcome by added TORC1 inhibition, but not PI3K or CDK4/6 inhibition. Conclusions: Collectively, these findings suggest that while FGFR1 amplification confers broad resistance to ER, PI3K, and CDK4/6 inhibitors, mTOR inhibitors might have a unique therapeutic role in the treatment of patients with ER+/FGFR1+ MBC.

Plasma genotyping identifies potentially actionable mutations at variable mutant allele frequencies, often admixed with multiple subclonal variants, highlighting the need for their clinical and functional validation. We prospectively monitored plasma genotypes in 143 women with endocrine-resistant metastatic breast cancer (MBC), identifying multiple novel mutations including HER2 mutations (8.4%), albeit at different frequencies highlighting clinical heterogeneity. To evaluate functional significance, we established ex vivo culture from circulating tumor cells (CTCs) from a patient with HER2-mutant MBC, which revealed resistance to multiple targeted therapies including endocrine and CDK 4/6 inhibitors, but high sensitivity to neratinib (IC50: 0.018 μM). Immunoblotting analysis of the HER2-mutant CTC culture line revealed high levels of HER2 expression at baseline were suppressed by neratinib, which also abrogated downstream signaling, highlighting oncogenic dependency with HER2 mutation. Furthermore, treatment of an index patient with HER2-mutant MBC with the irreversible HER2 inhibitor neratinib resulted in significant clinical response, with complete molecular resolution of two distinct clonal HER2 mutations, with persistence of other passenger subclones, confirming HER2 alteration as a driver mutation. Thus, driver HER2 mutant alleles that emerge during blood-based monitoring of endocrine-resistant MBC confer novel therapeutic vulnerability, and ex vivo expansion of viable CTCs from the blood circulation may broadly complement plasma-based mutational analysis in MBC.

Background: Although the androgen receptor (AR) is frequently co-expressed with ER and PR in hormone receptor-positive (HR+)/HER2- breast cancer, the biological significance of detectable AR alterations (ARalt) in metastatic disease (MBC) remains poorly understood. The primary objective of this study was to evaluate the association of ARalt status with clinical outcomes among women with HR+/HER2- MBC. Methods: Retrospective review was performed on patients with HR+/HER2- MBC treated at an academic institution, for whom genotyping information was available. ARalt status was determined using Guardant360, a 73-gene next-generation sequencing assay that detects both AR mutations and amplifications in circulating tumor DNA. Women with positive or unknown HER2 status and triple-negative breast cancer were excluded from analysis, as were cases of male breast cancer. Time-to-progression on the therapy initiated immediately following Guardant testing was compared based on ARalt status, excluding patients treated with androgen-directed therapies given potential for confounding. Cumulative incidence plots were generated and analyzed by Gray’s test, and propensity score-adjusted competing risk models were generated with the probability of treatment as a function of age at metastatic diagnosis, presence of visceral metastasis, presence of de novo metastases, as well as number of prior therapies. Additional analysis was performed to assess progression stratified by treatment type (endocrine or non-endocrine based). Results: Among women with HR+/HER2- MBC (n=222), 16 patients (7%) had detectable ARalt (12 point mutations, 4 amplifications). No baseline differences were observed between women with ARalt and those without AR alterations (ARwt), with respect to age at primary or metastatic diagnosis, menopause status, time to onset of metastasis or de novo metastatic disease, presence of visceral metastases, or number of endocrine/chemotherapies received prior to Guardant testing. ARalt tumors had a higher frequency of detected mutations (14% vs. 5%, p Conclusions: ARalt tumors demonstrate a higher rate of progression on endocrine-based therapy as compared to ARwt tumors, highlighting a potential role of AR in mediating resistance to endocrine therapy in HR+/HER2- disease. Further translational investigations are warranted to determine whether ARalt/HR+/HER2- disease represents a unique biological subtype that predominantly relies on AR signaling and may thus benefit from blockade with AR antagonists. Table 1. Multivariable competing risks model for endocrine progression.CovariatePFSMultivariableHR95% CIP-valuePositive ARalt status4.172.43-7.17 Citation Format: Charles Dai, Andrzej Niemierko, Neelima Vidula, Laura M Spring, Seth A Wander, Arielle J Medford, Katherine A Hesler, Giuliana Malvarosa, Jeffrey Peppercorn, Dejan Juric, Steven J Isakoff, Beverly Moy, Leif W Ellisen, Aditya Bardia. Molecular alterations in the androgen receptor and associated clinical outcomes in hormone receptor-positive/HER2- 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 PS17-02.

Background: Oncogenic mutations are potential targets for therapeutic intervention in metastatic breast cancer (MBC). While tumor genotyping (TG) has been viewed as the gold standard for identifying oncogenic mutations, cell-free circulating tumor DNA (cfDNA) is emerging as an alternate technique. We previously reported the selection of matched therapy targeted to an actionable mutation based on either TG or cfDNA testing (Vidula N, ASCO, 2018). Therefore, we are now comparing TG and cfDNA results in MBC patients undergoing both tests to examine their relative utility in the selection of matched therapy. Methods: Patients with MBC at an academic institution who underwent both TG (Next Generation Sequencing/NGS, institutional platform, 104 gene assay) and cfDNA testing (NGS/Guardant360, 73 gene assay) between 1/2016-10/2017 were identified. A chart review was conducted to identify tumor subtype, demographics, treatment, TG and cfDNA results, and clinical outcomes. The relative utility of these tests in the selection of matched therapy was determined, and linked with clinical outcomes (progression-free survival and overall survival). Results: Thirty patients who underwent both TG and cfDNA testing were identified. The median age was 60 years, the majority (97%) had hormone receptor (HR) positive/HER2 negative disease, and most patients had recurrent disease (83.3%) at MBC diagnosis. The median number of therapies prior to obtaining either test was 1 (cfDNA range 0-9, TG range 0-8). The majority had simultaneous cfDNA and tumor genotyping testing (83.3%) versus sequential testing (16.7%). Twenty-four (80%) patients had actionable mutations detected by cfDNA compared to 19 (63.3%) patients with actionable mutations detected by TG. The median number of actionable mutations detected by cfDNA was 2 (range 0-11) compared with a median of 1 (range 0-4) detected by TG. Failure of TG occurred in 2 of 30 patients (6.7%) but no test failures were seen with cfDNA. Eleven of 30 patients (36.7%) had ≥ 1 concordant mutation via cfDNA and TG. Altogether, 12 out of 30 (40%) patients received matched therapy, 5 of which were based on cfDNA actionable mutations alone (ESR1, ERBB2, CCND1, and PIK3CA), and 7 based on cfDNA and TG results (ESR1, PIK3CA, STK11, and BRCA). Twelve of 24 (50%) patients with actionable cfDNA mutations went on to receive matched therapy compared with 7 of 19 (36.8%) patients with actionable TG results. Matched therapies included SERDs, inhibitors of CDK 4/6, PI3K, mTOR, HER2 directed therapy, and DNA damaging chemotherapy. The impact of matched therapy on survival outcomes will be presented at the meeting. Conclusions: In patients undergoing both TG and cfDNA testing, both tests identify a significant cohort of HR+ MBC patients with actionable mutations, with greater detection of actionable mutations by cfDNA. Greater application of matched therapy occurred via cfDNA, which independently informed the selection of matched therapies. Further research is needed to prospectively evaluate the clinical utility of blood based genotyping assays versus TG for patients with MBC. Citation Format: Vidula N, Juric D, Niemierko A, Spring L, Moy B, Malvarosa G, Yuen M, Habin K, Shin J, Peppercorn J, Isakoff S, Ellisen L, Iafrate AJ, Bardia A. Comparison of tumor genotyping and cell-free circulating tumor DNA sequencing in metastatic breast cancer patients and their utility in the selection of matched therapy [abstract]. In: Proceedings of the 2018 San Antonio Breast Cancer Symposium; 2018 Dec 4-8; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2019;79(4 Suppl):Abstract nr P4-01-06.

Background: The combination of cyclin-dependent kinase 4 and 6 inhibitors (CDK 4/6 inh) and endocrine therapy have changed the treatment paradigm for hormone receptor-positive metastatic breast cancer (HR+ MBC). Three CDK 4/6 inhibitors (palbociclib, ribociclib, and abemaciclib) are now approved for HR+ MBC in combination with either an aromatase inhibitor (AI) or fulvestrant (a selective estrogen receptor degrader, SERD) after demonstrating significant improvement in progression-free survival compared to endocrine monotherapy. However, both primary and secondary resistance has been observed clinically, and the mechanisms of such resistance are not well understood. Here we describe emergence of molecular alterations following treatment with the combination of CDK 4/6 inhibitors and endocrine therapy. Methods: We evaluated molecular alterations by next-generation sequencing (NGS) prior to and following treatment with CDK 4/6 + endocrine therapy pre and post CDK inh among patients with HR+ MBC. We performed circulating tumor (ct) DNA analysis via the Guardant360 assay, an NGS-based assay that identifies potential tumor-related (somatic) genomic alterations through sequencing of the critical exons within 73 cancer-related genes, and/or tissue-based genomic analysis utilizing our institutional NGS platform (SNaPshot), which targets hotspots and exons in 89 genes. Results: A total of 33 HR+ MBC patients had paired pre/post CDK 4/6 tissue/liquid biopsies. 19 (57.6%) received palbociclib and 14 (42.4%) ribociclib, with 25 (75%) receiving an AI and 8 (24%) a SERD. Eight patients (24.2%) were first line. The median duration of response was 8 months (range 1-35 months). The most common acquired alterations at the time of progression that were not present in the pre-treatment specimens included mutations in ESR1 (30.3%), TP53 (30.3%), RB1 (12.1%) and PIK3CA (9.1%), and FGFR1 amplification (27.3%). We then evaluated these results in a subset of patients (N=17) who had paired pre/post liquid biopsies only and found similar results. We observed alterations in oncogenic pathways both upstream and downstream of CDK 4/6-cyclin-D complex (N=17), including upstream receptor tyrosine kinase alterations (29.4%), PI3K/AKT/mTOR alterations (5.9%), and MAPK alterations (17.6%), as well as downstream cell-cycle (35.3%) and DNA repair gene alterations (17.6%). Furthermore, 58.8% of patients had more than one acquired potential driver mutation, and 41.2% had both upstream and downstream alterations possibly reflecting emergence of different subclones and tumor heterogeneity under selective pressure from CDK 4/6 inhibitors. Conclusion: Both upstream and downstream genomic alterations may mediate acquired clinical resistance to CDK 4/6 inhibitors, reflecting genomic evolution and diversification after exposure to CDK 4/6 inhibitors. Additional studies are needed to confirm these findings and develop potential therapeutic strategies to overcome clinical resistance to CDK 4/6 inhibitors for patients with MBC. Citation Format: Spring LM, Haddad S, Malvarosa G, Vidula N, Juric D, Iafrate AJ, Ellisen LW, Moy B, Isakoff SJ, Bardia A. Paired pre- and post-treatment DNA sequencing identifies genomic alterations mediating clinical resistance to CDK 4/6 inhibitors in patients with metastatic breast cancer [abstract]. In: Proceedings of the 2018 San Antonio Breast Cancer Symposium; 2018 Dec 4-8; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2019;79(4 Suppl):Abstract nr PD9-06.

1021Background: While molecular alterations in PI3K pathway, including PIK3CA mutations, are common in HR+ breast cancer, molecular alterations in MAPK pathway, including KRAS and BRAF mutations, a...

Introduction: Genomic profiling of circulating tumor DNA (ctDNA) allows non-invasive monitoring of tumor genetic changes and molecular heterogeneity. In addition to actionable mutations, mutational landscape derived from ctDNA could provide a better representation of overall tumor burden and tumor heterogeneity, as well as potentially impact clinical outcomes. To evaluate this hypothesis, this study assessed the association of mutation burden and average mutant allele fraction (MAF) with tumor subtype, therapeutic response, and survival in patients with metastatic breast cancer. Methods: Whole blood samples from patients with metastatic breast cancer were collected during clinic visits before start of a new therapy. Plasma-derived cell-free DNA underwent complete next-generation sequencing of 73 cancer-related genes with the Guardant360 test. Mutation burden was defined as the number of genes with mutations, and average MAF was calculated as the sum of the highest MAF for each mutated gene divided by the number of genes with mutations. Time to progression was measured from the date of new treatment initiation after circulating tumor DNA collection to the date of progression. Multivariate cox proportional hazard models assessed the association of mutation burden and average MAF with progression free survival (PFS), adjusted for age, receptor subtype (hormone receptor positive, HR+; human epidermal growth factor 2 positive, HER2+; triple negative breast cancer, TNBC), treatment subtype (chemotherapy vs. targeted therapy), and number of prior metastatic breast cancer therapies. A p value of 0.05 was considered statistically significant. Results: The study population consisted of 158 women with metastatic breast cancer (108 HR+, 14 HER2+, 19 TNBC) with a median age of 59 years and a median of 2 prior metastatic breast cancer therapies. Median follow up time was 4.0 months, and median PFS was 15.7 months. Mutation burden was greater in triple negative compared to hormone receptor positive breast cancer (7.5 vs. 4.8, p = 0.02) but no different in patients with > 2 prior metastatic therapies vs. not (5.1 vs. 4.7, p = 0.60) and age >45 vs. not (5.0 vs. 3.9; p = 0.26). In univariate models, high mutation burden (> median of 2) and high MAF (> median of 1.4) were significantly associated with worse PFS (Table). These results were similar in effect size and significance when adjusted for age, receptor subtype, treatment subtype, and number of prior metastatic breast cancer therapies. Impact of mutation burden on response to specific therapies will be presented at the meeting. VariableHazard Ratio95% Confidence Intervalp valueHigh mutation burden1.991.12-3.540.02High mutant allele fraction1.881.06-3.330.03 Conclusions: Higher ctDNA mutation burden and average MAF is associated with worse progression free survival and possibly reflects a more treatment refractory phenotype. Whether immunotherapy, alone or in combination, could influence the clinical outcomes in metastatic breast cancer patients with high ctDNA mutation burden is unclear and warrants additional research. Citation Format: Keenan T, Juric D, Niemierko A, Spring L, Park H, Malvarosa G, Beeler M, Moy B, Ellisen L, Isakoff S, Bardia A. Higher mutation burden and mutant allele fraction of circulating tumor DNA corresponds to worse progression free survival in metastatic breast cancer patients [abstract]. In: Proceedings of the 2017 San Antonio Breast Cancer Symposium; 2017 Dec 5-9; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2018;78(4 Suppl):Abstract nr P2-02-18.