Circulating tumour DNA dynamics during alternating chemotherapy and hormonal therapy in metastatic breast cancer: the ALERT study.

PURPOSE: Although changes in circulating tumour DNA (ctDNA) in breast cancer are well described, the kinetics of their fluctuations has not been described over short timescales. We investigated ctDNA dynamics during alternating cycles of chemotherapy and hormonal treatment in pre-treated patients with oestrogen receptor-positive metastatic breast cancer. METHODS: Patients received alternating, 9-week cycles of eribulin and aromatase inhibitors (AIs). The clinical primary endpoint, progression-free survival (PFS), was monitored at 3, 6 and 9 months; secondary endpoints, clinical benefit rate (CBR), safety and tolerability profiles, were also assessed. Importantly, ctDNA fluctuations were monitored using the Oncomine™ Breast cfDNA assay to test whether biomarkers may change rapidly between chemotherapy and aromatase inhibitor (AI) treatment in the setting of advanced breast cancer, potentially reflecting disease dynamics. RESULTS: The median PFS was 202 days (95% CI: 135-undefined) and 235 days (95% CI: 235-undefined) at 6 and 9 months, respectively, with a 50% CBR at both 6 and 9 months. Dynamic changes in ctDNA were observed in short timescales between chemotherapy and AI treatment and support the clinical benefit (CB) seen in individual patients and, critically, appear informative of acquired resistance in real time. CONCLUSION: Changes in ctDNA can occur rapidly and reflect changes in patients' clinical tumour responses (NCT02681523).

The utility of ctDNA in detecting minimal residual disease following curative surgery in colorectal cancer: a systematic review and meta-analysis.

INTRODUCTION: Colorectal cancer is the fourth most common cancer in the UK. There remains a need for improved risk stratification following curative resection. Circulating-tumour DNA (ctDNA) has gained particular interest as a cancer biomarker in recent years. We performed a systematic review to assess the utility of ctDNA in identifying minimal residual disease in colorectal cancer. METHODS: Studies were included if ctDNA was measured following curative surgery and long-term outcomes were assessed. Studies were excluded if the manuscript could not be obtained from the British Library or were not available in English. RESULTS: Thirty-seven studies met the inclusion criteria, involving 3002 patients. Hazard ratios (HRs) for progression-free survival (PFS) were available in 21 studies. A meta-analysis using a random effects model demonstrated poorer PFS associated with ctDNA detection at the first liquid biopsy post-surgery [HR: 6.92 CI: 4.49-10.64 p < 0.00001]. This effect was also seen in subgroup analysis by disease extent, adjuvant chemotherapy and assay type. DISCUSSION: Here we demonstrate that ctDNA detection post-surgery is associated with a greater propensity to disease relapse and is an independent indicator of poor prognosis. Prior to incorporation into clinical practice, consensus around timing of measurements and assay methodology are critical. PROTOCOL REGISTRATION: The protocol for this review is registered on PROSPERO (CRD42021261569).

A Rapid, Shallow Whole Genome Sequencing Workflow Applicable to Limiting Amounts of Cell-Free DNA.

BACKGROUND: Somatic copy number alterations (sCNAs) acquired during the evolution of breast cancer provide valuable prognostic and therapeutic information. Here we present a workflow for screening sCNAs using picogram amounts of cell-free DNA (cfDNA) and single circulating tumor cells (CTCs). METHODS: We repurposed the Ion ReproSeq PGS™ preimplantation genetic testing kit to perform shallow whole genome sequencing on 178 cfDNA samples (300 pg) and individual CTCs from 10 MBC patients with metastatic breast cancer (MBC) recovered by CellSearch®/DEPArray™. Results were analyzed using a tailored ichorCNA workflow. RESULTS: sCNAs were detected in cfDNA of 41/105 (39%) patients with MBC and 3/23 (13%) primary breast cancers on follow-up (PBC FU), all of whom subsequently relapsed. In 8 of 10 MBCs, individual CTCs had a higher copy number count than matched cfDNA. The median tumor fraction detected by ichorCNA was 0.34 (range 0.17-0.58) for MBC and 0.36 (range 0.31-0.37) for PBC FU. Patients with detectable tumor fraction (≥ 0.1) and TFx and OncomineTM variants had significantly lower overall survival rates (P values P = 0.002 and P < 0.0001 for the log-rank test, respectively). CONCLUSIONS: The ReproSeq PGS assay is rapid, at approximately $120 per sample, providing both a sCNA profile and estimation of the tumor DNA fraction from limiting cfDNA template (300pg) and individual CTCs. The approach could be used to examine the copy number landscape over time to guide treatment decisions, support future trial designs, and be applied to low volume blood spot samples enabling remote monitoring.

Shallow WGS of individual CTCs identifies actionable targets for informing treatment decisions in metastatic breast cancer.

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.

Comparison of two targeted ultra-deep sequencing technologies for analysis of plasma circulating tumour DNA in endocrine-therapy-resistant breast cancer patients.

PURPOSE: There 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 (< 0.5%) variant allele fraction (VAF). METHODS: Ninety-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. RESULTS: We detected alterations in 59/96 samples from 34/50 patients analysed with the InVision assay, most frequently affecting ESR1, PIK3CA and TP53. 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 < 1%. In longitudinal samples from progressing patients with endocrine resistance, we detected consistent alterations in sequential samples, most commonly in ESR1 and PIK3CA. CONCLUSION: This 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.

Diagnostic accuracy of circulating-free DNA for the determination of MYCN amplification status in advanced-stage neuroblastoma: a systematic review and meta-analysis.

BACKGROUND: MYCN amplification (MNA) is the strongest indicator of poor prognosis in neuroblastoma (NB). This meta-analysis aims to determine the diagnostic accuracy of MNA analysis in circulating-free DNA (cfDNA) from advanced-stage NB patients. METHODS: A systematic review of electronic databases was conducted to identify studies exploring the detection of MNA in plasma/serum cfDNA from NB patients at diagnosis using PCR methodology. Pooled estimates for sensitivity, specificity and diagnostic odds ratio (DOR) were calculated by conducting a bivariate/HSROC random-effects meta-analysis. RESULTS: Seven studies, with a total of 529 advanced-stage patients, were eligible. The pooled sensitivity of cfDNA-based MNA analysis was 0.908 (95% CI, 0.818-0.956), the pooled specificity was 0.976 (0.940-0.991) and the DOR was 410.0 (-103.6 to 923.7). Sub-grouped by INSS stage, the sensitivity for stage 3 and 4 patients was 0.832 (0.677-0.921) and 0.930 (0.834-0.972), respectively. The specificity was 0.999 (0.109-1.000) and 0.974 (0.937-0.990), respectively, and the DOR was 7855.2 (-66267.0 to 81977.4) and 508.7 (-85.8 to 1103.2), respectively. CONCLUSIONS: MNA analysis in cfDNA using PCR methodology represents a non-invasive approach to rapidly and accurately determine MNA status in patients with advanced-stage NB. Standardised methodology must be developed before this diagnostic test can enter the clinic.

The Circulating Nucleic Acid Characteristics of Non-Metastatic Soft Tissue Sarcoma Patients.

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™ panels were developed to track single nucleotide variants identified in patients' STSs by whole exome sequencing (1-6 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.

Plasma cell-free DNA (cfDNA) as a predictive and prognostic marker in patients with metastatic breast cancer.

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.

Correction to: Association of invasion-promoting tenascin-C additional domains with breast cancers in young women.

After the publication of this work [1] an error was noticed in Fig. 6 (b). In the MCF-7/Vector columns, the same image was used accidentally for the 0 h and 24 h time points. Both images were taken from the 0 h time point.

Next Generation Sequencing of Circulating Cell-Free DNA for Evaluating Mutations and Gene Amplification in Metastatic Breast Cancer.

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.

Genomic analysis of circulating cell-free DNA infers breast cancer dormancy.

Biomarkers in breast cancer to monitor minimal residual disease have remained elusive. We hypothesized that genomic analysis of circulating free DNA (cfDNA) isolated from plasma may form the basis for a means of detecting and monitoring breast cancer. We profiled 251 genomes using Affymetrix SNP 6.0 arrays to determine copy number variations (CNVs) and loss of heterozygosity (LOH), comparing 138 cfDNA samples with matched primary tumor and normal leukocyte DNA in 65 breast cancer patients and eight healthy female controls. Concordance of SNP genotype calls in paired cfDNA and leukocyte DNA samples distinguished between breast cancer patients and healthy female controls (P < 0.0001) and between preoperative patients and patients on follow-up who had surgery and treatment (P = 0.0016). Principal component analyses of cfDNA SNP/copy number results also separated presurgical breast cancer patients from the healthy controls, suggesting specific CNVs in cfDNA have clinical significance. We identified focal high-level DNA amplification in paired tumor and cfDNA clustered in a number of chromosome arms, some of which harbor genes with oncogenic potential, including USP17L2 (DUB3), BRF1, MTA1, and JAG2. Remarkably, in 50 patients on follow-up, specific CNVs were detected in cfDNA, mirroring the primary tumor, up to 12 yr after diagnosis despite no other evidence of disease. These data demonstrate the potential of SNP/CNV analysis of cfDNA to distinguish between patients with breast cancer and healthy controls during routine follow-up. The genomic profiles of cfDNA infer dormancy/minimal residual disease in the majority of patients on follow-up.

Noninvasive detection of activating estrogen receptor 1 (ESR1) mutations in estrogen receptor-positive metastatic breast cancer.

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 <1%. In serial blood samples from 11 patients, 4 showed changes in cfDNA, 2 with emergence of a mutation in ESR1. We also detected a low frequency ESR1 mutation (1.3%) in cfDNA of 1 primary patient who was thought to have metastatic disease but was clear by scans. 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.

Hide and seek: tell-tale signs of breast cancer lurking in the blood.

Breast cancer treatment is improving due to the introduction of new drugs, guided by molecular testing of the primary tumour for mutations/oncogenic drivers (e.g. HER2 gene amplification). However, tumour tissue is not always available for molecular analysis, intra-tumoural heterogeneity is common and the "cancer genome" is known to evolve with time, particularly following treatment as resistance develops. After resection, those patients with only residual micrometastases are likely to be cured but those with radiologically detectable overt disease are not. Thus, the discovery of blood test(s) that could (1) alert clinicians to early primary or recurrent disease and (2) monitor response to treatment could impact significantly on mortality. Towards this, we and others have focused on molecular profiling of circulating nucleic acids isolated from plasma, both cell-free DNA (cfDNA) and microRNAs, and the relationship of these to circulating tumour cells (CTCs). This review considers the utility of each as circulating biomarkers in breast cancer with particular emphasis on the bioinformatic tools available to support molecular profiling.

The prognostic role of circulating tumor cells in heavily pretreated individuals with a low life expectancy.

AIMS: Studies of circulating tumor cells (CTCs) have generally recruited individuals with newly diagnosed metastatic cancer, with recent data also indicating their prognostic value in the adjuvant setting. Their role in dying patients has not been established. EXPERIMENTAL: CTCs were measured in 43 individuals with metastatic breast cancer estimated to have less than 6 months to live who had exhausted standard therapeutic options. RESULTS: Those with a CTC count of ≤ 100 had a median of 182 days to live, compared with those with a CTC count of >100 who had a median of 17 days until death (p = 0.009, log rank, HR: 3.1, 95% CI: 1.4-7.3). CONCLUSION: A CTC count of >100 is associated with imminent death. Provided external validity is demonstrated, such information would be useful for patients and their families who often request specific prognostic clarity and could improve the quality of end-of-life care.

Serial Postoperative Circulating Tumor DNA Assessment Has Strong Prognostic Value During Long-Term Follow-Up in Patients With Breast Cancer.

PURPOSE: Here, we report the sensitivity of a personalized, tumor-informed circulating tumor DNA (ctDNA) assay (Signatera) for detection of molecular relapse during long-term follow-up of patients with breast cancer. METHODS: A total of 156 patients with primary breast cancer were monitored clinically for up to 12 years after surgery and adjuvant chemotherapy. Semiannual blood samples were prospectively collected, and analyzed retrospectively to detect residual disease by ultradeep sequencing using ctDNA assays, developed from primary tumor whole-exome sequencing data. RESULTS: Personalized Signatera assays detected ctDNA ahead of clinical or radiologic relapse in 30 of the 34 patients who relapsed (patient-level sensitivity of 88.2%). Relapse was predicted with a lead interval of up to 38 months (median, 10.5 months; range, 0-38 months), and ctDNA positivity was associated with shorter relapse-free survival (P < .0001) and overall survival (P < .0001). All relapsing triple-negative patients (n = 7/23) had a ctDNA-positive test within a median of 8 months (range, 0-19 months), while the 16 nonrelapsed patients with triple-negative breast cancer remained ctDNA-negative during a median follow-up of 58 months (range, 8-99 months). The four patients who had negative tests before relapse all had hormone receptor-positive (HR+) disease and conversely, five of the 122 nonrelapsed patients (all HR+) had an occasional positive test. CONCLUSION: Serial postoperative ctDNA assessment has strong prognostic value, provides a potential window for earlier therapeutic intervention, and may enable more effective monitoring than current clinical tests such as cancer antigen 15-3. Our study provides evidence that those with serially negative ctDNA tests have superior clinical outcomes, providing reassurance to patients with breast cancer. For select cases with HR+ disease, decisions about treatment management might require serial monitoring despite the ctDNA-positive result.

Comparison of phenomics and cfDNA in a large breast screening population: the Breast Screening and Monitoring Study (BSMS).

To assess their roles in breast cancer diagnostics, we aimed to compare plasma cell-free DNA (cfDNA) levels with the circulating metabolome in a large breast screening cohort of women recalled for mammography, including healthy women and women with mammographically detected breast diseases, ductal carcinoma in situ and invasive breast cancer: the Breast Screening and Monitoring Study (BSMS). In 999 women, plasma was analyzed by nuclear magnetic resonance (NMR) and Ultra-Performance Liquid Chromatography-Mass Spectrometry (UPLC-MS) and then processed to isolate and quantify total cfDNA. NMR and UPLC-MS results were compared with data for 186 healthy women derived from the AIRWAVE cohort. Results showed no significant differences between groups for all metabolites, whereas invasive cancers had significantly higher plasma cfDNA levels than all other groups. When stratified the supervised OPLS-DA analysis and total cfDNA concentration showed high discrimination accuracy between invasive cancers and the disease/medication-free subjects. Furthermore, comparison of OPLS-DA data for invasive breast cancers with the AIRWAVE cohort showed similar discrimination between breast cancers and healthy controls. This is the first report of agreement between metabolomics and plasma cfDNA levels for discriminating breast cancer from healthy subjects in a true screening population. It also emphasizes the importance of sample standardization. Follow on studies will involve analysis of candidate features in a larger validation series as well as comparing results with serial plasma samples taken at the next routine screening mammography appointment. The findings here help establish the role of plasma analysis in the diagnosis of breast cancer in a large real-world cohort.

The Evolution of Affordable Technologies in Liquid Biopsy Diagnostics: The Key to Clinical Implementation.

Cancer remains a leading cause of death worldwide, despite many advances in diagnosis and treatment. Precision medicine has been a key area of focus, with research providing insights and progress in helping to lower cancer mortality through better patient stratification for therapies and more precise diagnostic techniques. However, unequal access to cancer care is still a global concern, with many patients having limited access to diagnostic tests and treatment regimens. Noninvasive liquid biopsy (LB) technology can determine tumour-specific molecular alterations in peripheral samples. This allows clinicians to infer knowledge at a DNA or cellular level, which can be used to screen individuals with high cancer risk, personalize treatments, monitor treatment response, and detect metastasis early. As scientific understanding of cancer pathology increases, LB technologies that utilize circulating tumour DNA (ctDNA) and circulating tumour cells (CTCs) have evolved over the course of research. These technologies incorporate tumour-specific markers into molecular testing platforms. For clinical translation and maximum patient benefit at a wider scale, the accuracy, accessibility, and affordability of LB tests need to be prioritized and compared with gold standard methodologies in current use. In this review, we highlight the range of technologies in LB diagnostics and discuss the future prospects of LB through the anticipated evolution of current technologies and the integration of emerging and novel ones. This could potentially allow a more cost-effective model of cancer care to be widely adopted.

Genome engineering for estrogen receptor mutations reveals differential responses to anti-estrogens and new prognostic gene signatures for breast cancer.

Mutations in the estrogen receptor (ESR1) gene are common in ER-positive breast cancer patients who progress on endocrine therapies. Most mutations localise to just three residues at, or near, the C-terminal helix 12 of the hormone binding domain, at leucine-536, tyrosine-537 and aspartate-538. To investigate these mutations, we have used CRISPR-Cas9 mediated genome engineering to generate a comprehensive set of isogenic mutant breast cancer cell lines. Our results confirm that L536R, Y537C, Y537N, Y537S and D538G mutations confer estrogen-independent growth in breast cancer cells. Growth assays show mutation-specific reductions in sensitivities to drugs representing three classes of clinical anti-estrogens. These differential mutation- and drug-selectivity profiles have implications for treatment choices following clinical emergence of ER mutations. Our results further suggest that mutant expression levels may be determinants of the degree of resistance to some anti-estrogens. Differential gene expression analysis demonstrates up-regulation of estrogen-responsive genes, as expected, but also reveals that enrichment for interferon-regulated gene expression is a common feature of all mutations. Finally, a new gene signature developed from the gene expression profiles in ER mutant cells predicts clinical response in breast cancer patients with ER mutations.

Visualization of exhaled breath metabolites reveals distinct diagnostic signatures for acute cardiorespiratory breathlessness.

Acute cardiorespiratory breathlessness accounts for one in eight of all emergency hospitalizations. Early, noninvasive diagnostic testing is a clinical priority that allows rapid triage and treatment. Here, we sought to find and replicate diagnostic breath volatile organic compound (VOC) biomarkers of acute cardiorespiratory disease and understand breath metabolite network enrichment in acute disease, with a view to gaining mechanistic insight of breath biochemical derangements. We collected and analyzed exhaled breath samples from 277 participants presenting acute cardiorespiratory exacerbations and aged-matched healthy volunteers. Topological data analysis phenotypes differentiated acute disease from health and acute cardiorespiratory exacerbation subtypes (acute heart failure, acute asthma, acute chronic obstructive pulmonary disease, and community-acquired pneumonia). A multibiomarker score (101 breath biomarkers) demonstrated good diagnostic sensitivity and specificity (≥80%) in both discovery and replication sets and was associated with all-cause mortality at 2 years. In addition, VOC biomarker scores differentiated metabolic subgroups of cardiorespiratory exacerbation. Louvain clustering of VOCs coupled with metabolite enrichment and similarity assessment revealed highly specific enrichment patterns in all acute disease subgroups, for example, selective enrichment of correlated C5-7 hydrocarbons and C3-5 carbonyls in heart failure and selective depletion of correlated aldehydes in acute asthma. This study identified breath VOCs that differentiate acute cardiorespiratory exacerbations and associated subtypes and metabolic clusters of disease-associated VOCs.