Progress in sensitive analytical approaches has opened new avenues for the detection of cells or products such as circulating cell-free DNA released by tumors. These 'liquid biopsies' are being ...explored in clinical trials for early cancer detection, prediction of recurrent disease, and assessment of therapeutic resistance mechanisms.
Circulating tumor cells (CTCs) are important clinical indicators for prognosis and treatment efficacy. However, CTC investigation is hampered by their low number, making the establishment of ...permanent CTC lines very challenging. We derived and characterized nine CTC lines using blood samples from a patient with metastatic colorectal cancer collected before and after chemotherapy and targeted therapy, and during cancer progression. These cell lines displayed an intermediate epithelial/mesenchymal phenotype, stem-cell like characteristics, angiogenesis potential, an osteomimetic signature and the capacity to escape from the immune system. Moreover, they showed changes in mRNA and protein expression (e.g., DEFA6, ABCB1 and GAL), whereas analysis of chromosomal copy number aberrations revealed no significant variation over time. These data indicate that although CTC lines derived from sequential blood samples during therapy have common traits, treatment-resistant CTC clones with distinct phenotypic characteristics are selected over time.
Endocrine treatment is the most preferable systemic treatment in metastatic breast cancer patients that have had an estrogen receptor (ER) positive primary tumor or metastatic lesions, however, ...approximately 20% of these patients do not benefit from the therapy and demonstrate further metastatic progress. One reason for failure of endocrine therapy might be the heterogeneity of ER expression in tumor cells spreading from the primary tumor to distant sites which is reflected in detectable circulating tumor cells (CTCs).
A sensitive and specific staining protocol for ER, keratin 8/18/19, CD45 was established. Peripheral blood from 35 metastatic breast cancer patients with ER-positive primary tumors was tested for the presence of CTCs. Keratin 8/18/19 and DAPI positive but CD45 negative cells were classified as CTCs and evaluated for ER staining. Subsequently, eight individual CTCs from four index patients (2 CTCs per patient) were isolated and underwent whole genome amplification and ESR1 gene mutation analysis.
CTCs were detected in blood of 16 from 35 analyzed patients (46%), with a median of 3 CTCs/7.5 ml. In total, ER-negative CTCs were detected in 11/16 (69%) of the CTC positive cases, including blood samples with only ER-negative CTCs (19%) and samples with both ER-positive and ER-negative CTCs (50%). No correlation was found between the intensity and/or percentage of ER staining in the primary tumor with the number and ER status of CTCs of the same patient. ESR1 gene mutations were not found.
CTCs frequently lack ER expression in metastatic breast cancer patients with ER-positive primary tumors and show a considerable intra-patient heterogeneity, which may reflect a mechanism to escape endocrine therapy. Provided single cell analysis did not support a role of ESR1 mutations in this process.
Circulating tumor cells (CTCs) hold great potential to answer key questions of how non-small cell lung cancer (NSCLC) evolves and develops resistance upon anti-PD-1/PD-L1 treatment. Currently, their ...clinical utility in NSCLC is compromised by a low detection rate with the established, Food and Drug Administration (FDA)-approved, EpCAM-based CellSearch
System. We tested an epitope-independent method (Parsortix
system) and utilized it to assess PD-L1 expression of CTCs from NSCLC patients. We prospectively collected 127 samples, 97 of which were analyzed with the epitope-independent system in comparison to the CellSearch system. CTCs were determined by immunocytochemistry as intact, nucleated, CD45
, pankeratins (K)
cells. PD-L1 status of CTCs was evaluated from 89 samples. With the epitope-independent system, ≥1 CTC per blood sample was detected in 59 samples (61%) compared to 31 samples (32%) with the EpCAM-based system. Upon PD-L1 staining, 47% of patients harbored only PD-L1
CTCs, 47% had PD-L1
and PD-L1
CTCs, and only 7% displayed exclusively PD-L1
CTCs. The percentage of PD-L1
CTCs did not correlate with the percentage of PD-L1
in biopsies determined by immunohistochemistry (
= 0.179). Upon disease progression, all patients showed an increase in PD-L1
CTCs, while no change or a decrease in PD-L1
CTCs was observed in responding patients (
= 11;
= 0.001). Our data show a considerable heterogeneity in the PD-L1 status of CTCs from NSCLC patients. An increase of PD-L1
CTCs holds potential to predict resistance to PD-1/PD-L1 inhibitors.
Whole genome amplification (WGA) is required for single cell genotyping. Effectiveness of currently available WGA technologies in combination with next generation sequencing (NGS) and material ...preservation is still elusive.
In respect to the accuracy of SNP/mutation, indel, and copy number aberrations (CNA) calling, the HiSeq2000 platform outperformed IonProton in all aspects. Furthermore, more accurate SNP/mutation and indel calling was demonstrated using single tumor cells obtained from EDTA-collected blood in respect to CellSave-preserved blood, whereas CNA analysis in our study was not detectably affected by fixation. Although MDA-based WGA yielded the highest DNA amount, DNA quality was not adequate for downstream analysis. PCR-based WGA demonstrates superiority over MDA-PCR combining technique for SNP and indel analysis in single cells. However, SNP calling performance of MDA-PCR WGA improves with increasing amount of input DNA, whereas CNA analysis does not. The performance of PCR-based WGA did not significantly improve with increase of input material. CNA profiles of single cells, amplified with MDA-PCR technique and sequenced on both HiSeq2000 and IonProton platforms, resembled unamplified DNA the most.
We analyzed the performance of PCR-based, multiple-displacement amplification (MDA)-based, and MDA-PCR combining WGA techniques (WGA kits Ampli1, REPLI-g, and PicoPlex, respectively) on single and pooled tumor cells obtained from EDTA- and CellSave-preserved blood and archival material. Amplified DNA underwent exome-Seq with the Illumina HiSeq2000 and ThermoFisher IonProton platforms.
We demonstrate the feasibility of single cell genotyping of differently preserved material, nevertheless, WGA and NGS approaches have to be chosen carefully depending on the study aims.
Despite remarkable progress in melanoma therapy, the exceptional heterogeneity of the disease has prevented the development of reliable companion biomarkers for the prediction or monitoring of ...therapy responses. Here, we show that difficulties in detecting blood-based markers, like circulating tumor cells (CTC), might arise from the translation of the mutational heterogeneity of melanoma cells towards their surface marker expression. We provide a unique method, which enables the molecular characterization of clinically relevant CTC subsets, as well as circulating tumor DNA (ctDNA), from a single blood sample. The study demonstrates the benefit of a combined analysis of ctDNA and CTC counts in melanoma patients, revealing that CTC subsets and ctDNA provide synergistic real-time information on the mutational status, RNA and protein expression of melanoma cells in individual patients, in relation to clinical outcome.
Among emerging circulating biomarkers, miRNA has the potential to detect lung cancer and follow the course of the disease. However, miRNA analysis deserves further standardization before ...implementation into clinical trials or practice. Here, we performed international ring experiments to explore (pre)-analytical factors relevant to the outcome of miRNA blood tests in the context of the EU network CANCER-ID.
Cell-free (cfmiRNA) and extracellular vesicle-derived miRNA (EVmiRNA) were extracted using the miRNeasy Serum/Plasma Advanced, and the ExoRNeasy Maxi kit, respectively, in a plasma cohort of 27 NSCLC patients and 20 healthy individuals. Extracted miRNA was investigated using small RNA sequencing and hybridization platforms. Validation of the identified miRNA candidates was performed using quantitative PCR.
We demonstrate the highest read counts in healthy individuals and NSCLC patients using QIAseq. Moreover, QIAseq showed 15.9% and 162.9% more cfmiRNA and EVmiRNA miRNA counts, respectively, in NSCLC patients compared to healthy control samples. However, a systematic comparison of selected miRNAs revealed little agreement between high-throughput platforms, thus some miRNAs are detected with one technology, but not with the other. Adding to this, 35% (9 of 26) of selected miRNAs in the cfmiRNA and 42% (11 of 26) in the EVmiRNA fraction were differentially expressed by at least one qPCR platform; about half of the miRNAs (54%) were concordant for both platforms.
Changing of (pre)-analytical methods of miRNA analysis has a significant impact on blood test results and is therefore a major confounding factor. In addition, to confirm miRNA biomarker candidates screening studies should be followed by targeted validation using an independent platform or technology.
Objectives/Hypothesis
This study aimed to evaluate the olfactory status in children with laboratory confirmed SARS‐CoV‐2 using subjective and psychophysical methods.
Study Design
Prospective clinical ...cross‐sectional study.
Methods
This is a prospective clinical cross‐sectional study of 79 children with COVID‐19. The 21st item of SNOT‐22 questionnaire and odor identification test were used for smell assessment. Children were examined twice during the hospitalization, and a telephone survey was conducted 60 days after hospital discharge.
Results
Immediately after confirmation of COVID‐19, smell impairment was detected in 86.1% of children by means of the Identification test and in 68.4% of children by means of the survey (P = .010). After 5 days survey revealed a statistically significant decrease in the number of patients with hyposmia (41 out of 79, 51.9%). On the first visit, the mean Identification test score corresponded to “hyposmia” (9.5 ± 2.7), while on the second visit, the average value was 13.1 ± 1.9, which corresponded to “normosmia.” According to the telephone survey, recovery of the olfactory function occurred within 10 days in 37 of 52 patients (71.2%), 11 to 29 days ‐ in 12 children (23.1%), and later than 30 days ‐ in three cases (5.7%).
Conclusions
In the pediatric population, olfactory dysfunction is an early and common symptom of COVID‐19. There is a trend to quick recovery of olfactory function in children with COVID‐19. The overwhelming majority of patients (94.3%) had no subjective olfactory complaints by the end of the first month.
Level of Evidence
4 Laryngoscope, 131:E2475–E2480, 2021
Abstract
BACKGROUND
In cancer patients, circulating cell-free DNA (ccfDNA) can contain tumor-derived DNA (ctDNA), which enables noninvasive diagnosis, real-time monitoring, and treatment ...susceptibility testing. However, ctDNA fractions are highly variable, which challenges downstream applications. Therefore, established preanalytical work flows in combination with cost-efficient and reproducible reference materials for ccfDNA analyses are crucial for analytical validity and subsequently for clinical decision-making.
METHODS
We describe the efforts of the Innovative Medicines Initiative consortium CANCER-ID (http://www.cancer-id.eu) for comparing different technologies for ccfDNA purification, quantification, and characterization in a multicenter setting. To this end, in-house generated mononucleosomal DNA (mnDNA) from lung cancer cell lines carrying known TP53 mutations was spiked in pools of plasma from healthy donors generated from 2 different blood collection tubes (BCTs). ccfDNA extraction was performed at 15 partner sites according to their respective routine practice. Downstream analysis of ccfDNA with respect to recovery, integrity, and mutation analysis was performed centralized at 4 different sites.
RESULTS
We demonstrate suitability of mnDNA as a surrogate for ccfDNA as a process quality control from nucleic acid extraction to mutation detection. Although automated extraction protocols and quantitative PCR-based quantification methods yielded the most consistent and precise results, some kits preferentially recovered spiked mnDNA over endogenous ccfDNA. Mutated TP53 fragments derived from mnDNA were consistently detected using both next-generation sequencing-based deep sequencing and droplet digital PCR independently of BCT.
CONCLUSIONS
This comprehensive multicenter comparison of ccfDNA preanalytical and analytical work flows is an important contribution to establishing evidence-based guidelines for clinically feasible (pre)analytical work flows.
Genomic information can help to identify colorectal tumors with high and low metastatic potential, thereby improving prediction of benefit of local and/or systemic treatment. Here we investigated ...chromosomal aberrations in relation to the different stages of the metastatic cascade: dissemination of tumor cells into the mesenteric vein, metastatic outgrowth in the liver, intravasation of the peripheral blood circulation, and development of further distant metastasis.
Peripheral and mesenteric blood from colorectal cancer patients (n = 72) were investigated for circulating tumor cells, and DNA extracted from their primary tumors was subjected to array comparative genomic hybridization profiling. The results were validated with an independent set of primary colorectal tumors (n = 53) by quantitative reverse transcription PCR.
Mesenteric intravasation and liver metastasis were correlated with losses of chromosomes 16p (72%), 16q (27%), and 19 (54%), gain along 1q31 (45%) and 20q (60%), tumor cell infiltration into the peripheral blood circulation, and further distant metastasis with gain of chromosome 8q (59%) and 12 (47%,
< 0.01). Chromosome 12 gain was associated with poor overall survival in the initial (2.8 vs >7 years) and validation cohort (3.3 vs >6 years). The prospective study presented here is a hypothesis-generating study and confirmation with larger cohorts is required.
This is the first study that investigated colorectal cancer in its different stages of metastasis in correlation with copy number changes of the primary tumor. This information might be helpful to identify patients with limited metastatic spread who may profit from liver metastasis resection and may lead to the discovery of new therapeutic targets.Microarray data have been deposited in NCBI's Gene Expression Omnibus and are accessible through GEO Series accession number GSE82228.
PDF
