The liquid biopsy: towards standardisation in preparation for prime time

The liquid biopsy holds potential as a more cost-effective, easier, less-invasive method for diagnosing and monitoring cancer, as well as predicting response to many currently available therapies. Circulating tumour cells (CTCs) and circulating free DNA (cfDNA) are currently the most intensely investigated analytes, with some tests already approved in clinical practice. Despite the potential of the liquid biopsy for managing patient therapy, there is currently no widely accepted consensus regarding pre-analytical blood sample handling and technologies used for extracting cfDNA and isolating CTCs, or optimum workflows for their molecular analyses – all of which are required before liquid biopsies can become routinely used in the clinic. Here, we discuss the current status of liquid biopsy testing in patients with solid tumours and the variation in blood sample handling and isolation/extraction methods, focusing on cfDNA and CTC

Racial differences in endometrial cancer molecular portraits in The Cancer Genome Atlas.

Endometrial cancer (EC) is now the most prevalent gynaecological malignancy in the Western world. Black or African American women (BoAA) have double the mortality of Caucasian women, and their tumours tend to be of higher grade. Despite these disparities, little is known regarding the mutational landscape of EC between races. Hence, we wished to investigate the molecular features of ECs within The Cancer Genome Atlas (TCGA) dataset by racial groupings. In total 374 Caucasian, 109 BoAA and 20 Asian patients were included in the analysis. Asian women were diagnosed at younger age, 54.2 years versus 64.5 years for Caucasian and 64.9 years for BoAA women (OR 3.432; p=0.011); BoAA women were more likely to have serous type tumors (OR 2.061; p=0.008). No difference in overall survival was evident. The most frequently mutated gene in Caucasian and Asian tumours was PTEN (63% and 85%), unlike BoAA cases where it was TP53 (49%). Mutation and somatic copy number alteration (SCNA) analysis revealed an enrichment of TP53 mutations in BoAAs; whereas POLE and RPL22 mutations were more frequent in Caucasians. Major recurrent SCNA racial differences were observed at chromosomes 3p, 8, 10, and 16, which clustered BoAA tumors into 4 distinct groups and Caucasian tumors into 5 groups. There was a significantly higher frequency of somatic mutations in DNA mismatch repair genes in Asian tumours, in particular PMS2 (p=0.0036). In conclusion, inherent racial disparities appear to be present in the molecular profile of EC, which could have potential implications on clinical management

Unique apicomplexan IMC sub-compartment proteins are early markers for apical polarity in the malaria parasite

The phylum Apicomplexa comprises over 5000 intracellular protozoan parasites, including Plasmodium and Toxoplasma, that are clinically important pathogens affecting humans and livestock. Malaria parasites belonging to the genus Plasmodium possess a pellicle comprised of a plasmalemma and inner membrane complex (IMC), which is implicated in parasite motility and invasion. Using live cell imaging and reverse genetics in the rodent malaria model P. berghei, we localise two unique IMC sub-compartment proteins (ISPs) and examine their role in defining apical polarity during zygote (ookinete) development. We show that these proteins localise to the anterior apical end of the parasite where IMC organisation is initiated, and are expressed at all developmental stages, especially those that are invasive. Both ISP proteins are N-myristoylated, phosphorylated and membrane-bound. Gene disruption studies suggest that ISP1 is likely essential for parasite development, whereas ISP3 is not. However, an absence of ISP3 alters the apical localisation of ISP1 in all invasive stages including ookinetes and sporozoites, suggesting a coordinated function for these proteins in the organisation of apical polarity in the parasite

Mutation analysis of cell-free DNA and single circulating tumor cells in metastatic breast cancer patients with high CTC counts.

PURPOSE: The purpose of this study was to directly compare mutation profiles in multiple single CTCs and cfDNA isolated from the same blood samples taken from patients with metastaic breast cancer (MBC). We aimed to determine whether cell-free DNA 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 metastatic breast cancer. In 5 patients with {greater than or equal to}100 CTCs, multiple individual EpCAM-positive CTCs were isolated by DEPArray and compared with matched cfDNA and primary tumour tissue by targeted next generation sequencing (NGS) of ~2200 mutations in 50 cancer genes. RESULTS: In the whole cohort, total cfDNA levels and cell counts ({greater than or equal to}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 tumour tissue and therefore likely reflect either a minor sub-clonal mutation or were acquired with disease progression. CONCLUSION: 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

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. Due to 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 hotspot mutations in ESR1, PIK3CA, TP53, FGFR1 and FGFR2 in 48 patients with ERα positive metastatic breast cancer who were receiving systemic therapy. Selected mutations were validated using digital droplet PCR (ddPCR). Results: Nine baseline cfDNA samples had an ESR1 mutation. NGS detected 3 activating mutations in ESR1, 3 hotspot mutations in PIK3CA and 3 in TP53 in baseline cfDNA, and the ESR1 p.D538G mutation in one 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 one primary patient thought to have metastatic disease, but was clear by scans. Conclusion: 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 prior to the emergence of metastatic disease

Next generation sequencing of circulating cell-free DNA 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