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Purpose: The purpose of this study was to directly compare mutation profiles in multiple single circulating tumor cells (CTC) and cell-free DNA (cfDNA) isolated from the same blood samples taken from patients with metastatic breast cancer (MBC). We aimed to determine whether cfDNA would reflect the heterogeneity observed in 40 single CTCs.Experimental Design: CTCs were enumerated by CELLSEARCH. CTC count was compared with the quantity of matched cfDNA and serum CA15-3 and alkaline phosphatase (ALP) in 112 patients with MBC. In 5 patients with ≥100 CTCs, multiple individual EpCAM-positive CTCs were isolated by DEPArray and compared with matched cfDNA and primary tumor tissue by targeted next-generation sequencing (NGS) of about 2,200 mutations in 50 cancer genes.Results: In the whole cohort, total cfDNA levels and cell counts (≥5 CTCs) were both significantly associated with overall survival, unlike CA15-3 and ALP. NGS analysis of 40 individual EpCAM-positive CTCs from 5 patients with MBC revealed mutational heterogeneity in PIK3CA, TP53, ESR1, and KRAS genes between individual CTCs. In all 5 patients, cfDNA profiles provided an accurate reflection of mutations seen in individual CTCs. ESR1 and KRAS gene mutations were absent from primary tumor tissue and therefore likely either reflect a minor subclonal mutation or were acquired with disease progression.Conclusions: Our results demonstrate that cfDNA reflects persisting EpCAM-positive CTCs in patients with high CTC counts and therefore may enable monitoring of the metastatic burden for clinical decision-making. Clin Cancer Res; 23(1); 88–96. ©2016 AACR.

Supplementary data, figures and tables Supplementary Fig. 1. Patient clinical timelines and samples taken. Supplementary Fig. 2. Total cfDNA levels track with CTC counts and CA15-3 in patient CTCM167. Supplementary Table 1: Summary of individual CTCs and WBCs, and respective pools isolated by DEPArray for NGS analysis Supplementary Table 2: Details of PCR amplicons for the custom 30 amplicon NGS panel Supplementary Table 3: Details of PCR amplicons for the Ampli1 Custom Cancer NGS panel showing confirmed mutations in cfDNA and CTCs Supplementary Table 4: Primer sequences of the ESR1 screening assays Supplementary Table 5: Patient details and variables for correlation and survival analyses Supplementary Table 6: Spearman's correlation of each circulating biomarker Supplementary Table 7: Sequencing metrics per panel for each sample Supplementary Table 8: Allelic dropout rate (%) for individual CTCs, WBCs and sample pools by patient Supplementary Table 9: Private mutations detected in single CTCs and WBCs Supplementary Table 10: AF and coverage for each mutation in patient CTCM155 Supplementary Table 11: AF and coverage for each mutation in patient CTCM138 Supplementary Table 12: AF and coverage for each variant in patient CTCM105 Supplementary Table 13: AF and coverage for each mutation in patient CTCM292

Background We report copy-number profiling by low-pass WGS (LP-WGS) in individual circulating tumour cells (CTCs) for guiding treatment in patients with metastatic breast cancer (MBC), comparing CTC results with mutations detected in circulating tumour DNA (ctDNA) in the same blood samples. Methods Across 10 patients with MBC who were progressing at the time of blood sampling and that had >20 CTCs detected by CellSearch®, 63 single cells (50 CTCs and 13 WBCs) and 16 cell pools (8 CTC pools and 8 WBC pools) were recovered from peripheral blood by CellSearch®/DEPArray™ and sequenced with Ampli1 LowPass technology (Menarini Silicon Biosystems). Copy-number aberrations were identified using the MSBiosuite software platform, and results were compared with mutations detected in matched plasma cfDNA analysed by targeted next-generation sequencing using the Oncomine™ Breast cfDNA Assay (Thermo Fisher). Results LP-WGS data demonstrated copy-number gains/losses in individual CTCs in regions including FGFR1, JAK2 and CDK6 in five patients, ERBB2 amplification in two HER2-negative patients and BRCA loss in two patients. Seven of eight matched plasmas also had mutations in ctDNA in PIK3CA, TP53, ESR1 and KRAS genes with mutant allele frequencies (MAF) ranging from 0.05 to 33.11%. Combining results from paired CTCs and ctDNA, clinically actionable targets were identified in all ten patients. Conclusion This combined analysis of CTCs and ctDNA may offer a new approach for monitoring of disease progression and to direct therapy in patients with advanced MBC, at a time when they are coming towards the end of other treatment options.

Aims: The decision to consider adjuvant chemotherapy (AC) for non‐small cell lung cancer is currently governed by clinical stage. This study aims to assess other routinely collected pathological variables related to metastasis and survival for their ability to predict the efficacy of AC in lung adenocarcinoma. Methods and results: A retrospective single‐centre series of 620 resected lung non‐mucinous adenocarcinoma cases from 2005 to 2015 was used. Digital images of all slides were subjected to central review, and data on tumour histopathology, AC treatment and patient survival were compiled. A statistical case matching approach was used to counter selection bias. Several high‐risk pathological criteria predict both pathological nodal involvement and early death: positive vascular invasion status (VI+) (HR = 2.10, P < 0.001), positive visceral pleural invasion status (VPI+) (HR = 2.16, P < 0.001), and solid/micropapillary‐predominant WHO tumour type (SPA/MPPA) (HR = 3.29, P < 0.001). Crucially, these criteria also identify patient groups benefiting from AC (VI + HR = 0.69, P = 0.167, VPI + HR = 0.44, P = 0.004, SPA/MPPA HR = 0.36, P = 0.006). Cases showing VI+/VPI+/SPA/MPPA histology in the absence of AC stage criteria were common (170 of 620 total), and 8 had actually received AC. This group showed much better outcomes than equivalent untreated cases in matched analysis (3‐year OS 100.0% versus 31.3%). Inclusion of patients with VI+/VPI+/SPA/MPPA histology would increase AC‐eligible patients from 51.0% to 84.0% of non‐mucinous tumours in our cohort. Conclusions: Our data provide preliminary evidence that the consideration of AC in patients with additional high‐risk pathological indicators may significantly improve outcomes in operable lung adenocarcinoma, and that AC may be currently underused.

Background Breast cancer (BC) is the most common cancer in women, and despite the introduction of new screening programmes, therapies and monitoring technologies, there is still a need to develop more useful tests for monitoring treatment response and to inform clinical decision making. The purpose of this study was to compare circulating cell-free DNA (cfDNA) and circulating tumour cells (CTCs) with conventional breast cancer blood biomarkers (CA15-3 and alkaline phosphatase (AP)) as predictors of response to treatment and prognosis in patients with metastatic breast cancer (MBC). Methods One hundred ninety-four female patients with radiologically confirmed MBC were recruited to the study. Total cfDNA levels were determined by qPCR and compared with CELLSEARCH® CTC counts and CA15-3 and alkaline phosphatase (AP) values. Blood biomarker data were compared with conventional tumour markers, treatment(s) and response as assessed by RECIST and survival. Non-parametric statistical hypothesis tests were used to examine differences, correlation analysis and linear regression to determine correlation and to describe its effects, logistic regression and receiver operating characteristic curve (ROC curve) to estimate the strength of the relationship between biomarkers and clinical outcomes and value normalization against standard deviation to make biomarker values comparable. Kaplan–Meier estimator and Cox regression models were used to assess survival. Univariate and multivariate models were performed where appropriate. Results Multivariate analysis showed that both the amount of total cfDNA (p value = 0.024, HR = 1.199, CI = 1.024–1.405) and the number of CTCs (p value = 0.001, HR = 1.243, CI = 1.088–1.421) are predictors of overall survival (OS), whereas total cfDNA levels is the sole predictor for progression-free survival (PFS) (p value = 0.042, HR = 1.193, CI = 1.007–1.415) and disease response when comparing response to non-response to treatment (HR = 15.917, HR = 12.481 for univariate and multivariate analysis, respectively). Lastly, combined analysis of CTCs and cfDNA is more informative than the combination of two conventional biomarkers (CA15-3 and AP) for prediction of OS. Conclusion Measurement of total cfDNA levels, which is a simpler and less expensive biomarker than CTC counts, is associated with PFS, OS and response in MBC, suggesting potential clinical application of a cheap and simple blood-based test.

PURPOSE We investigated the utility of the Oncomine Breast cfDNA Assay for detecting circulating tumor DNA (ctDNA) in women from a breast screening population, including healthy women with no abnormality detected by mammogram, and women on follow-up through to advanced breast cancer. MATERIALS AND METHODS Blood samples were taken from 373 women (127 healthy controls recruited through breast screening, 28 ductal carcinoma in situ, 60 primary breast cancers, 47 primary breast cancer on follow-up, and 111 metastatic breast cancers [MBC]) to recover plasma and germline DNA for analysis with the Oncomine Breast cfDNA Assay on the Ion S5 platform. RESULTS One hundred sixteen of 373 plasma samples had one or more somatic variants detected across eight of the 10 genes and were called ctDNA-positive; MBC had the highest proportion of ctDNA-positive samples (61; 55%) and healthy controls the lowest (20; 15.7%). ESR1, TP53, and PIK3CA mutations account for 93% of all variants detected and predict poor overall survival in MBC (hazard ratio = 3.461; 95% CI, 1.866 to 6.42; P = .001). Patients with MBC had higher plasma cell-free DNA levels, higher variant allele frequencies, and more polyclonal variants, notably in ESR1 than in all other groups. Only 15 individuals had evidence of potential clonal hematopoiesis of indeterminate potential mutations. CONCLUSION We were able detect ctDNA across the breast cancer spectrum, notably in MBC where variants in ESR1, TP53, and PIK3CA predicted poor overall survival. The assay could be used to monitor emergence of resistance mutations such as in ESR1 that herald resistance to aromatase inhibitors to tailor adjuvant therapies. However, we suggest caution is needed when interpreting results from a single plasma sample as variants were also detected in a small proportion of HCs., Detection of ctDNA increases as breast cancer progresses and predicts poor overall survival in metastatic patients. or ctDNA detection increases as breast cancer progresses and predicts poor overall survival in metastatic patients.

PurposeThere is growing interest in the application of circulating tumour DNA (ctDNA) as a sensitive tool for monitoring tumour evolution and guiding targeted therapy in patients with cancer. However, robust comparisons of different platform technologies are still required. Here we compared the InVisionSeq™ ctDNA Assay with the Oncomine™ Breast cfDNA Assay to assess their concordance and feasibility for the detection of mutations in plasma at low (MethodsNinety-six plasma samples from 50 patients with estrogen receptor (ER)-positive metastatic breast cancer (mBC) were profiled using the InVision Assay. Results were compared to the Oncomine assay in 30 samples from 26 patients, where there was sufficient material and variants were covered by both assays. Longitudinal samples were analysed for 8 patients with endocrine resistance.ResultsWe detected alterations in 59/96 samples from 34/50 patients analysed with the InVision assay, most frequently affectingESR1, PIK3CAandTP53. Complete or partial concordance was found in 28/30 samples analysed by both assays, and VAF values were highly correlated. Excellent concordance was found for most genes, and most discordant calls occurred at VAF ESR1andPIK3CA.ConclusionThis study shows that both ultra-deep next-generation sequencing (NGS) technologies can detect genomic alternations even at low VAFs in plasma samples of mBC patients. The strong agreement of the technologies indicates sufficient reproducibility for clinical use as prognosic and predictive biomarker.

Soft tissue sarcomas (STS) are rare, malignant tumours with a generally poor prognosis. Our aim was to explore the potential of cell free DNA (cfDNA) and circulating tumour DNA (ctDNA) analysis to track non-metastatic STS patients undergoing attempted curative treatment. The analysed cohort (n = 29) contained multiple STS subtypes including myxofibrosarcomas, undifferentiated pleomorphic sarcomas, leiomyosarcomas, and dedifferentiated liposarcomas amongst others. Perioperative cfDNA levels trended towards being elevated in patients (p = 0.07), although did not correlate with tumour size, grade, recurrence or subtype, suggesting a limited diagnostic or prognostic role. To characterise ctDNA, an amplicon panel covering three genes commonly mutated in STSs was first trialled on serial plasma collected from nine patients throughout follow-up. This approach only identified ctDNA in 2.5% (one in 40) of the analysed samples. Next custom-designed droplet digital PCR assays and Ion AmpliSeq&trade, panels were developed to track single nucleotide variants identified in patients&rsquo, STSs by whole exome sequencing (1&ndash, 6 per patient). These approaches identified ctDNA in 17% of patients. Although ctDNA was identified before radiologically detectable recurrence in two cases, the absence of demonstrable ctDNA in 83% of cases highlights the need for much work before circulating nucleic acids can become a useful means to track STS patients.

Little is known about the metastatic evolutionary dynamics of BRCA2-mutated cancers. Here, we applied whole-exome sequencing (WES) of primary tumor (PT), local relapse (LR) and eight serial plasma cfDNA samples collected from disease progression to depict the 12 years evolutionary trajectory of a metastatic BRCA2-mutated breast cancer. While longitudinal WES-cfDNA recapitulated clonal and subclonal mutations and copy number profiles detected in LR, emergence of plasma-exclusive mutations in TSC2 and HDAC9 cancer-related genes and loss of HLA loci as an immune escape mechanism were also detected. Lastly, mutation signature 3, associated with homologous recombination deficiency and response to platinum-based therapy raised profoundly from 19% in PT to 60% in late stage disease. In conclusion, we show for the first time that longitudinal WES-cfDNA enables the evolutionary trajectory of advanced cancer to be uncovered and that increment of MS3 and loss of HLA are key players in this BRCA2-mutated breast metastasis.

Following treatment 40% of soft tissue sarcoma (STS) patients suffer disease recurrence. In certain cancers circulating cell free DNA (cfDNA) and circulating tumour-derived DNA (ctDNA) characteristics correlate closely with disease burden, making them exciting potential sources of biomarkers. Despite this, the circulating nucleic acid characteristics of only 2 STS patients have been reported to date. To address this we used an Ion AmpliSeq™ panel custom specifically designed for STS patients to conduct a genetic characterisation of plasma cfDNA, buffy coat (germline) DNA and where available Formalin-Fixed Paraffin-Embedded (FFPE) primary STS tissue DNA in a cohort of 11 metastatic STS patients. We found that total cfDNA levels were significantly elevated in the STS patients analysed, and weakly correlated with disease burden. Using our Ion AmpliSeq™ panel we also successfully detected ctDNA in 4/11 (36%) patients analysed with a wide variety of STS subtypes and disease burdens. This evidence included the presence of cancer associated TP53 / PIK3CA mutations in 2 patients’ plasma and matched primary STS tumour tissue, and in the plasma alone for 2 patients. We also identified 2 potential examples of allelic loss of heterozygosity in an additional patient's STS DNA and cfDNA. This is the largest study performed characterising STS patient cfDNA/ctDNA and confirms that the field remains an attractive potential source of novel STS biomarkers. Further work is required to investigate the circulating nucleic acid characteristics of individual STS subtypes, and the potential prognostic or therapeutic roles that cfDNA/ctDNA may hold for patients with these complex tumours.

Introduction: Genomic profiling of circulating tumor cells (CTCs) is an approach to help to understand tumour heterogeneity and evolution in metastatic breast cancer (MBC) to inform prognosis and guide treatment options. We showed previously that single CTCs provide complementary information on hot spot mutations to those detected in circulating tumor DNA (ctDNA) and the primary tumour tissue, and now extend analyses to include genomic copy number changes. Herein, we report that identification of actionable targets through copy number profiling of single CTCs is achievable at any time point during metastatic progression and could be a valuable tool to guide treatment options. Methods: Fourteen patients (13 12 progressing and 1 2 responding) who were receiving treatment for MBC (12 13 HR pos, 1 HER2 pos, I1 TNBC) were recruited to the study with ethical consent and followed up with a single blood sample. A 7.5ml blood sample was collected into a CellSave Preservative tube and processed and counted within 96 hours of collection using the CELLSEARCH® system and the Circulating Tumor Cell Kit (Menarini Silicon Biosystems). The DEPArray™ System was used to identify and recover individual, intact CTCs of epithelial origin (Cytokeratin 8, 18, 19 +ve, CD45 -ve) and white blood cells (WBCs) as single cells or as a small pool of cells. Whole genome amplification was performed using the Ampli1 WGA kit. Libraries were generated using the Ampli1 LowPass Kit (Menarini Silicon Biosystems) and samples passing QC were analysed f by next generation sequencing on an S5 platform (Thermo fFisher). Results were analysed using the MSBiosuite software and data was compared with in- house informatics workfows. Results: From the 14 patients included in the study, we profiled a total of 97 samples using the Ampli1 LowPass sequencing workflow comprising 79 individual cells (across 14 patients) and 18 pools of approximately 10 cells. Successful genome- wide copy number profiles were generated in 12 of the 14 patients; samples from 2 patients had low QC scores and were inconclusive. In all, 12 patients individual CTCs from 12 patients showed similar copy number profiles, but with some heterogeneity, that revealed gene specific changes not detected by the cell pool. Moreover, 10 of the 12 patients had gene- specific changes (5 with CCND1 amplification, 2 with FGFR1 amplification, 1 with JAK2 amplification and 2 with STK11 deletion) that could have guided treatment decisions, for example offering an FGFR1 or JAK2 inhibitor. Conclusion: This study highlights the genomic heterogeneity in CTCs seen in MBC and the potential of monitoring gene specific changes to identify actionable targets in order to inform treatment decisions. The work requires further evaluation and validation but it may offer a new approach to managing treatment decisions in MBC for those patients with detectable CTCs. Citation Format: Daniel Fernandez Garcia, Georgios Nteliopoulos, Robert Hastings, Amelia Rushton, Karen Page, Luke J. Martinson, Molly Gray, David S. Guttery, Alberto Ferrarini, Nicolò Manaresi, Charles Coombes, Jacqui Shaw. Genomic copy number profiling of single CTCs reveals clonal evolution in metastatic breast cancer and identifies actionable targets for informing treatment decisions [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 587.

Purpose: The purpose of this study was to directly compare mutation profiles in multiple single circulating tumor cells (CTC) and cell-free DNA (cfDNA) isolated from the same blood samples taken from patients with metastatic breast cancer (MBC). We aimed to determine whether cfDNA would reflect the heterogeneity observed in 40 single CTCs. Experimental Design: CTCs were enumerated by CELLSEARCH. CTC count was compared with the quantity of matched cfDNA and serum CA15-3 and alkaline phosphatase (ALP) in 112 patients with MBC. In 5 patients with ≥100 CTCs, multiple individual EpCAM-positive CTCs were isolated by DEPArray and compared with matched cfDNA and primary tumor tissue by targeted next-generation sequencing (NGS) of about 2,200 mutations in 50 cancer genes. Results: In the whole cohort, total cfDNA levels and cell counts (≥5 CTCs) were both significantly associated with overall survival, unlike CA15-3 and ALP. NGS analysis of 40 individual EpCAM-positive CTCs from 5 patients with MBC revealed mutational heterogeneity in PIK3CA, TP53, ESR1, and KRAS genes between individual CTCs. In all 5 patients, cfDNA profiles provided an accurate reflection of mutations seen in individual CTCs. ESR1 and KRAS gene mutations were absent from primary tumor tissue and therefore likely either reflect a minor subclonal mutation or were acquired with disease progression. Conclusions: Our results demonstrate that cfDNA reflects persisting EpCAM-positive CTCs in patients with high CTC counts and therefore may enable monitoring of the metastatic burden for clinical decision-making. Clin Cancer Res; 23(1); 88–96. ©2016 AACR.

Background: Malignant pleural mesothelioma (MPM) is an aggressive tumor associated with exposure to asbestos and is rising in incidence worldwide. A lack of early detection methods and treatment successes have maintained a dismal prognosis of just 12 - 16 months. Circulating tumor DNA (ctDNA) is emerging as an important stratification biomarker in other thoracic malignancies, however research in this area has been lacking in MPM. Homozygous deletion of CDKN2A is one of the few established negatively prognostic molecular markers in MPM. Methods and Results: We conducted multiregional whole exome sequencing (m-WES) on tumor tissue (4 or 5 regions) and matched germline DNA from 11 patients with MPM that received surgery (extended pleurectomy decortication) as their primary treatment modality. Using a validated bioinformatics pipeline (developed as part of the TRACERx study), clonal single nucleotide variants (SNVs) and/or indels (present within all tumor regions) were selected from m-WES data for patient-specific assay design. Variants were prioritized for selection based on tumor driver status and higher mean variant allele frequencies (VAFs) amongst tumor regions. A minimum of 10 ng cfDNA isolated from pre-surgical blood was analyzed for ctDNA detection using Droplet Digital PCR (ddPCR), with positivity being defined as ≥ 3 mutant droplets and a plasma VAF ≥ 0.1%. Four out of eleven (36.4%) patients were ctDNA-positive using this patient-specific approach with VAFs ranging between 0.16% and 2.96%. Two additional patients had one or two positive mutant droplets, but these were excluded as potential false positives. In this pilot study, ctDNA-positive patients had significantly shorter survival than ctDNA-negative patients (P = 0.047), although this failed to reach significance by Cox regression analysis, likely due to a small sample size. Furthermore, in this cohort ctDNA status was a stronger prognostic biomarker than CDKN2A deletion status. Conclusions: Considering the short survival of patients with MPM despite surgery, we consider ctDNA-based analyses could be used to stratify patients regarding surgery, where risk-benefit would be marginal for those ctDNA-positive. This study provides proof-of-principle that the detection of tumor DNA from minimally invasive sources is possible in MPM and has alluded to how this could potentially impact clinical decision-making. Citation Format: Luke J. Martinson, Annabel J. Sharkey, Alan G. Dawson, Robert K. Hastings, Gareth Wilson, David Waller, Apostolos Nakas, Charles Swanton, Jacqui A. Shaw, Dean A. Fennell. Personalized circulating tumor DNA profiling in malignant pleural mesothelioma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 1349.

Background: Many breast cancer patients relapse after primary treatment but there are no reliable tests to detect distant metastases before they become overt. Here we show earlier identification of recurring patients through a scalable personalised ctDNA analysis. The method is applicable to all patients, and not limited to hot-spot mutations typically detected by gene panels. Methods: Forty-nine non-metastatic breast cancer patients were recruited following surgery and adjuvant therapy. Plasma samples (n=208) were serially collected semi-annually. Using the analytically validated SignateraTM workflow, we determined mutational signatures from primary tumour whole exome data and designed personalised assays targeting 16 variants with high sensitivity by ultra-deep sequencing (average >100,000X). The patient-specific assay was used to detect the presence of the mutational signature in the plasma. Results: In 16 of 18 (89%) clinically-relapsing patients, ctDNA was detected ahead of metastatic relapse being diagnosed by clinical examination, radiological and biochemical (CA15-3) measurements, and remained ctDNA-positive through follow-up. Of the 2 patients not detected by ctDNA, one had a small local recurrence only (now resected) and the other had three primary tumours. None of the 31 non-relapsing patients were ctDNA-positive at any time point (n=142). Metastatic relapse was predicted by Signatera with high accuracy and a lead time of up to 2 years (median=9.5 months). Conclusions: The use of a scalable patient-specific ctDNA-based validated workflow detects breast cancer recurrence ahead of clinical detection. Accurate and earlier prediction by ctDNA analysis could provide a means of monitoring breast cancer patients in need of second-line salvage adjuvant therapy in order to prevent overt life-threatening metastatic progression. Citation Format: Coombes RC, Armstrong A, Ahmed S, Page K, Hastings RK, Salari R, Sethi H, Boydell A-R, Shchegrova SV, Fernandez-Garcia D, Gleason KL, Goddard K, Guttery DS, Assaf ZJ, Balcioglu M, Moore DA, Primrose L, Navarro SL, Aleshin A, Rehman F, Toghill BJ, Louie MC, Zimmermann BG, Lin C-HJ, Shaw JA. Early detection of residual breast cancer through a robust, scalable and personalized analysis of circulating tumour DNA (ctDNA) antedates overt metastatic recurrence [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 P4-01-02.

BACKGROUND Breast cancer tissues are heterogeneous and show diverse somatic mutations and somatic copy number alterations (CNAs). We used a novel targeted next generation sequencing (NGS) panel to examine cell-free DNA (cfDNA) to detect somatic mutations and gene amplification in women with metastatic breast cancer (MBC). METHODS cfDNA from pretreated patients (n = 42) and 9 healthy controls were compared with matched lymphocyte DNA by NGS, using a custom 158 amplicon panel covering hot-spot mutations and CNAs in 16 genes, with further validation of results by droplet digital PCR. RESULTS No mutations were identified in cfDNA of healthy controls, whereas exactly half the patients with metastatic breast cancer had at least one mutation or amplification in cfDNA (mean 2, range 1–6) across a total of 13 genes. Longitudinal follow up showed dynamic changes to mutations and gene amplification in cfDNA indicating clonal and subclonal response to treatment that was more dynamic than cancer antigen 15-3 (CA15-3). Interestingly, at the time of blood sampling disease progression was occurring in 7 patients with erb-b2 receptor tyrosine kinase 2 (ERBB2) gene amplification in their cfDNA and 3 of these patients were human epidermal growth factor receptor 2 (HER2) negative at diagnosis, suggesting clonal evolution to a more aggressive phenotype. Lastly, 6 patients harbored estrogen receptor 1 (ESR1) mutations in cfDNA, suggesting resistance to endocrine therapy. Overall 9 of 42 patients (21%) had alterations in cfDNA that could herald a change in treatment. CONCLUSIONS Targeted NGS of cfDNA has potential for monitoring response to targeted therapies through both mutations and gene amplification, for analysis of dynamic tumor heterogeneity and stratification to targeted therapy.

Long interspersed element 1s (LINE-1s or L1s) are a family of non-long-terminal-repeat retrotransposons that predominate in the human genome. Active LINE-1 elements encode proteins required for their mobilization. L1-encoded proteins also act in trans to mobilize short interspersed elements (SINEs), such as Alu elements. L1 and Alu insertions have been implicated in many human diseases, and their retrotransposition provides an ongoing source of human genetic diversity. L1/Alu elements are expected to ensure their transmission to subsequent generations by retrotransposing in germ cells or during early embryonic development. Here, we determined that several subfamilies of Alu elements are expressed in undifferentiated human embryonic stem cells (hESCs) and that most expressed Alu elements are active elements. We also exploited expression from the L1 antisense promoter to map expressed elements in hESCs. Remarkably, we found that expressed Alu elements are enriched in the youngest subfamily, Y, and that expressed L1s are mostly located within genes, suggesting an epigenetic control of retrotransposon expression in hESCs. Together, these data suggest that distinct subsets of active L1/Alu elements are expressed in hESCs and that the degree of somatic mosaicism attributable to L1 insertions during early development may be higher than previously anticipated.

Successful early detection and intervention in cancer is key if we are to drastically improve cancer survivorship. Previous research by us and others has shown that copy number and mutational profiling of circulating cell-free DNA (cfDNA) can detect minimal residual disease and predict who will relapse after early stage breast cancer (Shaw et al. Genome Res 2012, doi: 10.1101, Garcia-Murillas et al. Sci Transl Med. 2015 doi: 10.1126). Here we used massively parallel sequencing with the Ion CHEF -S5 and Oncomine™ Breast cfDNA Assay (Thermofisher) for ultrasensitive detection of circulating tumour derived DNA (ctDNA) in 26 asymptomatic early stage breast cancer patients with no scan evidence of distant metastases on follow up after surgery, (all of whom were receiving adjuvant AI (PAI) at the time of blood sampling). We compared these early stage breast cancers with cfDNA isolated from 40 patients with metastatic breast cancer (MBC) and 92 healthy age matched female controls (HC) attending for a routine breast screening mammogram. The Oncomine™ Breast cfDNA Assay targets >150 hotspot mutations in 10 breast cancer genes and uses tag sequencing technology to achieve a limit of detection (LOD) down to 0.1%. One or more driver mutations were detected in cfDNA of 19 (73%) PAIs and 29 (72.5 %) MBCs, whereas just 15 HCs (16%) were positive. Applying this threshold of 1 or more driver mutations for detection of ctDNA (Cohen et al. Science. 2018 doi: 10.1126) there was a significant difference between ctDNA positive cancers and healthy controls, which also remained significant for 2 or more driver mutations (P < 0.0001 and P < 0.001 respectively, Fisher's exact test) (Abbosh et al. Nature 2017, doi: 10.1038). It is notable that >72% of breast cancers were positive with this test, both disease-free women on follow up and patients with MBC, suggesting a role for the test in routine monitoring. A second finding of clinical significance was that variant allele fraction of ESR1 mutant ctDNA was significantly higher in cancers than controls (PAIs than HCs (P = 0.034) and MBCs than HCs (P = 0.002); adjusted P value, Dunn's multiple comparison test) suggesting that ESR1 mutations distinguish disease-free breast cancer survivors receiving adjuvant aromatase inhibitors from normal women. Importantly, we are the first to report ultrasensitive evaluation of ctDNA alterations in a large number of healthy women in a true cancer screening setting. The 15 healthy women that were “positive” for ctDNA may be harbouring an early stage cancer not detectable by imaging. Research is ongoing to validate these results in a larger study, but our results suggest that the approach may have utility for early disease detection and monitoring to enable appropriate intervention at earlier stages. Citation Format: Jacqui A. Shaw, Karen Page, Daniel Fernandez-Garcia, Allison Hills, Anna R. Boydell, Lindsay Primrose, Bradley Toghill, Robert K. Hastings, Kelly Gleeson, Brenda M. Rosales, Kate Goddard, David S. Guttery, Simak Ali, Raoul C. Coombes. Circulating tumor DNA for early detection and intervention in breast cancer: ctDNA profiles discriminate between healthy women in a true cancer screening setting and disease-free women on follow up [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 LB-224.

The Human Genome Variation database of Genotype to Phenotype information (HGVbaseG2P) is a new central database for summary-level findings produced by human genetic association studies, both large and small. Such a database is needed so that researchers have an easy way to access all the available association study data relevant to their genes, genome regions or diseases of interest. Such a depository will allow true positive signals to be more readily distinguished from false positives (type I error) that fail to consistently replicate. In this paper we describe how HGVbaseG2P has been constructed, and how its data are gathered and organized. We present a range of user-friendly but powerful website tools for searching, browsing and visualizing G2P study findings. HGVbaseG2P is available at http://www.hgvbaseg2p.org.

BACKGROUND Activating mutations in the estrogen receptor 1 (ESR1) gene are acquired on treatment and can drive resistance to endocrine therapy. Because of the spatial and temporal limitations of needle core biopsies, our goal was to develop a highly sensitive, less invasive method of detecting activating ESR1 mutations via circulating cell-free DNA (cfDNA) and tumor cells as a “liquid biopsy.” METHODS We developed a targeted 23-amplicon next-generation sequencing (NGS) panel for detection of hot-spot mutations in ESR1, phosphatidylinositol-4,5-bisphosphate 3-kinase, catalytic subunit alpha (PIK3CA), tumor protein p53 (TP53), fibroblast growth factor receptor 1 (FGFR1), and fibroblast growth factor receptor 2 (FGFR2) in 48 patients with estrogen receptor-α–positive metastatic breast cancer who were receiving systemic therapy. Selected mutations were validated using droplet digital PCR (ddPCR). RESULTS Nine baseline cfDNA samples had an ESR1 mutation. NGS detected 3 activating mutations in ESR1, and 3 hot-spot mutations in PIK3CA, and 3 in TP53 in baseline cfDNA, and the ESR1 p.D538G mutation in 1 matched circulating tumor cell sample. ddPCR analysis was more sensitive than NGS and identified 6 additional baseline cfDNA samples with the ESR1 p.D538G mutation at a frequency of CONCLUSIONS Early identification of ESR1 mutations by liquid biopsy might allow for cessation of ineffective endocrine therapies and switching to other treatments, without the need for tissue biopsy and before the emergence of metastatic disease.

International audience; The BioMart Community Portal (www.biomart.org) is a community-driven effort to provide a unified interface to biomedical databases that are distributed worldwide. The portal provides access to numerous database projects supported by 30 scientific organizations. It includes over 800 different biological datasets spanning genomics, proteomics, model organisms, cancer data, ontology information and more. All resources available through the portal are independently administered and funded by their host organizations. The BioMart data federation technology provides a unified interface to all the available data. The latest version of the portal comes with many new databases that have been created by our ever-growing community. It also comes with better support and extensibility for data analysis and visualization tools. A new addition to our toolbox, the enrichment analysis tool is now accessible through graphical and web service interface. The BioMart community portal averages over one million requests per day. Building on this level of service and the wealth of information that has become available, the BioMart Community Portal has introduced a new, more scalable and cheaper alternative to the large data stores maintained by specialized organizations.

Using the current version of the Distributed Annotation System (DAS) protocol to obtain data from large regions of interest from remote DAS servers can be a time and resource consuming process. Therefore it would be useful to know the amounts and types of features that exist in a region of interest before the DAS request is made. In the current DAS protocol (1.6), the types of data that exist with a DAS source can be obtained before the complete set of features are requested for a specific region using the feature command. Depending on the implementation of the types command in the DAS server, the number of features across the segment can also be obtained. However, counting of features is computationally intensive for every user request and so most DAS servers do not include it in their implementation. For these DAS servers in order for the user to obtain counts of features, the complete set of features needs to be obtained and re-analysed. Additionally in the current DAS protocol no parameter exists to include or exclude the count of features per type when needed. In this paper, an addition to DAS protocol is proposed and implemented in order to request a summary of the features that exist within a region of interest when needed. The summary command was implemented in order to broaden the functionalities, and extend the flexibility of the current DAS protocol. The summary functionality can conserve time and resources, especially for large regions of interest.

To facilitate broad and convenient integrative visualization of and access to GWAS data, we have created the GWAS Central resource ( http://www.gwascentral.org ). This database seeks to provide a comprehensive collection of summary-level genetic association data, structured both for maximal utility and for safe open access (i.e., non-directional signals to fully preclude research subject identification). The resource emphasizes on advanced tools that allow comparison and discovery of relevant data sets from the perspective of genes, genome regions, phenotypes or traits. Tested markers and relevant genomic features can be visually interrogated across up to 16 multiple association data sets in a single view, starting at a chromosome-wide view and increasing in resolution down to individual bases. In addition, users can privately upload and view their own data as temporary files. Search and display utility is further enhanced by exploiting phenotype ontology annotations to allow genetic variants associated with phenotypes and traits of interest to be precisely identified, across all studies. Data submissions are accepted from individual researchers, groups and consortia, whereas we also actively gather data sets from various public sources. As a result, the resource now provides over 67 million P-values for over 1600 studies, making it the world’s largest openly accessible online collection of summary-level GWAS association information.

Circulating tumor cells (CTCs) are more technically challenging to isolate and characterize than circulating cell-free DNA (cfDNA). However, analysis of single CTCs may reveal more actionable information to guide targeted therapies than mutation tracking in cfDNA. Here, we compared CTC count with cfDNA levels in 113 patients with metastatic breast cancer. In 5 patients we compared mutations in 40 individual CTCs and matched cfDNA using targeted amplicon sequencing of 54 genes (∼2800 COSMIC mutations) at >2500x mean read depth. Total cfDNA levels were significantly associated with overall survival (hazard ratio (HR), 2.2; 95% CI, 1.1 to 4.3; P = 0.021) and correlated with CTC counts (P = 0.002). Four patients had mutations detected in cfDNA, the levels of which tracked with levels of circulating tumor DNA (ctDNA). Mutational heterogeneity was seen across individual CTCs (including mutations in PIK3CA, ESR1 and KRAS) and was reflected in matched cfDNA, but cfDNA had other mutations not seen in CTCs (including TP53). Therefore, cfDNA reflects persisting CTCs and enables monitoring of the metastatic burden for clinical decision-making when CTCs cannot be obtained. Moreover, monitoring of total cfDNA levels could provide an inexpensive and simple measure of treatment response. Citation Format: David S. Guttery, Jacqueline A. Shaw, Allison Hills, Daniel Fernandez-Garcia, Karen Page, Brenda Rosales, Kate Goddard, Robert Hastings, Jinli Luo, Olivia Ogle, Laura Woodley, Simak Ali, Justin Stebbing, Charles Coombes. Mutation analysis of cell-free DNA captures heterogeneity of individual circulating tumor cells in metastatic breast cancer. [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 LB-339.