Small-cell lung cancer (SCLC), an aggressive neuroendocrine tumor with early dissemination and dismal prognosis, accounts for 15-20% of lung cancer cases and ∼200,000 deaths each year. Most cases are ...inoperable, and biopsies to investigate SCLC biology are rarely obtainable. Circulating tumor cells (CTCs), which are prevalent in SCLC, present a readily accessible 'liquid biopsy'. Here we show that CTCs from patients with either chemosensitive or chemorefractory SCLC are tumorigenic in immune-compromised mice, and the resultant CTC-derived explants (CDXs) mirror the donor patient's response to platinum and etoposide chemotherapy. Genomic analysis of isolated CTCs revealed considerable similarity to the corresponding CDX. Most marked differences were observed between CDXs from patients with different clinical outcomes. These data demonstrate that CTC molecular analysis via serial blood sampling could facilitate delivery of personalized medicine for SCLC. CDXs are readily passaged, and these unique mouse models provide tractable systems for therapy testing and understanding drug resistance mechanisms.
The CellSearch® semiautomated CTC enrichment and staining system has been established as the ‘gold standard’ for CTC enumeration with CellSearch® CTC counts recognized by the FDA as prognostic for a ...number of cancers. We and others have gone on to show that molecular analysis of CellSearch® CTCs isolated shortly after CellSearch® enrichment provides another valuable layer of information that has potential clinical utility including predicting response to treatment. Although CellSearch® CTCs can be readily isolated after enrichment, the process of analysing a single CellSearch® patient sample, which may contain many CTCs, is both time‐consuming and costly. Here, we describe a simple process that will allow storage of all CellSearch®‐enriched cells in glycerol at −20 °C for up to 2 years without any measurable loss in the ability to retrieve single cells or in the genome integrity of the isolated cells. To establish the suitability of long‐term glycerol storage for single‐cell molecular analysis, we isolated individual CellSearch®‐enriched cells by DEPArray™ either shortly after CellSearch® enrichment or following storage of matched enriched cells in glycerol at −20 °C. All isolated cells were subjected to whole‐genome amplification (WGA), and the efficacy of single‐cell WGA was evaluated by multiplex PCR to generate a Genome Integrity Index (GII). The GII results from 409 single cells obtained from both ‘spike‐in’ controls and clinical samples showed no statistical difference between values obtained pre‐ and postglycerol storage and that there is no further loss in integrity when DEPArray™‐isolated cells are then stored at −80 °C for up to 2 years. In summary, we have established simple yet effective ‘stop‐off’ points along the CTC workflow enabling CTC banking and facilitating selection of suitable samples for intensive analysis once patient outcomes are known.
Here, we describe a simple process for the storage of CellSearch®‐enriched cells for up to 2 years without any measurable loss in the ability to retrieve single cells, or in the genome integrity of the isolated cells. This workflow enables CTC banking and facilitates the retrospective selection of samples for intensive analysis once patient outcomes are known.
The gilthead sea bream (Sparus aurata L.) is a marine fish of great importance for fisheries and aquaculture. It has also a peculiar sex-determination system, being a protandrous hermaphrodite. Here ...we report the construction of a first-generation genetic linkage map for S. aurata, based on 204 microsatellite markers. Twenty-six linkage groups (LG) were found. The total map length was 1241.9 cM. The ratio between sex-specific map lengths was 1:1.2 (male:female). Comparison with a preliminary radiation hybrid (RH) map reveals a good concordance, as all markers located in a single LG are located in a single RH group, except for Ad-25 and CId-31. Comparison with the Tetraodon nigroviridis genome revealed a considerable number of evolutionary conserved regions (ECRs) between the two species. The mean size of ECRs was 182 bp (sequence identity 60-90%). Forty-one ECRs have a known chromosomal location in the pufferfish genome. Despite the limited number of anchoring points, significant syntenic relationships were found. The linkage map presented here provides a robust comparative framework for QTL analysis in S. aurata and is a step toward the identification of genetic loci involved both in the determination of economically important traits and in the individual timing of sex reversal.
Toll-Like Receptors (TLRs) have recently emerged as key sensors of invading microbes, acting through recognition of pathogen-associated molecular patterns. It has been demonstrated that TLR9 is ...involved in the recognition of unmethylated CpG motifs in mice, humans, and pigs. We report here the full-length sequence of
TLR9 cDNA in the gilthead sea bream (
Sparus aurata L.). The predicted protein (1063 amino acids) was similar to mammalian TLR9s, showing 21 leucine-rich repeats in the extracellular region and a typical Toll/IL-1R (TIR) domain in the intracellular region. Comparative analysis of TLR9 sequences indicated that critical residues for ligand-binding are conserved across vertebrate lineages, although evidence of functional divergence was observed. Analysis of the genomic structure of sea bream
TLR9 gene revealed the presence of two intervening sequences. Retention of the second intron produced an alternatively spliced mRNA (
TLR9B) showing differential expression among tissues or developmental stages compared to the wild-type isoform (
TLR9A). RT-PCR analysis indicated a broad expression of
TLR9A, especially in immune-related organs (spleen, head-kidney) and mucosal–epithelial barriers (gills, gut, skin). Using quantitative Real-Time RT-PCR, no statistically significant variation was observed for
TLR9 mRNAs expression in the spleen of experimentally infected animals compared to healthy controls. Comparing sequence and expression profile of sea bream TLR9 with mammalian TLR9s suggested that the main function of TLR9 might be conserved across vertebrates, although species-specific features are present (modulation of ligand-binding specificity, alternative splicing).
In most patients with small-cell lung cancer (SCLC)-a metastatic, aggressive disease-the condition is initially chemosensitive but then relapses with acquired chemoresistance. In a minority of ...patients, however, relapse occurs within 3 months of initial treatment; in these cases, disease is defined as chemorefractory. The molecular mechanisms that differentiate chemosensitive from chemorefractory disease are currently unknown. To identify genetic features that distinguish chemosensitive from chemorefractory disease, we examined copy-number aberrations (CNAs) in circulating tumor cells (CTCs) from pretreatment SCLC blood samples. After analysis of 88 CTCs isolated from 13 patients (training set), we generated a CNA-based classifier that we validated in 18 additional patients (testing set, 112 CTC samples) and in six SCLC patient-derived CTC explant tumors. The classifier correctly assigned 83.3% of the cases as chemorefractory or chemosensitive. Furthermore, a significant difference was observed in progression-free survival (PFS) (Kaplan-Meier P value = 0.0166) between patients designated as chemorefractory or chemosensitive by using the baseline CNA classifier. Notably, CTC CNA profiles obtained at relapse from five patients with initially chemosensitive disease did not switch to a chemorefractory CNA profile, which suggests that the genetic basis for initial chemoresistance differs from that underlying acquired chemoresistance.
Circulating tumour cells (CTCs) have potential utility as minimally-invasive biomarkers to aid cancer treatment decision making. However, many current CTC technologies enrich CTCs using specific ...surface epitopes that do not necessarily reflect CTC heterogeneity. Here we evaluated the epitope-independent Parsortix system which enriches CTCs based on size and rigidity using both healthy normal volunteer blood samples spiked with tumour cells and blood samples from patients with small cell lung cancer (SCLC). Blood samples were maintained unfractionated at room temperature for up to 4 days followed by plasma removal for circulating free DNA (cfDNA) isolation and direct application of the remaining cell component to the Parsortix system. For tumour cells expressing the EpCAM cell surface marker the numbers of spiked cells retained using the Parsortix system and by EpCAM-positive selection using CellSearch® were not significantly different, whereas only the Parsortix system showed strong enrichment of cells with undetectable EpCAM expression. In a pilot clinical study we banked both enriched CTCs as well as plasma from SCLC patient blood samples. Upon retrieval of the banked Parsortix cellular samples we could detect cytokeratin positive CTCs in all 12 SCLC patients tested. Interestingly, processing parallel samples from the same patients by EpCAM enrichment using CellSearch® revealed only 83% (10/12) with cytokeratin positive CTCs indicating the Parsortix system is enriching for EpCAM negative SCLC CTCs. Our combined results indicate the Parsortix system is a valuable tool for combined cfDNA isolation and CTC enrichment that enables CTC analysis to be extended beyond dependence on surface epitopes.
Molecular information obtained from cancer patients' blood is an emerging and powerful research tool with immense potential as a companion diagnostic for patient stratification and monitoring. Blood, ...which can be sampled routinely, provides a means of inferring the current genetic status of patients' tumours via analysis of circulating tumour cells (CTCs) or circulating tumour DNA (ctDNA). However, accurate assessment of both CTCs and ctDNA requires all blood cells to be maintained intact until samples are processed. This dictates for ctDNA analysis EDTA blood samples must be processed with 4 h of draw, severely limiting the use of ctDNA in multi-site trials. Here we describe a blood collection protocol that is amenable for analysis of both CTCs and ctDNA up to four days after blood collection. We demonstrate that yields of circulating free DNA (cfDNA) obtained from whole blood CellSave samples are equivalent to those obtained from conventional EDTA plasma processed within 4 h of blood draw. Targeted and genome-wide NGS revealed comparable DNA quality and resultant sequence information from cfDNA within CellSave and EDTA samples. We also demonstrate that CTCs and ctDNA can be isolated from the same patient blood sample, and give the same patterns of CNA enabling direct analysis of the genetic status of patients' tumours.
In summary, our results demonstrate the utility of a simple approach that enabling robust molecular analysis of CTCs and cfDNA for genotype-directed therapies in multi-site clinical trials and represent a significant methodological improvement for clinical benefit.
•Demonstrated collection of blood in CellSave tubes preserves cfDNA up to 96 h post draw.•No significant difference between standard EDTA cfDNA and preserved cfDNA.•Preserved cfDNA applicable to both targeted and genome wide NGS analysis.•CTCs and cfDNA can be isolated from same CellSave blood sample.•Comparable copy number aberrations seen in both CTCs and ctDNA from same blood sample.
The prostate cancer (PCa) diagnostic pathway is undergoing a radical change with the introduction of multiparametric magnetic resonance imaging (mpMRI), genomic testing, and different prostate biopsy ...techniques. It has been proposed that these tests should be used in a sequential manner to optimise risk stratification.
To characterise the genomic, epigenomic, and transcriptomic features of mpMRI-visible and -nonvisible PCa in clinically localised disease.
Multicore analysis of fresh prostate tissue sampled immediately after radical prostatectomy was performed for intermediate- to high-risk PCa.
Low-pass whole-genome, exome, methylation, and transcriptome profiling of patient tissue cores taken from microscopically benign and cancerous areas in the same prostate. Circulating free and germline DNA was assessed from the blood of five patients.
Correlations between preoperative mpMRI and genomic characteristics of tumour and benign prostate samples were assessed. Gene profiles for individual tumour cores were correlated with existing genomic classifiers currently used for prognostication.
A total of 43 prostate cores (22 tumour and 21 benign) were profiled from six whole prostate glands. Of the 22 tumour cores, 16 were tumours visible and six were tumours nonvisible on mpMRI. Intratumour genomic, epigenomic, and transcriptomic heterogeneity was found within mpMRI-visible lesions. This could potentially lead to misclassification of patients using signatures based on copy number or RNA expression. Moreover, three of the six cores obtained from mpMRI-nonvisible tumours harboured one or more genetic alterations commonly observed in metastatic castration-resistant PCa. No circulating free DNA alterations were found. Limitations include the small cohort size and lack of follow-up.
Our study supports the continued use of systematic prostate sampling in addition to mpMRI, as avoidance of systematic biopsies in patients with negative mpMRI may mean that clinically significant tumours harbouring genetic alterations commonly seen in metastatic PCa are missed. Furthermore, there is inconsistency in individual genomics when genomic classifiers are applied.
Our study shows that tumour heterogeneity within prostate tumours visible on multiparametric magnetic resonance imaging (mpMRI) can lead to misclassification of patients if only one core is used for genomic analysis. In addition, some cancers that were missed by mpMRI had genomic aberrations that are commonly seen in advanced metastatic prostate cancer. Avoiding biopsies in mpMRI-negative cases may mean that such potentially lethal cancers are missed.
Our study supports the continued use of systematic prostate sampling in addition to multiparametric magnetic resonance imaging (mpMRI), as avoidance of systematic biopsies in patients with negative mpMRI can mean that clinically significant tumours harbouring genetic alterations commonly seen in metastatic prostate cancer can be missed. Furthermore, there is inconsistency in individual genomics when genomic classifiers are applied.
Fish pasteurellosis is an infectious disease that affects several fish species living in marine temperate waters. Its causative agent is the Gram-negative bacterium
Photobacterium damselae subsp.
...piscicida (
Phdp). Fish pasteurellosis represents a serious health problem for the majority of intensive sea bream hatcheries, with 90–100% mortality during disease outbreaks. Larvae and juveniles are the most susceptible stages. A potential strategy to prevent fish pasteurellosis is to select for animals that are genetically resistant to it. The aim of this work was to evaluate the genetic variation of disease resistance and growth in the gilthead sea bream (
Sparus aurata L.). An experimental population, approximately 3500 animals originating from mass spawning of four broodstocks, was experimentally infected with a highly virulent strain of
Phdp. Mortality was monitored daily for 19
days. Upon completion of the challenge experiment, genetic profiles at nine microsatellite
loci were obtained for 1753 animals and for all (256) broodstock fish, for parentage assignment. A subset of families defined by means of genetic analysis was used to calculate heritability for survival to infection. For body length (measured
post-mortem), heritability was 0.38
±
0.07. The heritability for disease resistance varied depending on how resistance was defined. Heritability was 0.12
±
0.04 for days of survival post challenge, defined as a continuous trait, while it ranged from 0.45
±
0.04 to 0.18
±
0.08 for the binary trait dead/alive at a specific day. The genetic correlation between body length and survival was positive and significant (
r
=
0.61
±
0.16). These results confirm the existence of genetic variation for growth and resistance against
Phpd and highlight also the potential for selective breeding programs to improve these traits.
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