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PURPOSE As the continuation beyond progression (BP) of cyclin-dependent kinase 4/6 inhibitors (CDK4/6i) is becoming increasingly attractive for the treatment of patients with hormone receptor (HR)–positive, human epidermal growth factor receptor 2 (HER2)–negative metastatic breast cancer (MBC), the definition of resistance factors is crucial. The aim of the study was to investigate the impact of CDK 4/6i BP and to explore potential genomic stratification factors. MATERIALS AND METHODS We retrospectively analyzed a multi-institutional cohort of patients with HR-positive HER2-negative MBC characterized for circulating tumor DNA through next-generation sequencing before treatment start. Differences across subgroups were analyzed by chi-square test, and survival was tested by univariable and multivariable Cox regression. Further correction was applied by propensity score matching. RESULTS Among the 214 patients previously exposed to CDK4/6i, 172 were treated with non–CDK4/6i-based treatment (non-CDK) and 42 with CDK4/6i BP. Multivariable analysis showed a significant impact of CDK4/6i BP, TP53 single-nucleotide variants, liver involvement, and treatment line on both progression-free survival (PFS) and overall survival (OS). Propensity score matching confirmed the prognostic role of CDK4/6i BP both for PFS and OS. The favorable impact of CDK4/6i BP was consistent across all subgroups, and a differential benefit was suggested for ESR1-mutated patients. ESR1 and RB1 mutations were more represented in the CDK4/6i BP subgroup with respect to CDK4/6i upfront. CONCLUSION The study highlighted a significant prognostic impact of the CDK4/6i BP strategy with a potential added benefit in patients with ESR1 mutations suggesting the need for an extensive biomarker characterization.

Purpose: Mutations in the estrogen receptor (ER)α gene, ESR1, have been identified in breast cancer metastases after progression on endocrine therapies. Because of limitations of metastatic biopsies, the reported frequency of ESR1 mutations may be underestimated. Here, we show a high frequency of ESR1 mutations using circulating plasma tumor DNA (ptDNA) from patients with metastatic breast cancer.Experimental Design: We retrospectively obtained plasma samples from eight patients with known ESR1 mutations and three patients with wild-type ESR1 identified by next-generation sequencing (NGS) of biopsied metastatic tissues. Three common ESR1 mutations were queried for using droplet digital PCR (ddPCR). In a prospective cohort, metastatic tissue and plasma were collected contemporaneously from eight ER-positive and four ER-negative patients. Tissue biopsies were sequenced by NGS, and ptDNA ESR1 mutations were analyzed by ddPCR.Results: In the retrospective cohort, all corresponding mutations were detected in ptDNA, with two patients harboring additional ESR1 mutations not present in their metastatic tissues. In the prospective cohort, three ER-positive patients did not have adequate tissue for NGS, and no ESR1 mutations were identified in tissue biopsies from the other nine patients. In contrast, ddPCR detected seven ptDNA ESR1 mutations in 6 of 12 patients (50%).Conclusions: We show that ESR1 mutations can occur at a high frequency and suggest that blood can be used to identify additional mutations not found by sequencing of a single metastatic lesion. Clin Cancer Res; 22(4); 993–9. ©2015 AACR.

Supplementary Methods Figure S1 ESR1 Y537SY537N and D538G probes are mutation specific. Figure S2 Patient 1 and Patient 14's plasma DNA. Table S1 Additional patient characteristics. Table S2 Primers used in this study.

A Woman with Nausea, Diarrhea, and Acute Kidney Failure A 76-year-old woman with a history of type 2 diabetes, obstructive sleep apnea, coronary artery disease, and heart failure with reduced eject...

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.

Antibody-drug conjugates (ADCs) are composed of monoclonal antibodies linked to a cytotoxic payload, enabling targeted delivery of more potent chemotherapy. In the past decade, there has been rapid development of ADCs aimed at different types of breast cancer. The success of the monoclonal antibody trastuzumab has led to the evolution of several antibody-drug conjugates targeting HER2-positive breast cancer. Trastuzumab-emtansine (T-DM1), the first approved ADC targeting HER2-positive breast cancer, has become standard of care for patients with high-risk early-stage HER2-positive breast cancer who have residual disease after neoadjuvant chemotherapy. More recently, the observation of the bystander effect, in which ADCs target both antigen-positive cells and adjacent antigen-negative cells, has led to the reclassification of “HER2-low” breast cancer and the development of trastuzumab-deruxtecan (T-DXd) to target this population. This article reviews the history of HER2-directed ADCs in breast cancer as well as ongoing ADCs in development.

Cancer-associated mutations in the spliceosome gene SF3B1 create a neomorphic protein that produces aberrant mRNA splicing in hundreds of genes, but the ensuing biologic and therapeutic consequences of this missplicing are not well understood. Here we have provided evidence that aberrant splicing by mutant SF3B1 altered the transcriptome, proteome, and metabolome of human cells, leading to missplicing-associated downregulation of metabolic genes, decreased mitochondrial respiration, and suppression of the serine synthesis pathway. We also found that mutant SF3B1 induces vulnerability to deprivation of the nonessential amino acid serine, which was mediated by missplicing-associated downregulation of the serine synthesis pathway enzyme PHGDH. This vulnerability was manifest both in vitro and in vivo, as dietary restriction of serine and glycine in mice was able to inhibit the growth of SF3B1(MUT) xenografts. These findings describe a role for SF3B1 mutations in altered energy metabolism, and they offer a new therapeutic strategy against SF3B1(MUT) cancers.

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.

The receptor-based classification of breast cancer predicts its optimal therapy. Hormone Receptor (HR) positive breast cancer is treated with endocrine therapy, and HER2+ disease is treated with HER2-targeted therapy. Triple negative breast cancer (TNBC), defined as tumors lacking HR and HER2, represents an aggressive subtype of breast cancer associated with poor prognosis. Development of targeted therapy for this subtype has been challenging since TNBC usually lacks targetable genomic alterations. However, the advent of antibody drug conjugates (ADC) to target antigens overexpressed in breast cancer has opened the door to a new class of breast cancer therapeutics. In this review, we describe the current FDA-approved ADC therapies for breast cancer, including sacituzumab govitecan, as well as agents currently in advanced stages of investigation. In addition, we review the potential therapeutic application of ADCs across different breast cancer subtypes. In the future, therapeutic advances in ADCs targeting different antigens could redefine the current receptor-based classification of breast cancer.

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.

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.

Purpose: Mutations in the estrogen receptor (ER)α gene, ESR1, have been identified in breast cancer metastases after progression on endocrine therapies. Because of limitations of metastatic biopsies, the reported frequency of ESR1 mutations may be underestimated. Here, we show a high frequency of ESR1 mutations using circulating plasma tumor DNA (ptDNA) from patients with metastatic breast cancer. Experimental Design: We retrospectively obtained plasma samples from eight patients with known ESR1 mutations and three patients with wild-type ESR1 identified by next-generation sequencing (NGS) of biopsied metastatic tissues. Three common ESR1 mutations were queried for using droplet digital PCR (ddPCR). In a prospective cohort, metastatic tissue and plasma were collected contemporaneously from eight ER-positive and four ER-negative patients. Tissue biopsies were sequenced by NGS, and ptDNA ESR1 mutations were analyzed by ddPCR. Results: In the retrospective cohort, all corresponding mutations were detected in ptDNA, with two patients harboring additional ESR1 mutations not present in their metastatic tissues. In the prospective cohort, three ER-positive patients did not have adequate tissue for NGS, and no ESR1 mutations were identified in tissue biopsies from the other nine patients. In contrast, ddPCR detected seven ptDNA ESR1 mutations in 6 of 12 patients (50%). Conclusions: We show that ESR1 mutations can occur at a high frequency and suggest that blood can be used to identify additional mutations not found by sequencing of a single metastatic lesion. Clin Cancer Res; 22(4); 993–9. ©2015 AACR.

Ki-67 expression is correlated with cell proliferation and is a prognostic marker for various cancers; however, its function is unknown. Here we demonstrate that genetic disruption of Ki-67 in human epithelial breast and colon cancer cells depletes the cancer stem cell niche. Ki-67 null cells had a proliferative disadvantage compared to wildtype controls in colony formation assays and displayed increased sensitivity to various chemotherapies. Ki-67 null cancer cells showed decreased and delayed tumor formation in xenograft assays, which was associated with a reduction in cancer stem cell markers. Immunohistochemical analyses of human breast cancers revealed that Ki-67 expression is maintained at equivalent or greater levels in metastatic sites of disease compared to matched primary tumors, suggesting that maintenance of Ki-67 expression is associated with metastatic/clonogenic potential. These results elucidate Ki-67's role in maintaining the cancer stem cell niche, which has potential diagnostic and therapeutic implications for human malignancies.

Breast cancer is the leading cause of cancer in women and the second leading cause of cancer-related death. There are many subtypes of breast cancer, which can be identified through the process of molecular and genetic profiling. While the current standard of care utilizes tumor tissue biopsy to subclassify breast cancer, plasma tumor DNA (ptDNA) can be detected through droplet digital PCR (ddPCR) of plasma obtained from a simple blood draw. Tissue biopsy is not only more invasive but because tumors exhibit heterogeneity it can be less accurate. Blood collects DNA shed from normal and cancerous cells alike, thus ddPCR of plasma offers a broader picture of a cancer's genetic makeup. This chapter summarizes how patients with breast cancer can be screened for specific cancerous mutations in both tissue and plasma through the use of ddPCR.

Background Genomic testing is often limited by the exhaustible nature of human tissue and blood samples. Here we describe biotinylated amplicon sequencing (BAmSeq), a method that allows for the creation of PCR amplicon based next-generation sequencing (NGS) libraries while retaining the original source DNA. Design and methods Biotinylated primers for different loci were designed to create NGS libraries using human genomic DNA from cell lines, plasma, and formalin-fixed paraffin embedded (FFPE) tissues using the BAmSeq protocol. DNA from the original template used for each BAmSeq library was recovered after separation with streptavidin magnetic beads. The recovered DNA was then used for end-point, quantitative and droplet digital PCR (ddPCR) as well as NGS using a cancer gene panel. Results Recovered DNA was analyzed and compared to the original DNA after one or two rounds of BAmSeq. Recovered DNA revealed comparable genomic distributions and mutational allelic frequencies when compared to original source DNA. Sufficient quantities of recovered DNA after BAmSeq were obtained, allowing for additional downstream applications. Conclusions We demonstrate that BAmSeq allows original DNA template to be recovered with comparable quality and quantity to the source DNA. This recovered DNA is suitable for many downstream applications and may prevent sample exhaustion, especially when DNA quantity or source material is limiting., Highlights • Modification of targeted panel sequencing allows for recovery of original DNA template. • Protocol provides value in the setting of scarce DNA template. • Recovered DNA is suitable for NGS, ddPCR and qPCR. • Recovered DNA shows no loss of genomic regions.

Mutations in the SF3B1 gene occur in multiple cancers, creating a neomorphic spliceosome protein that induces aberrant mRNA splicing in thousands of genes. Little is known about the downstream oncogenic or therapeutic consequences of this missplicing. To study this, we engineered knockins of SF3B1 mutation in untransformed breast epithelial cells, as well as “reverse” knockins in which SF3B1-mutant uveal melanoma cells were corrected to wild type. Transcriptomic analysis of these isogenic pairs showed cryptic splicing induced by mutant SF3B1, and SILAC proteomics demonstrated a broad reshaping of the proteome dominated by downregulation of misspliced genes. Interestingly, pathway analysis revealed a suppression of proteins involved in energy metabolism. This included mitochondrial complex III, which rescue experiments showed was downregulated through missplicing of its assembly factor UQCC1, and PHGDH, a serine synthesis enzyme likewise misspliced and downregulated. Metabolomic analysis also showed evidence of metabolic reprogramming by mutant SF3B1, including suppression of serine, glycine, and aspartate levels. Because serine and glycine (SG) starvation can have anticancer activity in vivo—and because PHGDH expression modulates this effect—we tested SG starvation on SF3B1-mutant cells. Growth of our SF3B1-mutant isogenic cells was differentially suppressed by SG starvation, and two independent AML cell lines with endogenous SF3B1 mutation demonstrated significant cell death upon SG starvation, in contrast to multiple AML cell lines wild type for SF3B1. These data suggest impaired serine synthesis may be a therapeutic vulnerability in SF3B1-mutant cancers. In addition to downregulation of genes induced by mutant SF3B1, our proteomic data also revealed a novel cryptic protein that was highly enriched in SF3B1-mutant cells. This protein, CD98, is a surface membrane protein, and the cryptic peptide change occurred in its extracellular domain. Additional transcriptome analysis revealed eleven more candidate surface proteins with mutant SF3B1-induced cryptic isoforms containing in-frame peptide changes in extracellular domains, and overexpression studies demonstrated at least two of these cryptic proteins can traffic to the cell surface. We have named these putative proteins MASAs, for Missplicing-Associated Surface Antigens, and we believe they represent a novel kind of tumor surface antigen that may be targetable with antibody-based immunotherapeutics. In summary, our data provide evidence that SF3B1 mutations induce proteome alterations, metabolic reprogramming, and a new kind of tumor surface antigen in human cells. Citation Format: William B. Dalton, Daniel Shinn, Noel Walsh, Eric Christenson, Taylor Groginski, Dhanashree Kelkar, Anil Magugundu, Arun Patil, Daniel Zabransky, Arielle Medford, Justin Lee, Alex Cole, Josh Donaldson, Amy DeZern, Karen Cravero, David Chu, Natasha Hunter, Akhilesh Pandey, Josh Lauring, Ben Park. SF3B1 mutations induce proteome remodeling, metabolic reprogramming, and a novel kind of tumor surface antigen in human cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 1408.

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...