Despite advances in early detection and therapies, cancer is still one of the most common causes of death worldwide. Since each tumor is unique, there is a need to implement personalized care and ...develop robust tools for monitoring treatment response to assess drug efficacy and prevent disease relapse.
Recent developments in liquid biopsies have enabled real-time noninvasive monitoring of tumor burden through the detection of molecules shed by tumors in the blood. These molecules include circulating tumor nucleic acids (ctNAs), comprising cell-free DNA or RNA molecules passively and/or actively released from tumor cells. Often highlighted for their diagnostic, predictive, and prognostic potential, these biomarkers possess valuable information about tumor characteristics and evolution. While circulating tumor DNA (ctDNA) has been in the spotlight for the last decade, less is known about circulating tumor RNA (ctRNA). There are unanswered questions about why some tumors shed high amounts of ctNAs while others have undetectable levels. Also, there are gaps in our understanding of associations between tumor evolution and ctNA characteristics and shedding kinetics. In this review, we summarize current knowledge about ctNA biology and release mechanisms and put this information into the context of tumor evolution and clinical utility.
A deeper understanding of the biology of ctDNA and ctRNA may inform the use of liquid biopsies in personalized medicine to improve cancer patient outcomes.
Summary Background Telomere shortness in human beings is a prognostic marker of ageing, disease, and premature morbidity. We previously found an association between 3 months of comprehensive ...lifestyle changes and increased telomerase activity in human immune-system cells. We followed up participants to investigate long-term effects. Methods This follow-up study compared ten men and 25 external controls who had biopsy-proven low-risk prostate cancer and had chosen to undergo active surveillance. Eligible participants were enrolled between 2003 and 2007 from previous studies and selected according to the same criteria. Men in the intervention group followed a programme of comprehensive lifestyle changes (diet, activity, stress management, and social support), and the men in the control group underwent active surveillance alone. We took blood samples at 5 years and compared relative telomere length and telomerase enzymatic activity per viable cell with those at baseline, and assessed their relation to the degree of lifestyle changes. Findings Relative telomere length increased from baseline by a median of 0·06 telomere to single-copy gene ratio (T/S)units (IQR–0·05 to 0·11) in the lifestyle intervention group, but decreased in the control group (−0·03 T/S units, −0·05 to 0·03, difference p=0·03). When data from the two groups were combined, adherence to lifestyle changes was significantly associated with relative telomere length after adjustment for age and the length of follow-up (for each percentage point increase in lifestyle adherence score, T/S units increased by 0·07, 95% CI 0·02–0·12, p=0·005). At 5 years, telomerase activity had decreased from baseline by 0·25 (–2·25 to 2·23) units in the lifestyle intervention group, and by 1·08 (–3·25 to 1·86) units in the control group (p=0·64), and was not associated with adherence to lifestyle changes (relative risk 0·93, 95% CI 0·72–1·20, p=0·57). Interpretation Our comprehensive lifestyle intervention was associated with increases in relative telomere length after 5 years of follow-up, compared with controls, in this small pilot study. Larger randomised controlled trials are warranted to confirm this finding. Funding US Department of Defense, NIH/NCI, Furlotti Family Foundation, Bahna Foundation, DeJoria Foundation, Walton Family Foundation, Resnick Foundation, Greenbaum Foundation, Natwin Foundation, Safeway Foundation, Prostate Cancer Foundation.
Circulating biomarkers are urgently needed in hepatocellular carcinoma (HCC). The aims of this study were to determine the feasibility of detecting and isolating circulating tumor cells (CTCs) in HCC ...patients using enrichment for epithelial cell adhesion molecule (EpCAM) expression, to examine their prognostic value, and to explore CTC-based DNA sequencing in metastatic HCC patients compared to a control cohort with non-malignant liver diseases (NMLD).
Whole blood was obtained from patients with metastatic HCC or NMLD. CTCs were enumerated by CellSearch then purified by immunomagnetic EpCAM enrichment and fluorescence-activated cell sorting. Targeted ion semiconductor sequencing was performed on whole genome-amplified DNA from CTCs, tumor specimens, and peripheral blood mononuclear cells (PBMC) when available.
Twenty HCC and 10 NMLD patients enrolled. CTCs ≥ 2/7.5 mL were detected in 7/20 (35%, 95% confidence interval: 12%, 60%) HCC and 0/9 eligible NMLD (p = 0.04). CTCs ≥ 1/7.5 mL was associated with alpha-fetoprotein ≥ 400 ng/mL (p = 0.008) and vascular invasion (p = 0.009). Sequencing of CTC DNA identified characteristic HCC mutations. The proportion with ≥ 100x coverage depth was lower in CTCs (43%) than tumor or PBMC (87%) (p < 0.025). Low frequency variants were higher in CTCs (p < 0.001).
CTCs are detectable by EpCAM enrichment in metastatic HCC, without confounding false positive background from NMLD. CTC detection was associated with poor prognostic factors. Sequencing of CTC DNA identified known HCC mutations but more low-frequency variants and lower coverage depth than FFPE or PBMC.
Recent technological advancements in rare cell analysis have facilitated the detection of circulating tumor cells (CTCs) in the blood of patients diagnosed with breast and other types of cancers. ...Numerous clinical studies involving the enumeration of CTCs in breast cancer patients have unequivocally demonstrated the prognostic value of these cells. Evidence from recent molecular studies indicates that CTCs may be potential surrogate markers for systemic disease. As such, real-time assessment of therapeutic biomarkers in breast CTCs, such as the estrogen receptor (ER) and the human epidermal growth factor receptor 2 (HER2), may have a tremendous impact in guiding-targeted cancer therapy. In this review, we discuss the clinical implications of CTC detection and its potential utility for personalized medicine in breast cancer.
Much effort has been dedicated to developing circulating tumor cells (CTC) as a noninvasive cancer biopsy, but with limited success as yet. In this study, we combine a method for isolation of highly ...pure CTCs using immunomagnetic enrichment/fluorescence-activated cell sorting with advanced whole genome sequencing (WGS), based on long fragment read technology, to illustrate the utility of an accurate, comprehensive, phased, and quantitative genomic analysis platform for CTCs. Whole genomes of 34 CTCs from a patient with metastatic breast cancer were analyzed as 3,072 barcoded subgenomic compartments of long DNA. WGS resulted in a read coverage of 23× per cell and an ensemble call rate of >95%. These barcoded reads enabled accurate detection of somatic mutations present in as few as 12% of CTCs. We found in CTCs a total of 2,766 somatic single-nucleotide variants and 543 indels and multi-base substitutions, 23 of which altered amino acid sequences. Another 16,961 somatic single nucleotide variant and 8,408 indels and multi-base substitutions, 77 of which were nonsynonymous, were detected with varying degrees of prevalence across the 34 CTCs. On the basis of our whole genome data of mutations found in all CTCs, we identified driver mutations and the tissue of origin of these cells, suggesting personalized combination therapies beyond the scope of most gene panels. Taken together, our results show how advanced WGS of CTCs can lead to high-resolution analyses of cancers that can reliably guide personalized therapy.
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Summary Background Telomeres are protective DNA–protein complexes at the end of linear chromosomes that promote chromosomal stability. Telomere shortness in human beings is emerging as a prognostic ...marker of disease risk, progression, and premature mortality in many types of cancer, including breast, prostate, colorectal, bladder, head and neck, lung, and renal cell. Telomere shortening is counteracted by the cellular enzyme telomerase. Lifestyle factors known to promote cancer and cardiovascular disease might also adversely affect telomerase function. However, previous studies have not addressed whether improvements in nutrition and lifestyle are associated with increases in telomerase activity. We aimed to assess whether 3 months of intensive lifestyle changes increased telomerase activity in peripheral blood mononuclear cells (PBMC). Methods 30 men with biopsy-diagnosed low-risk prostate cancer were asked to make comprehensive lifestyle changes. The primary endpoint was telomerase enzymatic activity per viable cell, measured at baseline and after 3 months. 24 patients had sufficient PBMCs needed for longitudinal analysis. This study is registered on the ClinicalTrials.gov website, number NCT00739791. Findings PBMC telomerase activity expressed as natural logarithms increased from 2·00 (SD 0·44) to 2·22 (SD 0·49; p=0·031). Raw values of telomerase increased from 8·05 (SD 3·50) standard arbitrary units to 10·38 (SD 6·01) standard arbitrary units. The increases in telomerase activity were significantly associated with decreases in low-density lipoprotein (LDL) cholesterol ( r =−0·36, p=0·041) and decreases in psychological distress ( r =−0·35, p=0·047). Interpretation Comprehensive lifestyle changes significantly increase telomerase activity and consequently telomere maintenance capacity in human immune-system cells. Given this finding and the pilot nature of this study, we report these increases in telomerase activity as a significant association rather than inferring causation. Larger randomised controlled trials are warranted to confirm the findings of this study. Funding US Department of Defense (US Army Medical Research Acquisition Activity W81XWH-05-1-0375, Fort Detrick, Frederick, MD, USA); Henry M Jackson Foundation for the Advancement of Military Medicine (contract 56422; Rockville MD, USA) from the National Center for Complementary and Alternative Medicine (NCCAM) of the National Institutes of Health (grant number K01AT004199; Bethesda, MD, USA); Bahna Foundation (Stamford, CT, USA); DeJoria Foundation (Los Angeles, CA, USA); Kerzner Foundation (New York, NY, USA); Bernard Osher Foundation (San Francisco, CA, USA); Walton Family Foundation (Bentonville, AK, USA); Jeff Walker Family Foundation (Wilton, CT, USA); Safeway Foundation (Pleasanton, CA, USA).
We profiled circulating tumor cells (CTCs) to study the biology of blood-borne metastasis and to monitor biomarker status in metastatic breast cancer (MBC).
CTCs were isolated from 105 patients with ...MBC using EPCAM-based immunomagnetic enrichment and fluorescence-activated cells sorting (IE/FACS), 28 of whom had serial CTC analysis (74 samples, 2-5 time points). CTCs were subjected to microfluidic-based multiplex QPCR array of 64 cancer-related genes (
= 151) and genome-wide copy-number analysis by array comparative genomic hybridization (aCGH;
= 49).
Combined transcriptional and genomic profiling showed that CTCs were 26%
, 48%
, and 27%
Serial testing showed that
status was more stable over time compared with
and proliferation (
) status. While cell-to-cell heterogeneity was observed at the single-cell level, with increasingly stable expression in larger pools, patient-specific CTC expression "fingerprints" were also observed. CTC copy-number profiles clustered into three groups based on the extent of genomic aberrations and the presence of large chromosomal imbalances. Comparative analysis showed discordance in
/ER (27%) and
/HER2 (23%) status between CTCs and matched primary tumors. CTCs in 65% of the patients were considered to have low proliferation potential. Patients who harbored CTCs with high proliferation (
) status had significantly reduced progression-free survival (
= 0.0011) and overall survival (
= 0.0095) compared with patients with low proliferative CTCs.
We demonstrate an approach for complete isolation of EPCAM-positive CTCs and downstream comprehensive transcriptional/genomic characterization to examine the biology and assess breast cancer biomarkers in these cells over time.
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Epidemiological and prospective studies indicate that comprehensive lifestyle changes may modify the progression of prostate cancer. However, the molecular mechanisms by which improvements in diet ...and lifestyle might affect the prostate microenvironment are poorly understood. We conducted a pilot study to examine changes in prostate gene expression in a unique population of men with low-risk prostate cancer who declined immediate surgery, hormonal therapy, or radiation and participated in an intensive nutrition and lifestyle intervention while undergoing careful surveillance for tumor progression. Consistent with previous studies, significant improvements in weight, abdominal obesity, blood pressure, and lipid profile were observed (all P < 0.05), and surveillance of low-risk patients was safe. Gene expression profiles were obtained from 30 participants, pairing RNA samples from control prostate needle biopsy taken before intervention to RNA from the same patient's 3-month postintervention biopsy. Quantitative real-time PCR was used to validate array observations for selected transcripts. Two-class paired analysis of global gene expression using significance analysis of microarrays detected 48 up-regulated and 453 down-regulated transcripts after the intervention. Pathway analysis identified significant modulation of biological processes that have critical roles in tumorigenesis, including protein metabolism and modification, intracellular protein traffic, and protein phosphorylation (all P < 0.05). Intensive nutrition and lifestyle changes may modulate gene expression in the prostate. Understanding the prostate molecular response to comprehensive lifestyle changes may strengthen efforts to develop effective prevention and treatment. Larger clinical trials are warranted to confirm the results of this pilot study.
Circulating tumor cells are commonly observed in the peripheral blood of advanced breast cancer patients. We tested the feasibility of tumor cell detection in the cerebrospinal fluid (CSF) and ...studied its clinical relevance in leptomeningeal metastasis (LM) of breast cancer. CSF samples were collected from 38 metastatic breast cancer patients known or suspected to have LM. Control CSF samples were collected from 14 individuals without solid tumor malignancy. We used a modified CellSearch™ assay and an alternative EPCAM-based method involving immunomagnetic enrichment followed by flow cytometry (IE/FC) to enumerate CSF tumor cells (CSFTCs). CSFTCs were assayed at time of LM diagnosis and over the course of LM-directed therapy. We analyzed a total of 102 CSF samples with modified CellSearch™. The CSFTC counts were strongly correlated with the corresponding IE/FC results (Pearson’s
r
= 0.94). Twenty-eight out of 30 samples in which malignant cells were identified by CSF cytology were CSFTC-positive by modified CellSearch™. Baseline CSFTC levels from 21 patients eventually diagnosed with LM were significantly higher than the controls (
p
= 0.0202), whereas 13 patients deemed not to have LM showed CSFTC results indistinguishable from the controls. In patients with serial samples, it was possible to monitor CSFTC levels as a potential biomarker of treatment response. CSFTC detection using a modified CellSearch™ assay demonstrated high sensitivity in detecting malignant cells in CSF and may be a promising method for diagnosing LM and monitoring LM during treatment.
Molecular characterization of circulating tumor cells (CTCs) found in the blood of cancer patients offers the potential to provide new insights into the biology of cancer metastasis. However, since ...they are rare and difficult to isolate, the molecular nature of CTCs remains poorly understood. In this paper, we reviewed a decade’s worth of scientific literature (2003–2013) describing efforts on isolation and genomic analysis of CTCs. The limited number of CTC genomic studies we found attested to the infancy of this field of study. These initial reports, however, provide an important framework for future comprehensive exploration of CTC biology. For CTCs to be broadly accepted as therapeutic targets and biomarkers of metastatic spread, further in-depth molecular characterization is warranted.
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