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<0.001) and increasing centrifugation force, with depletion of platelet associated miRNAs, whereas cfDNA was unaffected. However, sample replicates showed excellent reproducibility on TaqMan low density arrays (ρ = 0.96, p<0.0001). We also successfully generated miRNA profiles for plasma samples stored > 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.
Celotno besedilo
Dostopno za:
DOBA, IZUM, KILJ, NUK, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK
Up to 30% of patients with breast cancer relapse after primary treatment. There are no sensitive and reliable tests to monitor these patients and detect distant metastases before overt recurrence. ...Here, we demonstrate the use of personalized circulating tumor DNA (ctDNA) profiling for detection of recurrence in breast cancer.
Forty-nine primary patients with breast cancer were recruited following surgery and adjuvant therapy. Plasma samples (
= 208) were collected every 6 months for up to 4 years. Personalized assays targeting 16 variants selected from primary tumor whole-exome data were tested in serial plasma for the presence of ctDNA by ultradeep sequencing (average >100,000X).
Plasma ctDNA was detected ahead of clinical or radiologic relapse in 16 of the 18 relapsed patients (sensitivity of 89%); metastatic relapse was predicted with a lead time of up to 2 years (median, 8.9 months; range, 0.5-24.0 months). None of the 31 nonrelapsing patients were ctDNA-positive at any time point across 156 plasma samples (specificity of 100%). Of the two relapsed patients who were not detected in the study, the first had only a local recurrence, whereas the second patient had bone recurrence and had completed chemotherapy just 13 days prior to blood sampling.
This study demonstrates that patient-specific ctDNA analysis can be a sensitive and specific approach for disease surveillance for patients with breast cancer. More importantly, earlier detection of up to 2 years provides a possible window for therapeutic intervention.
The
aim of this study was to analyze plasma DNA from primary and metastatic
breast cancer cases for tumor-specific alterations and to compare these
findings with immunocytochemistry and estimation of ...cytokeratin 19
(CK19) mRNA for detection of micrometastases. DNA was extracted from
plasma, lymphocytes, and microdissected tumor tissue sections obtained
from 71 patients with breast cancer and 9 controls. DNA samples were
analyzed for loss of heterozygosity (LOH) and/or microsatellite
instability (MI) by PCR with two polymorphic markers (DM-1 and
D16S400). Reverse transcription-quantitative PCR (QPCR) and
immunocytochemistry were used for detection of CK19 mRNA and protein.
Breast cancer plasma DNA displayed frequent LOH (31.3%) and MI
(11.6%) supported by the same alteration in microdissected tumor DNA.
Most notably, 10 of the 39 patients with primary breast cancer showed
LOH ( n = 6) or MI ( n = 4). We
compared plasma tumor DNA, plasma and bone marrow QPCR, and blood and
bone marrow immunocytochemistry in 32 of the patients with primary
cancer. Of these, only one patient had immunocytochemically detectable
carcinoma cells in the blood, and three showed abnormally high levels
of plasma CK19 mRNA. All four of these patients had plasma DNA
alterations. We then compared bone marrow findings: of the 10 primary
breast cancers that showed LOH or MI, 6 had elevated CK19 mRNA and 5
had immunocytochemically positive cells. Tumor DNA is readily
detectable in plasma of primary and metastatic breast cancer patients,
and plasma DNA alterations (LOH and MI) reflect those seen in the
tumor. The application of microsatellite analyses to plasma DNA may be
useful in assessing tumor burden in breast cancer patients,
particularly when combined with QPCR, and is preferable for patients
with breast cancer, for whom sequential bone marrow aspiration is
undesirable.
To assess their roles in breast cancer diagnostics, we aimed to compare plasma cell-free DNA (cfDNA) levels with the circulating metabolome in a large breast screening cohort of women recalled for ...mammography, including healthy women and women with mammographically detected breast diseases, ductal carcinoma in situ and invasive breast cancer: the Breast Screening and Monitoring Study (BSMS). In 999 women, plasma was analyzed by nuclear magnetic resonance (NMR) and Ultra-Performance Liquid Chromatography-Mass Spectrometry (UPLC-MS) and then processed to isolate and quantify total cfDNA. NMR and UPLC-MS results were compared with data for 186 healthy women derived from the AIRWAVE cohort. Results showed no significant differences between groups for all metabolites, whereas invasive cancers had significantly higher plasma cfDNA levels than all other groups. When stratified the supervised OPLS-DA analysis and total cfDNA concentration showed high discrimination accuracy between invasive cancers and the disease/medication-free subjects. Furthermore, comparison of OPLS-DA data for invasive breast cancers with the AIRWAVE cohort showed similar discrimination between breast cancers and healthy controls. This is the first report of agreement between metabolomics and plasma cfDNA levels for discriminating breast cancer from healthy subjects in a true screening population. It also emphasizes the importance of sample standardization. Follow on studies will involve analysis of candidate features in a larger validation series as well as comparing results with serial plasma samples taken at the next routine screening mammography appointment. The findings here help establish the role of plasma analysis in the diagnosis of breast cancer in a large real-world cohort.
Abstract
Successful early detection and intervention in cancer is key if we are to drastically improve cancer survivorship. Previous research by us and others has shown that copy number and ...mutational profiling of circulating cell-free DNA (cfDNA) can detect minimal residual disease and predict who will relapse after early stage breast cancer (Shaw et al. Genome Res 2012, doi: 10.1101, Garcia-Murillas et al. Sci Transl Med. 2015 doi: 10.1126). Here we used massively parallel sequencing with the Ion CHEF -S5 and Oncomine™ Breast cfDNA Assay (Thermofisher) for ultrasensitive detection of circulating tumour derived DNA (ctDNA) in 26 asymptomatic early stage breast cancer patients with no scan evidence of distant metastases on follow up after surgery, (all of whom were receiving adjuvant AI (PAI) at the time of blood sampling). We compared these early stage breast cancers with cfDNA isolated from 40 patients with metastatic breast cancer (MBC) and 92 healthy age matched female controls (HC) attending for a routine breast screening mammogram. The Oncomine™ Breast cfDNA Assay targets >150 hotspot mutations in 10 breast cancer genes and uses tag sequencing technology to achieve a limit of detection (LOD) down to 0.1%. One or more driver mutations were detected in cfDNA of 19 (73%) PAIs and 29 (72.5 %) MBCs, whereas just 15 HCs (16%) were positive. Applying this threshold of 1 or more driver mutations for detection of ctDNA (Cohen et al. Science. 2018 doi: 10.1126) there was a significant difference between ctDNA positive cancers and healthy controls, which also remained significant for 2 or more driver mutations (P < 0.0001 and P < 0.001 respectively, Fisher's exact test) (Abbosh et al. Nature 2017, doi: 10.1038). It is notable that >72% of breast cancers were positive with this test, both disease-free women on follow up and patients with MBC, suggesting a role for the test in routine monitoring. A second finding of clinical significance was that variant allele fraction of ESR1 mutant ctDNA was significantly higher in cancers than controls (PAIs than HCs (P = 0.034) and MBCs than HCs (P = 0.002); adjusted P value, Dunn's multiple comparison test) suggesting that ESR1 mutations distinguish disease-free breast cancer survivors receiving adjuvant aromatase inhibitors from normal women. Importantly, we are the first to report ultrasensitive evaluation of ctDNA alterations in a large number of healthy women in a true cancer screening setting. The 15 healthy women that were “positive” for ctDNA may be harbouring an early stage cancer not detectable by imaging. Research is ongoing to validate these results in a larger study, but our results suggest that the approach may have utility for early disease detection and monitoring to enable appropriate intervention at earlier stages.
Citation Format: Jacqui A. Shaw, Karen Page, Daniel Fernandez-Garcia, Allison Hills, Anna R. Boydell, Lindsay Primrose, Bradley Toghill, Robert K. Hastings, Kelly Gleeson, Brenda M. Rosales, Kate Goddard, David S. Guttery, Simak Ali, Raoul C. Coombes. Circulating tumor DNA for early detection and intervention in breast cancer: ctDNA profiles discriminate between healthy women in a true cancer screening setting and disease-free women on follow up abstract. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr LB-224.
We investigated the utility of the Oncomine Breast cfDNA Assay for detecting circulating tumor DNA (ctDNA) in women from a breast screening population, including healthy women with no abnormality ...detected by mammogram, and women on follow-up through to advanced breast cancer.
Blood samples were taken from 373 women (127 healthy controls recruited through breast screening, 28 ductal carcinoma in situ, 60 primary breast cancers, 47 primary breast cancer on follow-up, and 111 metastatic breast cancers MBC) to recover plasma and germline DNA for analysis with the Oncomine Breast cfDNA Assay on the Ion S5 platform.
One hundred sixteen of 373 plasma samples had one or more somatic variants detected across eight of the 10 genes and were called ctDNA-positive; MBC had the highest proportion of ctDNA-positive samples (61; 55%) and healthy controls the lowest (20; 15.7%).
,
, and
mutations account for 93% of all variants detected and predict poor overall survival in MBC (hazard ratio = 3.461; 95% CI, 1.866 to 6.42;
= .001). Patients with MBC had higher plasma cell-free DNA levels, higher variant allele frequencies, and more polyclonal variants, notably in
than in all other groups. Only 15 individuals had evidence of potential clonal hematopoiesis of indeterminate potential mutations.
We were able detect ctDNA across the breast cancer spectrum, notably in MBC where variants in
,
, and
predicted poor overall survival. The assay could be used to monitor emergence of resistance mutations such as in
that herald resistance to aromatase inhibitors to tailor adjuvant therapies. However, we suggest caution is needed when interpreting results from a single plasma sample as variants were also detected in a small proportion of HCs.
Over 30% of ERα breast cancer patients develop relapses and progress to metastatic disease despite treatment with endocrine therapies. The pioneer factor PBX1 translates epigenetic cues and mediates ...estrogen induced ERα binding. Here we demonstrate that PBX1 plays a central role in regulating the ERα transcriptional response to epidermal growth factor (EGF) signaling. PBX1 regulates a subset of EGF-ERα genes highly expressed in aggressive breast tumours. Retrospective stratification of luminal patients using PBX1 protein levels in primary cancer further demonstrates that elevated PBX1 protein levels correlate with earlier metastatic progression. In agreement, PBX1 protein levels are significantly upregulated during metastatic progression in ERα-positive breast cancer patients. Finally we reveal that PBX1 upregulation in aggressive tumours is partly mediated by genomic amplification of the PBX1 locus. Correspondingly, ERα-positive breast cancer patients carrying PBX1 amplification are characterized by poor survival. Notably, we demonstrate that PBX1 amplification can be identified in tumor derived-circulating free DNA of ERα-positive metastatic patients. Metastatic patients with PBX1 amplification are also characterized by shorter relapse-free survival. Our data identifies PBX1 amplification as a functional hallmark of aggressive ERα-positive breast cancers. Mechanistically, PBX1 amplification impinges on several critical pathways associated with aggressive ERα-positive breast cancer.
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 registered 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(TM)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 mu l of plasma. miRNA profiles showed significant variation with increasing time before centrifugation (p<0.001) and increasing centrifugation force, with depletion of platelet associated miRNAs, whereas cfDNA was unaffected. However, sample replicates showed excellent reproducibility on TaqMan low density arrays ( rho = 0.96, p<0.0001). We also successfully generated miRNA profiles for plasma samples stored > 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.
Celotno besedilo
Dostopno za:
DOBA, IZUM, KILJ, NUK, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK
An in situ hybridization technique has been developed for assessing poly(A)+ RNA preservation in routine pathology specimens. The method detects poly-adenylated RNA sequences in tissue sections using ...a biotinylated polydeoxythymidine (poly d(T)) probe. The probe was prepared from single-stranded 25-30 base oligo d(T) and was biotinylated using the enzyme terminal deoxynucleotide transferase with biotin-11-dUTP and dTTP in the ratio 1:4. The hybridization protocol uses varying concentrations of proteinase K to unmask mRNA sequences and the biotin-labelled hybrids are demonstrated after hybridization under standard conditions by the application of streptavidin and biotinylated alkaline phosphatase. Alkaline phosphatase was visualized using a Fast Red naphthol-capture method and the sections were counterstained with haematoxylin. The results have confirmed that the method is specific for poly(A)+ RNA and shows that poly(A)+ RNA can be demonstrated in routine formalin-fixed sections using non-radioactive techniques with retention of morphology. It also provides a means of optimizing the hybridization conditions for specific mRNA probes and produces a staining pattern demonstrating the relative level of poly(A)+ RNA per cell which may reveal new information about cell activity and tissue function.
An in situ hybridization technique has been developed for the detection of immunoglobulin light chain mRNA in routine pathology specimens. The method detects kappa or lambda constant region sequences ...using a cocktail of synthetic oligonucleotide probes labelled with biotin or fluorescein 5-isothiocyanate (FITC) reporter molecules. The probes were labelled at flanking sites chemically by primary amine directed acylation and by 'homopolymer tailing' with terminal deoxynucleotidyl transferase using non-radioactive nucleotide analogues. The mRNA was unmasked in the formalin-fixed tissue sections by digestion with varying concentrations of proteinase K, and the hybrids were demonstrated using alkaline phosphatase with either a streptavidin/biotin based four-stage system or an anti-FITC antibody based detection system. Alkaline phosphatase was visualized using a Fast Red naphthol-capture method and the sections were counterstained with haematoxylin. The results confirm that the method is specific for kappa or lambda mRNA and show that specific mRNAs can be detected in routine formalin-fixed sections using non-radioactive techniques with retention of good morphology. The method reliably detects light chain mRNA in cells expressing secretory immunoglobulin. The protocol can also be applied to tissue rich in endogenous biotin by using hapten-labelled probes.
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