Laura Austin

TP53 mutations detected in circulating tumor cells present in the blood of metastatic triple negative breast cancer patients

IntroductionCirculating tumor cells (CTCs) are tumor cells shed from either primary tumors or its metastases that circulate in the peripheral blood of patients with metastatic cancers. The molecular characterization of the CTCs is critical to identifying the key drivers of cancer metastasis and devising therapeutic approaches. However, the molecular characterization of CTCs is difficult to achieve because their isolation is a major technological challenge.MethodsCTCs from two triple negative breast cancer patients were enriched using CellSearch and single cells selected by DEPArray™. A TP53 R110 fs*13 mutation identified by next generation sequencing in the breast and chest skin biopsies of both patients was studied in single CTCs.ResultsFrom 6 single CTC isolated from one patient, 1 CTC had TP53 R110 delC, 1 CTC showed the TP53 R110 delG mutation, and the remaining 4 single CTCs showed the wild type p53 sequence; a pool of 14 CTCs isolated from the same patient also showed TP53 R110 delC mutation. In the tumor breast tissue of this patient, only the TP53 R110 delG mutation was detected. In the second patient a TP53 R110 delC mutation was detected in the chest wall skin biopsy; from the peripheral blood of this patient, 5 single CTC and 6 clusters of 2 to 6 CTCs were isolated; 3 of the 5 single CTCs showed the TP53 R110 delC mutation and 2 CTCs showed the wild type TP53 allele; from the clusters, 5 showed the TP53 R110 delC mutation, and 1 cluster the wild type TP53 allele. Single white blood cells isolated as controls from both patients only showed the wild type TP53 allele.ConclusionsWe are able to isolate uncontaminated CTCs and achieve single cell molecular analysis. Our studies showed the presence of different CTC sub-clones in patients with metastatic breast cancer. Some CTCs had the same TP53 mutation as their matching tumor samples although others showed either a different TP53 mutation or the wild type allele. Our results indicate that CTCs could represent a non-invasive source of cancer cells from which to determine genetic markers of the disease progression and potential therapeutic targets.

Detection and Characterization of Circulating Tumor Associated Cells in Metastatic Breast Cancer.

The availability of blood-based diagnostic testing using a non-invasive technique holds promise for real-time monitoring of disease progression and treatment selection. Circulating tumor cells (CTCs) have been used as a prognostic biomarker for the metastatic breast cancer (MBC). The molecular characterization of CTCs is fundamental to the phenotypic identification of malignant cells and description of the relevant genetic alterations that may change according to disease progression and therapy resistance. However, the molecular characterization of CTCs remains a challenge because of the rarity and heterogeneity of CTCs and technological difficulties in the enrichment, isolation and molecular characterization of CTCs. In this pilot study, we evaluated circulating tumor associated cells in one blood draw by size exclusion technology and cytological analysis. Among 30 prospectively enrolled MBC patients, CTCs, circulating tumor cell clusters (CTC clusters), CTCs of epithelial–mesenchymal transition (EMT) and cancer associated macrophage-like cells (CAMLs) were detected and analyzed. For molecular characterization of CTCs, size-exclusion method for CTC enrichment was tested in combination with DEPArray™ technology, which allows the recovery of single CTCs or pools of CTCs as a pure CTC sample for mutation analysis. Genomic mutations of TP53 and ESR1 were analyzed by targeted sequencing on isolated 7 CTCs from a patient with MBC. The results of genomic analysis showed heterozygous TP53 R248W mutation from one single CTC and pools of three CTCs, and homozygous TP53 R248W mutation from one single CTC and pools of two CTCs. Wild-type ESR1 was detected in the same isolated CTCs. The results of this study reveal that size-exclusion method can be used to enrich and identify circulating tumor associated cells, and enriched CTCs were characterized for genetic alterations in MBC patients, respectively.

Cell-free DNA and circulating tumor cells: Comprehensive liquid biopsy analysis in advanced breast cancer

Purpose: Liquid biopsy provides a real-time assessment of metastatic breast cancer (MBC). We evaluated the utility of combining circulating tumor cells (CTC) and circulating tumor DNA (ctDNA) to predict prognosis in MBC. Experimental Design: We conducted a retrospective study of 91 patients with locally advanced breast cancer and MBC. CTCs were enumerated by CellSearch; the plasma-based assay was performed utilizing Guardant360 and the survival analysis using Kaplan–Meier curves. Results: Eighty-four patients had stage IV cancer, and 7 patients had no metastases. Eighty patients had CTC analysis: median number 2 (0–5,612). Blood samples [232 of 277 (84%)] had mutations. The average ctDNA fraction was 4.5% (0–88.2%) and number of alterations 3 (0–27); the most commonly mutated genes were TP53 (52%), PIK3CA (40%), and ERBB2 (20%). At the time of analysis, 36 patients (39.6%) were dead. The median follow-up for CTCs was 9 months; for ctDNA, it was 9.9 months. For CTCs and ctDNA, respectively, progression-free survival (PFS) was 4.2 and 5.2 months and overall survival (OS) was 18.7 and 21.5 months. There was a statistically significant difference in PFS and OS for baseline CTCs < 5 versus CTCs ≥ 5 (P = 0.021 and P = 0.0004, respectively); %ctDNA < 0.5 versus ≥ 0.5 (P = 0.003 and P = 0.012); number of alterations < 2 versus ≥ 2 (P = 0.059 borderline and P = 0.0015). A significant association by Fisher exact test was found between the number of alterations and the %ctDNA in the baseline sample (P < 0.0001). Conclusions: The study demonstrated that liquid biopsy is an effective prognostic tool. Clin Cancer Res; 24(3); 560–8. ©2017 AACR.

Analysis of tumor template from multiple compartments in a blood sample provides complementary access to peripheral tumor biomarkers

Targeted cancer therapeutics are promised to have a major impact on cancer treatment and survival. Successful application of these novel treatments requires a molecular definition of a patients disease typically achieved through the use of tissue biopsies. Alternatively, allowing longitudinal monitoring, biomarkers derived from blood, isolated either from circulating tumor cell derived DNA (ctcDNA) or circulating cell-free tumor DNA (ccfDNA) may be evaluated. In order to use blood derived templates for mutational profiling in clinical decisions, it is essential to understand the different template qualities and how they compare to biopsy derived template DNA as both blood-based templates are rare and distinct from the gold-standard. Using a next generation re-sequencing strategy, concordance of the mutational spectrum was evaluated in 32 patient-matched ctcDNA and ccfDNA templates with comparison to tissue biopsy derived DNA template. Different CTC antibody capture systems for DNA isolation from patient blood samples were also compared. Significant overlap was observed between ctcDNA, ccfDNA and tissue derived templates. Interestingly, if the results of ctcDNA and ccfDNA template sequencing were combined, productive samples showed similar detection frequency (56% vs 58%), were temporally flexible, and were complementary both to each other and the gold standard. These observations justify the use of a multiple template approach to the liquid biopsy, where germline, ctcDNA, and ccfDNA templates are employed for clinical diagnostic purposes and open a path to comprehensive blood derived biomarker access.

Detection of Activating Estrogen Receptor Gene (ESR1) Mutations in Single Circulating Tumor Cells

Purpose: Early detection is essential for treatment plans before onset of metastatic disease. Our purpose was to demonstrate feasibility to detect and monitor estrogen receptor 1 (ESR1) gene mutations at the single circulating tumor cell (CTC) level in metastatic breast cancer (MBC). Experimental Design: We used a CTC molecular characterization approach to investigate heterogeneity of 14 hotspot mutations in ESR1 and their correlation with endocrine resistance. Combining the CellSearch and DEPArray technologies allowed recovery of 71 single CTCs and 12 WBC from 3 ER-positive MBC patients. Forty CTCs and 12 WBC were subjected to whole genome amplification by MALBAC and Sanger sequencing. Results: Among 3 selected patients, 2 had an ESR1 mutation (Y537). One showed two different ESR1 variants in a single CTC and another showed loss of heterozygosity. All mutations were detected in matched cell-free DNA (cfDNA). Furthermore, one had 2 serial blood samples analyzed and showed changes in both cfDNA and CTCs with emergence of mutations in ESR1 (Y537S and T570I), which has not been reported previously. Conclusions: CTCs are easily accessible biomarkers to monitor and better personalize management of patients with previously demonstrated ER-MBC who are progressing on endocrine therapy. We showed that single CTC analysis can yield important information on clonal heterogeneity and can be a source of discovery of novel and potential driver mutations. Finally, we also validate a workflow for liquid biopsy that will facilitate early detection of ESR1 mutations, the emergence of endocrine resistance and the choice of further target therapy. Clin Cancer Res; 23(20); 6086–93. ©2017 AACR.

Abstract P1-01-05: Prognostic values of circulating tumor cells (CTC) and cancer associated macrophage-like cells (CAML) enumerations in metastatic breast cancer: The role for innate immunity in the metastatic process

Background: The enumeration of circulating tumor cells (CTCs) using the CellSearch assay is a well-established prognostic and predictive marker for metastatic breast cancer (MBC). However, additional prognostic markers are lacking in patients with ≥ 5 CTCs in 7.5 ml of blood. Tumor-associated macrophages (TAMs) are derived from circulating monocytes or tissue-resident macrophages. TAMs have a controversial role in metastasis and anti-tumor processes. Recent studies showed that circulating cancer associated macrophage-like cells (CAMLs) are specialized phagocytic myeloid cells and found in the peripheral blood of patients with solid tumors including breast cancer, but not in healthy individuals. The presence of CAMLs may indicate the activation of innate immunity in cancer patients. The function and prognostic value of CAMLs in MBC is unknown. In the current study, we measured CTCs and CAMLs on the CellSearch™ platform and investigated their prognostic values in MBC. Methods: Peripheral blood samples from 127 stages IV breast cancer patients were collected at baseline before starting first-line therapy. The detection and enumeration of CTCs and CAMLs in 7.5 ml blood sample were performed on the CellSearch™ system. CTCs were identified by cytokeratins (CK-8, 18, and 19) positive and CD45 negative staining. CAMLs were defined by positive staining for cytokeratins and CD45 (Adams et al, PNAS,111(9):3514-9, 2014). CTCs and CAMLs enumeration in associations with the progression-free survival (PFS) and overall survival (OS) of patients were evaluated using Kaplan Meier curves and Cox proportional hazards modeling. Results: The image review of CAMLs by using CellSearch analysis showed heterogeneous morphological phenotypes. CAMLs are large cells presenting enlarged nuclei or multiple individual nuclei, and both cytokeratin and CD45 positive with diffused cytoplasmic staining. Among the 127 MBC patients, 38 (29.9%) had elevated CTCs (≥5 CTCs), and 21 (16.5%) had at least one CAML detected. Patients with CAMLs had a significantly increased PFS (p=0.0374) and OS (p=0.0042), compared to patients without CAMLs at baseline. Patients with elevated baseline CTCs and CAMLs had worse PFS with a hazard ratio (HR) of 4.04 (95% CI 2.16 -7.56, P Conclusion: Baseline enumerations of both individual CTCs and CAMLs are feasible and increase our ability to accurately predict outcome in MBC patients. Evaluation of CAMLs in peripheral blood may be a marker of innate immunity and provide additional prognostic values for MBC. Citation Format: Mu Z, Wang C, Ye Z, Rossi G, Austin L, Yang H, Cristofanilli M. Prognostic values of circulating tumor cells (CTC) and cancer associated macrophage-like cells (CAML) enumerations in metastatic breast cancer: The role for innate immunity in the metastatic process [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 P1-01-05.

Abstract 1388: Multi-template analysis in metastatic breast cancer blood samples

Introduction The presence of Circulating Tumor Cells (CTC) has been observed in advanced cancer patients and studies have indicated that these cells contribute to the process of metastasis. Historically CTCs with metastatic potential have been characterized as cells that are EpCAM positive, Cytokeratin positive (CK+), and CD45 negative. We report the results of a prospective study challenging these historical definitions of circulating tumor cells. Description In a prospective study, patients with metastatic breast cancer there were about to start a new systemic therapy were enrolled The samples and representative tissue were collected at baseline. The patients were predominantly female (97%) and all had stage IV disease. From these whole blood samples a LiquidBiopsy® was performed using the “MultiTemplate” format which directly compares CTC, circulating cell free (CCF), and WBC patient-matched DNA in a NGS based resequencing test. Of 22 patients, 18 biopsy samples were successfully evaluated; the balance had insufficient tissue or DNA for analysis. Summary The LiquidBiopsy compared two phenotypic definitions for CTC capture. Tumor cell populations were enriched using epithelial targeted capture and compared to a novel cocktail of antibodies. The cocktail of reagents gave 77% average recovery of engineered samples when a target receptor was present on cell lines representing the spectrum of breast cancer subtypes. This enrichment protocol was applied to serial patient samples. Epithelial based enrichment served as a control and showed an average recovered purity of 10.7% CK+ cells. By comparison, cocktail selection enriched populations with on average 8.8% CK+ cell populations but a larger range of cells than epithelial selection. Importantly, the median number of CK+ cells recovered from cocktail capture was almost twice that of epithelial capture alone. (median of 35.8 vs 22.1 for cocktail and epithelial respectively). The median CD45+ non-target cells background was 80 and 155 cells respectively. This background supports sequencing detection of mutations present at >1%. ccfDNA sample was purified from the same tube of blood. The average concentration of patient matched ccfDNA recovered was 7.3 ng/mL. The combined results of ctcDNA and ccfDNA template sequencing gave productive samples showed similar detection frequency (55% vs 58%), were temporally flexible, and were complementary both to each other and the “gold standard” (FFPE). Conclusions The data from this prospective clinical study reveals subsets of CTC, including an important cohort that is not detected using the standard definition for epithelial CTCs. Furthermore, we present evidence for the successful use of a “Multi-Template” approach to the liquid biopsy, where germline, ctcDNA, and ccfDNA templates are employed for clinical diagnostic purposes and justifies the redefinition of CTC phenotype based upon cell surface biomarkers and mutation content. Citation Format: William M. Strauss, Chris Carter, Erich Klem, Jill Simmons, Keerthi Gogineni, Ruth O’Regan, Laura Austin, Paul W. Dempsey, Massimo Cristofanilli. Multi-template analysis in metastatic breast cancer blood samples. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 1388.

Abstract 172: Managing metastatic breast cancer via serial monitoring with circulating cell-free tumor DNA next generation sequencing testing: Table 1.

Background: Metastatic breast cancer (MBC) is an incurable disease with complex molecular features including somatic mutations that evolve in relation to genomic instability and selective treatment pressure. Patients with treatment-refractory MBC may benefit from tumor genomic evaluation using next generation sequencing (NGS). Furthermore, analysis of circulating tumor DNA (ctDNA) in patients with advanced disease offers the possibility of non-invasive molecular monitoring. Methods: A patient with MBC was tested at each progression with a ctDNA NGS panel (Guardant360™) that includes all NCCN-recommended somatic genomic variants for solid tumors and sequences complete exons of >50 genes to report single nucleotide variants (SNVs), fusions, amplifications, and indels with high sensitivity and ultra-high specificity (>99.9999%). The patient was diagnosed with invasive breast cancer at age 44 and treated with surgery and hormonal therapy. At age 61, she had axillary adenopathy and liver metastases. Treatment details are in Table 1. Results: ctDNA analysis was performed at the time of metastatic diagnosis and at 5 additional time points over the course of treatment. All samples revealed an ERBB2 exon 19 indel (p.Leu755_Glu757delinsSer), and multiple SNVs and gene amplifications. ERBB2 amplification was seen in 4 of 6 samples. Mutant allele fractions (Table 1) correlated with clinical response to treatment and progression. Conclusions: Analysis of ctDNA in this patient identified an ERBB2 exon 19 indel, which are present in 2-4% of non-small cell lung cancers but 1-2% in breast cancer. Treatment with anti-HER2 monoclonal antibody or dual anti-EGFR/ERBB2 tyrosine kinase inhibitor therapies may show clinical benefit. ctDNA analysis can detect emergence of actionable resistance mutations with the advantage of serial evaluation, allowing capture of inter- and intra-tumor heterogeneity and illustration of molecular progression and response. Citation Format: Laura Austin, Rebecca Nagy, Oliver Zill, Richard B. Lanman, AmirAli Talasaz, Massimo Cristofanilli. Managing metastatic breast cancer via serial monitoring with circulating cell-free tumor DNA next generation sequencing testing. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 172.

Abstract P6-05-07: Improving personalized management of primary breast cancer: Mammaprint® risk stratification and blueprint® molecular subtyping

Background: Historically, breast cancer (BC) patients were offered cytotoxic or endocrine therapy based on factors such as tumor size, stage, and immunohistochemistry (IHC) markers for estrogen receptor (ER) and HER2-positivity. In 2010 the College of American Pathologists revised the breast cancer guidelines on endocrine therapy (ET) to include a lower threshold of ER positivity by immunohistochemistry, changing the definition from 10% to 1% [Hammond et al]. As a result, although a larger number of patients are offered ET, not all may benefit from this expanded definition of ER positivity if their disease is not truly estrogen driven. More recently, sensitive gene profiling assays, such as Blueprint®, can determine intrinsic molecular subtype which may be more sensitive in predicting which patients will benefit from ET. Additionally, Mammaprint® provides risk stratification which can aid in determining which patients could benefit from neoadjuvant therapy. Methods This is an observational analysis of 60 patients with stage I-IV BC. Tissue analysis for ER, PR and HER2 status were determined by IHC/FISH. mRNA expression profiles of 80 genes for Blueprint® (Agendia) analysis provided molecular subtyping: luminal, basal or her2. Moreover, Mammaprint® (Agendia) analysis of 70 genes subdivided patients into low risk or high risk providing further stratification for Luminal-type. Results By IHC staining, 48% of patients were ER+/HER2-, 10% were ER+/HER2+, 8.3% were ER-/HER2+, and the remaining patients (20%) were triple negative (TN) BC. By comparison, molecular profiling classified 21% as luminal A, 18% luminal B, 11.6% Her2 and 35% basal subtype. The 35 ER+ patients were heterogeneous by subtype: 13 were classified as molecular luminal A, 16 were luminal B, 4 were reclassified as HER2 and 2 were basal-like (one of whom had 40% ER positivity). Of the ER+ patients whose IHC quantitative staining was known, 29% with low positivity (less than 10%) were reclassified as basal subtype. Of the 5 patients who are ER+/HER2+, 2 were luminal B and 3 were of the HER2-subtype. Two patients who were TN were reclassified as luminal B, and an ER-/HER2+ was classified as a basal subtype. One patient with ER+/HER2- disease had evidence of both HER2 and luminal B subtype. Of the patients who received neo-adjuvant therapy, pCR was obtained in 33% of luminal, 60% of HER2 and 50% of basal-type patients. Conclusions BluePrint® and Mammaprint® Molecular profiling are useful diagnostic tools which further characterize tumors to predict risk of recurrence and response to treatment. About one third of ER+ patients with low positivity (less than 10%) were reclassified as basal subtype, suggesting that there is a proportion of patients who are exposed to the morbidity of hormonal therapy with little therapeutic benefit. Additionally, the test is predictive of pCR, with the highest rates in the basal and Her2 subtypes, thus enabling clinicians to predict and improve clinical outcomes through more personalized treatment decisions. Citation Format: Mikkilineni L, Austin LK, Limentani K, Jaslow RJ, Avery TP, Palazzo J, Cristofanilli M. Improving personalized management of primary breast cancer: Mammaprint® risk stratification and blueprint® molecular subtyping. [abstract]. In: Proceedings of the Thirty-Eighth Annual CTRC-AACR San Antonio Breast Cancer Symposium: 2015 Dec 8-12; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2016;76(4 Suppl):Abstract nr P6-05-07.

Abstract P2-02-14: Detection and characterization of CTCs isolated by ScreenCell®-Filtration in metastatic breast cancer

Background: Circulating Tumor cells (CTCs) detection has prognostic and predictive implications in patients with metastatic breast cancer (MBC). Genomic and phenotypic analysis of CTCs hold enormous promise as blood-based molecular characterization and monitoring disease progression and treatment benefit with a strong potential to be translated into more individualized targeted treatments. FDA-approved CellSearch™ detection allows only enumeration of CTCs expressing EpCAM without molecular characterization. CTCs represent very heterogeneous populations of tumorigenic cancer cells and some subpopulations have undergone epithelial-Mesenchymal transition (EMT), which is associated metastasis process and an unfavourable outcome. EpCAM-based enrichment technique has failed to detect EMT subpopulations due to the decreased expression or loss of epithelial markers. Non-EpCAM-based approaches are needed for identifying EMT CTCs. The ScreenCell® devices are single-use and low-cost innovative devices that use a filter for enrichment-free isolation of CTCs by a two-steps combining size-based separation and staining using different markers. The DEPArray™ system is the ideal downstream isolation system to collect single or pooled CTCs for molecular and genetic analysis. In this study, we evaluated the feasibility of achieving CTCs detection/enumeration using ScreenCell® filtration followed by single cell isolation with the DEPArray™ in MBC patients. Methods: The first part of the study consisted in evaluating CTCs detection/enumeration in 30 patients with stage III and stage IV breast cancer. 3 mL of whole blood in an EDTA or Transfix tubes was collected and processed on the ScreenCell® Cyto device following the instructions of the supplier. CTCs were stained with cytokeratin (CK-8, 18, and 19), leukocyte antigen (CD45), and a nuclear dye (DAPI) and counted under fluorescence microscope. CTCs were identified as positive staining for CK and DAPI and negative staining for CD45 (CK+/DAPI+CD45-). In the second part, After enrichment, CTCs were stained with CK, CD45, and DAPI and sorted with DEPArray™ Platform (Silicon Biosystems, Inc). Single CTCs were collected and the DNA of each single CTCs was amplified with Ampli1™ WGA kit, and the genome integrity index (GII) was assessed by Ampli1™ QC kit (Silicon Biosystems, Inc). Library was constructed and whole exome sequencing (WES) of DNA mutations was conducted. Results: Twenty patient samples had CTCs detected (66.7%), the number of CTCs was 1 to 347 per 3.0 ml of whole blood. CTC-clusters were detected in 7 patient samples (23.3%). Single CTCs were collected on DEPArray™ platform after enrichment with ScreenCell filtration. GII was confirmed with the presence of short, medium, and long DNA fragments (3 to 4 PCR bands) in the WGA library by PCR-based assay. All collected CTCs showed high GII as measured by Ampli1™ QC kit (GII ≥ 3) for WES of DNA mutations. The data analysis of WES results is under processing. Conclusions: ScreenCell® filtration is simple and effective devices to isolate CTCs and identify CTC-clusters. Isolation of single cells for molecular analysis using the combination of ScreenCell® filtration and DEPArray™ Platform is feasible for genetic characterization of CTCs. Citation Format: Mu Z, Benali-Furet N, Uzan G, Ye Z, Austin L, Wang C, Nguyen1 T, Avery T, Jaslow R, Yang H, Cristofanilli M. Detection and characterization of CTCs isolated by ScreenCell®-Filtration in metastatic breast cancer. [abstract]. In: Proceedings of the Thirty-Eighth Annual CTRC-AACR San Antonio Breast Cancer Symposium: 2015 Dec 8-12; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2016;76(4 Suppl):Abstract nr P2-02-14.