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Purpose: Oral administration of the alkylating agent cyclophosphamide at low doses, metronomic dosing, is well tolerated, with efficacy in multiple tumor types. PARP inhibition potentiates effects of cyclophosphamide in preclinical models. We conducted a phase I trial of the PARP inhibitor veliparib and metronomic cyclophosphamide in patients with refractory solid tumors and lymphoid malignancies.Experimental Design: Objectives were to establish the safety and maximum tolerated dose (MTD) of the combination; characterize veliparib pharmacokinetics (PK); measure poly(ADP-ribose) (PAR), a product of PARP, in tumor biopsies and peripheral blood mononuclear cells (PBMC); and measure the DNA-damage marker γH2AX in PBMCs and circulating tumor cells (CTC). Cyclophosphamide was administered once daily in 21-day cycles in combination with veliparib administered once daily for 7, 14, or 21 days.Results: Thirty-five patients were enrolled. The study treatment was well tolerated, and the MTD was established as veliparib 60 mg with cyclophosphamide 50 mg given once daily. Seven patients had partial responses; an additional six patients had disease stabilization for at least six cycles. PAR was significantly decreased in PBMCs (by at least 50%) and tumor biopsies (by at least 80%) across dose levels (DL); γH2AX levels were increased in CTCs from seven of nine patients evaluated after drug administration.Conclusions: The combination of veliparib with metronomic cyclophosphamide is well tolerated and shows promising activity in a subset of patients with BRCA mutations. A phase II trial of the combination compared with single-agent cyclophosphamide is ongoing in BRCA-positive ovarian cancer, triple-negative breast cancer, and low-grade lymphoma. Clin Cancer Res; 18(6); 1726–34. ©2012 AACR.

Table S1. Sequence of targeted amplification primers. Table S2. Sequence of ddPCR primer and probes. Table S3. Cellularity and location of bone metastases.

Supplementary Figure S1A. Pre-amplification preserves mutant allele frequency and maintains sensitivity of ESR1-D538G mutation detection by ddPCR. Supplementary Figure S1B. Pre-amplification preserves mutant allele frequency and maintains sensitivity of PIK3CA-E545K mutation detection by ddPCR. Supplementary Figure S2. ESR1 Y537C/N/S and D538G mutation probes are specific to their corresponding mutations. Supplementary Figure S3. Specificity of Probes Supplementary Figure S4. LLoD determination of ddPCR on A) Frozen tissues, B) cfDNA, and C) FFPE tissues. Supplementary Figure S5. Mutant allele frequency of PIK3CA H1047R mutation in 12 bone metastases. Supplementary Figure S6. The D538G ER mutation in 3 brain mets was confirmed by Sanger sequencing. Supplementary Figure S7. ESR1 Y537S and D538G observed in the same specimens are not mutated on the same alleles. Supplementary Figure S8. Clinical timelines and allele frequency of ESR1 mutations in multiple blood draws and matched metastatic lesions from two patients.

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Purpose: Given the clinical relevance of ESR1 mutations as potential drivers of resistance to endocrine therapy, this study used sensitive detection methods to determine the frequency of ESR1 mutations in primary and metastatic breast cancer, and in cell-free DNA (cfDNA).Experimental Design: Six ESR1 mutations (K303R, S463P, Y537C, Y537N, Y537S, D538G) were assessed by digital droplet PCR (ddPCR), with lower limits of detection of 0.05% to 0.16%, in primary tumors (n = 43), bone (n = 12) and brain metastases (n = 38), and cfDNA (n = 29). Correlations between ESR1 mutations in metastatic lesions and single (1 patient) or serial blood draws (4 patients) were assessed.Results: ESR1 mutations were detected for D538G (n = 13), Y537S (n = 3), and Y537C (n = 1), and not for K303R, S463P, or Y537N. Mutation rates were 7.0% (3/43 primary tumors), 9.1% (1/11 bone metastases), 12.5% (3/24 brain metastases), and 24.1% (7/29 cfDNA). Two patients showed polyclonal disease with more than one ESR1 mutation. Mutation allele frequencies were 0.07% to 0.2% in primary tumors, 1.4% in bone metastases, 34.3% to 44.9% in brain metastases, and 0.2% to 13.7% in cfDNA. In cases with both cfDNA and metastatic samples (n = 5), mutations were detected in both (n = 3) or in cfDNA only (n = 2). Treatment was associated with changes in ESR1 mutation detection and allele frequency.Conclusions: ESR1 mutations were detected at very low allele frequencies in some primary breast cancers, and at high allele frequency in metastases, suggesting that in some tumors rare ESR1-mutant clones are enriched by endocrine therapy. Further studies should address whether sensitive detection of ESR1 mutations in primary breast cancer and in serial blood draws may be predictive for development of resistant disease. Clin Cancer Res; 22(5); 1130–7. ©2015 AACR.See related commentary by Gu and Fuqua, p. 1034

Breast cancer is categorized by the molecular and histologic presentation of the tumor; with the major histologic subtypes observed in patients being Invasive Ductal Carcinoma (IDC) and Invasive Lobular Carcinoma (ILC). ILC are characterized by growth in a single file discohesive manner with stromal infiltration attributed to their hallmark loss of E-cadherin. The lower incidence of ILC, relative to IDC, resulted in this subtype being understudied in the field. The Cancer Cell Line Encyclopedia (CCLE) currently only lists 2 ILC cell lines; emphasizing the need to expand the inventory of models available to researchers. To that end, here we report the successful establishment and characterization of a novel ILC cell line from a metastatic pleural aspirate from a postmenopausal Caucasian woman with ER+ metastatic ILC. The patient underwent a bilateral mastectomy, several pleural fluid aspirations and multiple treatment changes as her disease progressed until she succumbed to the disease. Various cell culture methods were attempted with the pleural aspirate and a standard practice of DMEM supplemented with 10% FBS in normoxic conditions was deemed effective. A cell line was developed, WCRC-25 which is ER-. WCRC-25 appears to be E2 unresponsive in vitro and is morphologically larger than other ILC cells currently in use, namely, MDA-MB-134, BCK4 and IPH-926, while exhibiting better adherence to 2D plates and a less rounded morphology. Sequencing revealed a CDH1 Q706 truncating mutation in the cells, which was also observed in the patient’s cfDNA with its enrichment increasing over time, supporting elevated disease burden. RNA-seq analysis comparing the primary tumor to metastases and the cell line reveal a signature for cell cycle progression and Akt signaling. To assess targetability, we subjected WCRC-25 to AZD5363 and Alpelisib which confirmed WCRC-25 as susceptible to PI3K/Akt signaling inhibition. Finally, we generated mouse xenograft models of WCRC-25 through mammary fat pad injection and observed small, but palpable tumors and metastasis to several sites. In conclusion, we report WCRC-25 as a novel ILC cell line with much promise as an invaluable research tool to advance our understanding of ILC and its therapeutic vulnerabilities. Citation Format: Ashuvinee Elangovan, Emily A Bossart, Ahmed Basudan, Nilgun Tasdemir, Osama Shiraz Shah, Kai Ding, Carolin Meier, Tanya Heim, Lori Miller, Tiantong Liu, Shannon L Puhalla, Grzegorz Gurda, Peter C Lucas, Priscilla F McAuliffe, Jennifer M Atkinson, Adrian V Lee, Steffi Oesterreich. Wcrc-25: A novel luminal invasive lobular carcinoma cell line model [abstract]. In: Proceedings of the 2021 San Antonio Breast Cancer Symposium; 2021 Dec 7-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2022;82(4 Suppl):Abstract nr P5-12-03.

TPS284 Background: Detectable ctDNA after resection of early-stage solid tumors has been associated with very high risk of recurrence, suggesting ctDNA is evidence of minimal residual disease (MRD). Several studies are ongoing to investigate the role of ctDNA in the optimal management of pts with colorectal cancer using different assay technologies. Methods: This is a prospective, observational, multicenter study in the United States and Canada of 750 patients who have undergone complete surgical resection for stage II or III colorectal cancer, who have FFPE tissue available from the primary resection sufficient for a novel bespoke MRD assay and are willing to provide serial whole blood specimens for ctDNA analysis. Participants are asked to provide study specimens after definitive surgical resection, pre-recurrence follow-up, and clinical recurrence (if applicable). Recently amended eligibility criteria include inclusion of rectal cancer patients who have completed neo-adjuvant therapy and surgical resection, as well as enrollment of all stage II and III patients regardless of microsatellite stability status. The Oncotype Colon Recurrence Score will be assessed on all patients from their surgical specimen if criteria are met for this testing. ctDNA will be analyzed with an NGS-based tumor-informed MRD assay that identifies somatic genomic alterations from DNA derived from the patient’s tumor tissue, subtracts germline variants, and detects a selected subset of tumor-specific (bespoke) ctDNA in their blood. All primary tumor specimens will undergo full exome and transcriptome sequencing using the Oncomap ExTra assay. If there is evidence of disease recurrence, the metastatic tissue will also undergo Oncomap ExTra testing, which will be shared with participants. The primary objective is to validate the association of post-definitive therapy and pre-recurrence follow-up ctDNA positivity with recurrence-free interval (RFI). Further objectives are to assess the: sensitivity and specificity of ctDNA positivity for subsequent clinical recurrence; contribution of post-surgery baseline, post-adjuvant therapy, and pre-recurrence follow-up ctDNA results on RFI; time from positive ctDNA to clinical recurrence in participants who had a positive ctDNA result; and compare the Oncotype Colon Recurrence Score estimate of 3-year recurrence risk with the observed 3-year recurrence rate. The primary analysis will use a Cox proportional hazards regression applied to the RFI with ctDNA result (positive or negative) measured at post-surgical baseline (or end of adjuvant therapy if used) and serially after that as a single, time-dependent covariate. Protocol#: NSABP C-14 / ES 16-002. Support: NSABP Foundation, ExactSciences Clinical trial information: 05210283 .

BackgroundCyclin-dependent kinases (CDK) 4 and 6 regulate G1 to S cell cycle progression and are often altered in cancers. Abemaciclib is a selective inhibitor of CDK4 and CDK6 approved for administration on a continuous dosing schedule as monotherapy or as combination therapy with an aromatase inhibitor or fulvestrant in patients with advanced or metastatic breast cancer. This Phase 1b study evaluated the safety and tolerability, pharmacokinetics, and antitumor activity of abemaciclib in combination with endocrine therapy for metastatic breast cancer (MBC), including aromatase inhibitors (letrozole, anastrozole, or exemestane) or tamoxifen.Patients and MethodsWomen ≥18 years old with hormone receptor positive (HR+), human epidermal growth factor receptor 2 negative (HER2-) MBC were eligible for enrollment. Eligibility included measurable disease or non-measurable but evaluable bone disease by Response Evaluation Criteria in Solid Tumours (RECIST) v1.1, Eastern Cooperative Oncology Group performance status 0–1, and no prior chemotherapy for metastatic disease. Adverse events were graded by the National Cancer Institute Common Terminology Criteria for Adverse Events v4.0 and tumor response were assessed by RECIST v1.1.ResultsSixty-seven patients were enrolled and received abemaciclib 200 mg every 12 hours in combination with letrozole (Part A, n=20), anastrozole (Part B, n=16), tamoxifen (Part C, n=16), or exemestane (Part D, n=15). The most common treatment-emergent adverse events (TEAE) were diarrhea, fatigue, nausea, and abdominal pain. Grade 4 TEAEs were reported in five patients (one each with hyperglycemia, hypertension, neutropenia, procedural hemorrhage, and sepsis). There was no effect of abemaciclib or endocrine therapy on the pharmacokinetics of any combination study drug. Across all treated patients, the median progression-free survival was 25.4 months (95% confidence interval: 18.0, 35.8). The objective response rate was 38.9% in 36 patients with measurable disease.ConclusionsAbemaciclib in combination with multiple endocrine therapy options exhibited manageable safety and promising antitumor activity in patients with HR+, HER2- MBC.Clinical Trial Registrationhttps://clinicaltrials.gov/, identifier NCT02057133

Pathologic complete response (pCR) rate after neoadjuvant chemotherapy was compared between 141 estrogen receptor (ER)-negative (43%), 41 low ER+ (13%), 47 moderate ER+ (14%), and 98 high ER+ (30%) tumors.Human epidermal growth factor receptor 2-positive cases, cases without semiquantitative ER score, and patients treated with neoadjuvant endocrine therapy alone were excluded.The pCR rate of low ER+ tumors was similar to the pCR rate of ER- tumors (37% and 26% for low ER and ER- respectively, P = .1722) but significantly different from the pCR rate of moderately ER+ (11%, P = .0049) and high ER+ tumors (4%, P.0001). Patients with pCR had an excellent prognosis regardless of the ER status. In patients with residual disease (no pCR), the recurrence and death rate were higher in ER- and low ER+ cases compared with moderate and high ER+ cases.Low ER+ breast cancers are biologically similar to ER- tumors. Semiquantitative ER H-score is an important determinant of response to neoadjuvant chemotherapy.

DNA structural variations (SVs) are a major source of genetic instability in cancer, but they remain understudied. Large-insert mate-pair sequencing (MPS) is a powerful method designed to detect SVs, even in highly repetitive regions. Using MPS and other methods, we performed a comprehensive analysis of genomic alterations in breast cancer progression. Matched primary/recurrent frozen tumor samples from 6 patients, including two patients from our rapid autopsy program with multiple metastatic tissues (20 total samples; average 5.5 years to recurrence) were examined by multiple large-insert library (3-5, 5-8, 8-12kb) MPS to identify metastatic acquired SVs. This was supplemented with RNAseq (n=15), whole exome sequencing (n=18;~75x), whole genome sequencing (n=3; 40-65x), and SNP arrays. A relatively small fraction (~10%) of somatic single nucleotide variants (SNVs) in the primary tumor were identified in matched metastatic samples, and the majority of metastatic SNVs were not found in the matched primary tumor. This indicates that a rare sub-clone colonizes the metastatic site and evolves extensively before becoming clinically evident. For example, in one patient with an ER+ tumor who initially declined anti-estrogen therapy, the recently described ESR1 Y537S mutation was not present in the primary tumor or in metastatic disease 5 years later. However, after extensive anti-estrogen treatment for metastatic disease, the mutation was identified at rapid autopsy, indicating that this mutation can be acquired even after initial metastatic spread. Chromatin immunoprecipitation assays in metastatic tissue from tumors with mutant ERα show strong enrichment for ERα at classical ERα target genes and we are currently assessing the genome-wide binding pattern of ERα to identify novel binding sites. We observed extensive patient-to-patient variability in the number and types of SVs. In general, the overall patterns of SVs were remarkably similar between matched primary and metastatic samples indicating that these events likely occurred early in tumorigenesis and are stable throughout disease progression. We identified a number of metastatic specific SVs that likely contribute to disease progression. Specifically, in one patient with an ER+ primary tumor treated with adjuvant Tamoxifen, we identified a novel fusion gene between ESR1 (estrogen receptor-α, ERα) and DAB2 (disabled-2) only in a lymph node recurrence. RT-PCR and western blot analysis confirmed that the fusion RNA/protein was expressed/translated only in the recurrent disease. The fusion retains the DNA-binding domain (DBD) and hinge region of ERα while the ligand-binding domain (LBD) is replaced with the majority of DAB2. We hypothesized that this is a functional genetic alteration conferring ligand-independent ERα-mediated signaling and growth. Confirming this, in vitro ERE-Tk-luc reporter assays showed that the ESR1-DAB2 fusion has ligand-independent activity that is 13-290x higher than wild-type ERα. We are currently assessing the genome-wide binding of ESR1-DAB2 and the functional contribution of DAB2 to the fusion protein. This study represents the most comprehensive analysis to date of genomic changes in breast cancer progression and indicates extensive changes occur during metastatic spread. A number of acquired changes likely represent therapeutically targetable metastatic dependencies. Citation Format: Ryan James Hartmaier, Amir Bahreini, Shannon L. Puhalla, Steffi Oesterreich, Aju Mathew, Nancy E. Davidson, Adam M. Brufsky, Adrian V. Lee. Identification of base pair mutations and structural rearrangements acquired in breast cancer metastases including a novel hyperactive ESR1-DAB2 fusion gene in hormone-resistant progression. [abstract]. In: Proceedings of the AACR Special Conference on Translation of the Cancer Genome; Feb 7-9, 2015; San Francisco, CA. Philadelphia (PA): AACR; Cancer Res 2015;75(22 Suppl 1):Abstract nr A1-07.

Metastatic breast cancer is often intractable due to its inherent ability to overcome current therapies. Genomic alterations are frequently responsible for therapeutic resistance. To better understand genomic mechanisms of acquired resistance in breast cancer we undertook a detailed characterization of single nucleotide variation (SNV) and structural variation (SV) in paired primary-metastasis metachronous tumors from 6 breast cancer patients (median time to recurrence 7.3 years). In ER-positive recurrent tumors treated with endocrine therapies, we identified multiple metastatic-acquired variants in ESR1 including a novel constitutively active, ligand-independent ESR1-DAB2 gene fusion. Importantly, this fusion resulted from a breakpoint in intron 4, retaining the DNA-binding domain but eliminating the ligand-binding domain (LBD), concordant to a similar fusion reported previously in a xenograft model. Hybrid capture based genomic profiling from >7,800 breast cancers identified similar exon/intron 4 fusions in 5 tumors with direct paired-read evidence. Using a novel copy number shift detection strategy, 58 additional tumors showed indirect evidence of a rearrangement at exon 4 based on a novel copy number shift detection strategy. ESR1 fusion and copy number shift positive tumors are strongly enriched in metastatic disease (78%; p Citation Format: Ryan J. Hartmaier, Nolan Priedigkeit, Laurie Gay, Michael E. Goldberg, James Suh, Siraj Ali, Jeffery Ross, Michaela Tsai, Barbara Haley, Julio Peguero, Rena D. Callahan, Irina Sachelarie, John Cho, Amir Bahreini, Shannon L. Puhalla, Steffi Oesterreich, Aju Mathew, Peter C. Lucas, Nancy E. Davidson, Adam M. Brufsky, Philip J. Stephens, Juliann Chmielecki, Adrian V. Lee. Comprehensive genomic analysis of metastatic breast cancers reveals ESR1 fusions as a recurrent mechanism of endocrine therapy resistance [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 421. doi:10.1158/1538-7445.AM2017-421