The Plasmodium berghei Ca(2+)/H(+) exchanger, PbCAX, is essential for tolerance to environmental Ca(2+) during sexual development.

Ca(2+) contributes to a myriad of important cellular processes in all organisms, including the apicomplexans, Plasmodium and Toxoplasma. Due to its varied and essential roles, free Ca(2+) is tightly regulated by complex mechanisms. These mechanisms are therefore of interest as putative drug targets. One pathway in Ca(2+) homeostatic control in apicomplexans uses a Ca(2+)/H(+) exchanger (a member of the cation exchanger family, CAX). The P. falciparum CAX (PfCAX) has recently been characterised in asexual blood stage parasites. To determine the physiological importance of apicomplexan CAXs, tagging and knock-out strategies were undertaken in the genetically tractable T. gondii and P. berghei parasites. In addition, a yeast heterologous expression system was used to study the function of apicomplexan CAXs. Tagging of T. gondii and P. berghei CAXs (TgCAX and PbCAX) under control of their endogenous promoters could not demonstrate measureable expression of either CAX in tachyzoites and asexual blood stages, respectively. These results were consistent with the ability of parasites to tolerate knock-outs of the genes for TgCAX and PbCAX at these developmental stages. In contrast, PbCAX expression was detectable during sexual stages of development in female gametocytes/gametes, zygotes and ookinetes, where it was dispersed in membranous networks within the cytosol (with minimal mitochondrial localisation). Furthermore, genetically disrupted parasites failed to develop further from "round" form zygotes, suggesting that PbCAX is essential for ookinete development and differentiation. This impeded phenotype could be rescued by removal of extracellular Ca(2+). Therefore, PbCAX provides a mechanism for free living parasites to multiply within the ionic microenvironment of the mosquito midgut. Ca(2+) homeostasis mediated by PbCAX is critical and suggests plasmodial CAXs may be targeted in approaches designed to block parasite transmission

Detection of HER2 amplification in circulating free DNA in patients with breast cancer.

BACKGROUND: Human epidermal growth factor receptor 2 (HER2) is amplified and overexpressed in 20-25% of breast cancers. This study investigated circulating free DNA (cfDNA) for detection of HER2 gene amplification in patients with breast cancer. METHODS: Circulating free DNA was extracted from plasma of unselected patients with primary breast cancer (22 before surgery and 68 following treatment), 30 metastatic patients and 98 female controls using the QIAamp Blood DNA Mini Kit (Qiagen). The ratio of HER2 to an unamplified reference gene (contactin-associated protein 1 (CNTNAP1)) was measured in cfDNA samples by quantitative PCR (qPCR) using SK-BR-3 cell line DNA as a positive control. RESULTS: We validated the qPCR assay with DNA extracted from 23 HER2 3+ and 40 HER2-negative tumour tissue samples; the results agreed for 60 of 63 (95.2%) tumours. Amplification was detected in cfDNA for 8 of 68 patients following primary breast cancer treatment and 5 of 30 metastatic patients, but was undetected in 22 patients with primary breast cancer and 98 healthy female controls. Of the patients with amplification in cfDNA, 10 had HER2 3+ tumour status by immunohistochemistry. CONCLUSIONS: The results demonstrate for the first time the existence of amplified HER2 in cfDNA in the follow-up of breast cancer patients who are otherwise disease free. This approach could potentially provide a marker in patients with HER2-positive breast cancer

Influence of plasma processing on recovery and analysis of circulating nucleic acids.

Circulating nucleic acids (CNAs) are under investigation as a liquid biopsy in cancer. However there is wide variation in blood processing and methods for isolation of circulating free DNA (cfDNA) and microRNAs (miRNAs). Here we compare the extraction efficiency and reproducibility of 4 commercially available kits for cfDNA and 3 for miRNA using spike-in of reference templates. We also compare the effects of increasing time between venepuncture and centrifugation and differential centrifugation force on recovery of CNAs. cfDNA was quantified by TaqMan qPCR and targeted deep sequencing. miRNA profiles were assessed with TaqMan low-density arrays and assays. The QIAamp(®) DNA Blood Mini and Circulating nucleic acid kits gave the highest recovery of cfDNA and efficient recovery (>90%) of a 564bp spike-in. Moreover, targeted sequencing revealed overlapping cfDNA profiles and variant depth, including detection of HER2 gene amplification, using the Ion AmpliSeq™Cancer Hotspot Panel v2. Highest yields of miRNA and the synthetic Arabidopsis thaliana miR-159a spike-in were obtained using the miRNeasy Serum/Plasma kit, with saturation above 200 µl of plasma. miRNA profiles showed significant variation with increasing time before centrifugation (p 12 years, highlighting the potential for analysis of stored sample biobanks. In the era of the liquid biopsy, standardisation of methods is required to minimise variation, particularly for miRNA

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. Experimental Design: DNA methylation was investigated in independent tumor cohorts using Illumina HumanMethylation arrays and gene expression by Affymetrix arrays and qRT-PCR. The role of Msh homeobox 1 (MSX1) in drug sensitivity was investigated by gene reintroduction and siRNA knockdown of ovarian cancer cell lines. Results: CpG sites at contiguous genomic locations within the MSX1 gene have significantly lower levels of methylation in independent cohorts of HGSOC patients, which recur by 6 months compared with after 12 months (P < 0.05, q < 0.05, n = 78), have poor RECIST response (P < 0.05, q < 0.05, n = 61), and are associated with PFS in an independent cohort (n = 146). A decrease in methylation at these CpG sites correlates with decreased MSX1 gene expression. MSX1 expression is associated with PFS (HR, 0.92; 95% CI, 0.85–0.99; P = 0.029; n = 309). Cisplatin-resistant ovarian cancer cell lines have reduced MSX1 expression, and MSX1 overexpression leads to cisplatin sensitization, increased apoptosis, and increased cisplatin-induced p21 expression. Conclusions: Hypomethylation of CpG sites within the MSX1 gene is associated with resistant HGSOC disease at presentation and identifies expression of MSX1 as conferring platinum drug sensitivity

Plasmodium APC3 mediates chromosome condensation and cytokinesis during atypical mitosis in male gametogenesis

The anaphase promoting complex/cyclosome (APC/C) is a highly conserved multi-subunit E3 ubiquitin ligase that controls mitotic division in eukaryotic cells by tagging cell cycle regulators for proteolysis. APC3 is a key component that contributes to APC/C function. Plasmodium, the causative agent of malaria, undergoes atypical mitotic division during its life cycle. Only a small subset of APC/C components has been identified in Plasmodium and their involvement in atypical cell division is not well understood. Here, using reverse genetics we examined the localisation and function of APC3 in Plasmodium berghei. APC3 was observed as a single focus that co-localised with the centriolar plaque during asexual cell division in schizonts, whereas it appeared as multiple foci in male gametocytes. Functional studies using gene disruption and conditional knockdown revealed essential roles of APC3 during these mitotic stages with loss resulting in a lack of chromosome condensation, abnormal cytokinesis and absence of microgamete formation. Overall, our data suggest that Plasmodium utilises unique cell cycle machinery to coordinate various processes during ndomitosis, and this warrants further investigation in future studies

Plasmodium P-Type Cyclin CYC3 Modulates Endomitotic Growth during Oocyst Development in Mosquitoes

Cell-cycle progression and cell division in eukaryotes are governed in part by the cyclin family and their regulation of cyclin-dependent kinases (CDKs). Cyclins are very well characterised in model systems such as yeast and human cells, but surprisingly little is known about their number and role in Plasmodium, the unicellular protozoan parasite that causes malaria. Malaria parasite cell division and proliferation differs from that of many eukaryotes. During its life cycle it undergoes two types of mitosis: endomitosis in asexual stages and an extremely rapid mitotic process during male gametogenesis. Both schizogony (producing merozoites) in host liver and red blood cells, and sporogony (producing sporozoites) in the mosquito vector, are endomitotic with repeated nuclear replication, without chromosome condensation, before cell division. The role of specific cyclins during Plasmodium cell proliferation was unknown. We show here that the Plasmodium genome contains only three cyclin genes, representing an unusual repertoire of cyclin classes. Expression and reverse genetic analyses of the single Plant (P)-type cyclin, CYC3, in the rodent malaria parasite, Plasmodium berghei, revealed a cytoplasmic and nuclear location of the GFP-tagged protein throughout the lifecycle. Deletion of cyc3 resulted in defects in size, number and growth of oocysts, with abnormalities in budding and sporozoite formation. Furthermore, global transcript analysis of the cyc3-deleted and wild type parasites at gametocyte and ookinete stages identified differentially expressed genes required for signalling, invasion and oocyst development. Collectively these data suggest that cyc3 modulates oocyst endomitotic development in Plasmodium berghei

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

The presence of disseminated tumour cells in the bone marrow is inversely related to circulating free DNA in plasma in breast cancer dormancy

Background: The aim of this study was to gain insight into breast cancer dormancy by examining different measures of minimal residual disease (MRD) over time in relation to known prognostic factors. Methods: Sixty-four primary breast cancer patients on follow-up (a median of 8.3 years post surgery) who were disease free had sequential bone marrow aspirates and blood samples taken for the measurement of disseminated tumour cells (DTCs), circulating tumour cells (CTCs) by CellSearch and qPCR measurement of overlapping (96-bp and 291-bp) amplicons in circulating free DNA (cfDNA). Results: The presence of CTCs was correlated with the presence of DTCs measured by immunocytochemistry (P=0.01) but both were infrequently detected. Increasing cfDNA concentration correlated with ER, HER2 and triple-negative tumours and high tumour grade, and the 291-bp amplicon was inversely correlated with DTCs measured by CK19 qRT-PCR (P=0.047). Conclusion: Our results show that breast cancer patients have evidence of MRD for many years after diagnosis despite there being no overt evidence of disease. The inverse relationship between bone marrow CK19 mRNA and the 291-bp amplicon in cfDNA suggests that an inverse relationship between a measure of cell viability in the bone marrow (DTCs) and cell death in the plasma occurs during the dormancy phase of breast cancer

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

SAS6-like protein in Plasmodium indicates that conoid-associated apical complex proteins persist in invasive stages within the mosquito vector

The SAS6-like (SAS6L) protein, a truncated paralogue of the ubiquitous basal body/centriole protein SAS6, has been characterised recently as a flagellum protein in trypanosomatids, but associated with the conoid in apicomplexan Toxoplasma. The conoid has been suggested to derive from flagella parts, but is thought to have been lost from some apicomplexans including the malaria-causing genus Plasmodium. Presence of SAS6L in Plasmodium, therefore, suggested a possible role in flagella assembly in male gametes, the only flagellated stage. Here, we have studied the expression and role of SAS6L throughout the Plasmodium life cycle using the rodent malaria model P. berghei. Contrary to a hypothesised role in flagella, SAS6L was absent during gamete flagellum formation. Instead, SAS6L was restricted to the apical complex in ookinetes and sporozoites, the extracellular invasive stages that develop within the mosquito vector. In these stages SAS6L forms an apical ring, as we show is also the case in Toxoplasma tachyzoites. The SAS6L ring was not apparent in blood-stage invasive merozoites, indicating that the apical complex is differentiated between the different invasive forms. Overall this study indicates that a conoid-associated apical complex protein and ring structure is persistent in Plasmodium in a stage-specific manner