Liquid Biopsies - ctDNA Flashcards
1
Q
What is a Biopsy?
A
A biopsy is a medical test commonly performed by a surgeon involving extraction of sample cells or tissues for examination
A “Primary biopsy” is the gold standard for cancer diagnosis
2
Q
What are the limitations of taking primary biopsies in cancer patients?
A
They have limited effectiveness as diagnostic tool because;
- Invasive nature means cannot easily take repeated samples
- Doesn’t enable early detection
- Vulnerable to tumour heterogeneity i.e. could miss lesions if non cancerous section of organ is biopsied
- Expensive and time-consuming
- Historically processed in FFPE is bad for DNA analysis
3
Q
What is a Liquid Biopsy?
A
- A liquid biopsy is a non-invasive alternative to a surgical biopsies which enables the identification of information about a tumour
- Often a simple blood sample but could also be other non-invasive specifiment e.g. urine.
- Traces of the cancer’s DNA in the blood can give clues about which treatments are most likely to work for that patient
4
Q
What is the potential clinical impact of the liquid biopsy in cancer care?
A
Cancers are often detected at a late stage
- LB could function as a vastly improved screening test
- to detect typically terminal malignancy at an earlier, potentially curable stage
- LB could enable monitoring cancer progression in real time
- to avoiding the significant morbidity and cost of repeat tissue biopsies
5
Q
What is the origin of the traces of tumour DNA in the blood steam?
A
- Fragments of DNA are constantly shed into the bloodstream during cell death, but the levels of cell-free DNA are kept relatively low due to the rapid clearance by the liver, kidney, and spleen.
- Two mechanisms lead to release of ctDNA into the bloodstream
- Passive: by necrotic cancer cells
- Active: actively released by tumour to condition target cellular niches at distant locations throughout the body
6
Q
Why do cancer patients have a higher quantity of cfDNA in their curculation?
A
- Fragments of DNA are constantly shed into the bloodstream during cell death, but the levels of cell-free DNA are kept relatively low due to the rapid clearance by the liver, kidney, and spleen.
- In general, patients with cancer have significantly higher levels of cell-free DNA as compared to healthy individuals because tumors tend to have elevated cell turnover rates and a l_arge number of necrotic cells_ relative to normal tissue
7
Q
What factors affect the rate of shedding of cfDNA by a tumour?
A
Rate of shedding affected by the tumour;
- location
- size
- vascularity
Leads to variability in levels across patients
8
Q
What other non-cancer causes can lead to increased cfDNA in the plasma?
A
infectious and autoimmune diseases
stroke
infarction
trauma
Pregnancy
9
Q
What are the properties ctDNA?
A
- Very low concentrations
- Highly fragmented
- Vast majority if fragments <150bp
- Tumour-specific methylation markers
- half-life of less than two hours
10
Q
What information can be gained from assessing the fragmentation of cfDNA?
A
Fragmentation pattern can reflect the tumor biology and stage;
- Apoptosis, which usually takes place in normal tissues, results mainly in DNA fragments of 180 bp
- Solid cancers, tumor necrosis creates a spectrum of DNA fragments due to the random digestion by nucleases
- Ratio of long:short ctDNA fragments (“DNA integrity”) was shown significantly correlate with colorectal cancer progression
11
Q
What is the clinical utility of methylation profiling of ctDNA?
A
- Methylation profiling of ctDNA provides another potential biomarker for cancer screening and surveillance
- Differential methylation levels of three promoters
- RASSF1A, CALCA, and EP300
- Detected ovarian cancer from healthy controls with
- sensitivity of 90%
- specificity of 86.7%
12
Q
What are the applications of liquid biopsies?
A
- Disease detection (potentially early)
- Future cancer screening programme (Aravanis et al 2017)
- Prognosis
- ctDNA correlates with poor prognosis in CRC (Fan et al 2017)
- Selection of correct therapy
- RAS mutation detection for anti-EGFR therapy
- Understanding mechanism of resistance
- Disease monitoring
- Detection of residual disease after BrCa surgery and likely relapse
- progression over time (MRD monitoring)
13
Q
What technologies underpin liquid biopsy?
A
- PCR based – Low cost and sensitivity
- Digital PCR – Detects known point mutations in ctDNA at low allele fractions
- NGS – Plasma DNA analysis, high throughput, detects chromosomal rearrangements and CNVs, though requires high coverage
- CellSearch system, FDA approved kit for enumeration of CTCs
- BEAMing
14
Q
What is the concordance between mutations detected in the tumour and via ctDNA analysis?
A
- In studies pairing plasma and tumor tissue, there was >80% concordance in tumor DNA aberrations
- Some results suggesting that the blood sample provided a more complete tumor profile than the tissue biopsy due to heterogeneity within primary tumors and between metastatic sites
15
Q
What challenges associated with liquid biopsies?
A
- Is ctDNA representative of all relevant metastatic clones located at different sites?
- Does ctDNA only it represent distinct sub-clones?
- Standardisations of pre-analytical procedures
- Collection of samples, storage, extraction, costs
- Standardisations of Analytical procedures
- Techniques, data analysis
16
Q
Early challenges of liquid biopsy?
A
Low quantity of ctDNA was not reliably detectibly with early techniques limiting clinical impact.
17
Q
What is the standard clinical practice for WGS of tumours and what are the liitations to this method?
A
- Paired tumor tissue/normal tissue sequencing from biopsy
- The fraction of tumor cells relative to normal cells in each biopsy can be varied
- Sampling of a single tumor region further limits the comprehensiveness of cancer WGS
- Intratumoral and intermetastatic tumor heterogeneity potentially leads to an incomplete picture
- ctDNA can overcome these barriers
18
Q
What is the most likely explanation for discordance between tumour WGS and ctDNA WGS?
A
Discordance is likely the result of a l_ack of sensitivity from tissue biopsies_
i.e. intratumoral and intermetastatic heterogeneity
Perkins et al. performed a large concordance study to assess this
19
Q
How does ctDNA compare to CTC as an analyt for liquid biopsy?
A
- A major hurdle in CTC analysis is a limited presence in the bloodstream constituting as few as one cell per 1 × 10^9.
- Their isolation is technical very difficult/expensive and may require lots of blood to be taken.
- In a recent trial to detect KRAS in lung cancer;
- ctDNA = sensitivities of 96%
- CTCs = sensitivities of 52%
20
Q
How does ctDNA compare to protein biomarkers e.g. cancer antigens?
A
Currently several protein biomarkers used to help detect malignancy and assess a therapeutic response;
- Prostate = PSA
- Pancreatic = CA19-9
- Gastrointestinal = CEA
- Ovarian cancers = CA-125
- Breat cancer = CA15-3
Generally perform poorly as screening assays and evidence mounting that ctDNA is more accurate
- Study in metastatic breast cancer detection of ctDNA in 85% vs. only CA 15-3 in 59% of patients
21
Q
How can ctDNA facilitate personalised cancer treatment?
A
- Personalized cancer therapy is the treatment of cancer based on the tumor’s unique genetic makeup
- Defining these mutations through a ctDNA liquid biopsy holds particular promise for prescribing personalized tumor therapy in cases in which;
- tumor heterogeneity might not be fully represented with tissue biopsy
- when a specimen is insufficient for all testing desired
drugs designed to interfere with the hyper-growth signals of specific driver mutations
22
Q
Can ctDNa be used a marker for Prognosis and Relapse risk?
A
- With its half-life of less than two hours, ctDNA is dynamic and can be used to discover changes in evolving tumor genomes in real time.
- Early-stage, non-metastatic breast cancer patients underwent ctDNA testing
- Positive ctDNA assays post-operatively signaled MRD, and these patients were more than four times as likely to relapse as patients with undetectable post-surgery ctDNA
- Serial ctDNA testing every six months further predicted recurrence;
- 93% ctDNA+ relapsed
- 10% ctDNA- relapsed
- ctDNA has outperformed many CA for detecting MRD and predicting relapse after surgery
23
Q
Can ctDNa be used a marker for resistance?
A
- Mutation burden tends to increase with serial ctDNA testing after relapse
- The mutation spectrum more closely matches the relapsed tumor biopsy DNA sequence than the pretreatment tumor sequence
- Thus, ctDNA profiling has clear potential not only for the prioritization of initial therapy but also for suggestion of second line therapeutics, e.g’s
- KRAS mutations promote resistance to EGFR-targeted therapies
- MEK1 activating mutations can be blocked with the MEK inhibitor, trametinib
24
Q
What alternative specimens can be used for ctDNA analysis?
A
- Central nervous system malignancies have been difficult to detect in the blood stream but are more readily detected in cerebrospinal fluid.
- Urine cfDNA exceeds plasma sensitivity in studies of renal, bladder, and prostate cancer, but surprisingly also in some series of lung and colon cancers.
- Tumor DNA has been detected in saliva, bronchoalveolar washings, pleural fluid, ascites, endocervical samplings, and stool.
- One can envision cell-free DNA diagnostics using the body fluid most proximate to a tumor site or even a pan-fluid screening assay, as test sensitivities continue to improve.
25
Are there any FDA approved liquid biopsy testscurrently available?
* Roche’s ctDNA-based test was the first liquid biopsy to garner FDA approval.
* EGFR mutations in lung cancer patients
* It is a high-specificity companion diagnostic for erlotinib
* Eliminates the need for EGFR tissue testing when this blood test is positive
26
Give examples of other non-FDA approved liquid biopsy tests
* Diagnostic labs are beginning ot offer 'panel tests' for a range of actionable cancer mutations e.g. Pathway Genomics CancerInterceptTM Detect
* Con's are that requires lab testing which can be slow, onerous
* POC devise manufacturers are also making products targetted at specific mutations to provide quick results at the bedside
* BioCartis IdyllaTM ctBRAF Mutation assay
* Assesses BRAF V600E/E/DK/R/M mutations from plasma directly pipetted into a proprietary cartridge and system. 85-130 minutes from blood draw to report.
* FDA currently assessing the validity of these tests
27
What are the key limitations of liquid biopsy?
* The quantity of ctDNA is the main limitation.
* Very low levels of mutated DNA can show as false-positive results, when the occasional DNA aberrancy does not represent a cancer clone, and as false-negative results when the level is below assay detection limits.
* The need to confirm the tissue of origin will limit liquid biopsy’s complete replacement of tumor biopsy
* The control of different parameters in all steps—from blood drawing to ctDNA analysis—has a significant impact on the quality and accuracy of the data - thus the current lack of standardisation is a limitation e.g.
* A recent evaluation into the feasibility and effectiveness of ctDNA in a large clinical study concluded that it resulted in low sensitivity and a low positive predictive value
* Which was most likely due to the diversity of settings in which the liquid biopsy was employed, and to the various in-house laboratory techniques used to test for the EGFR mutation
28
What are the major benefits to patient care delivered by ctDNA?
* The minimally invasive nature of ctDNA enables profiling without the delay, cost, and risk associated with tissue biopsy
* Early metastatic detection potentially at a microscopic stage before radiologic detectability.
* For cancers detected at a late stage a ctDNA assay could detect a typically terminal malignancy at an earlier, more treatable, even curable stage.
* Potential of dynamic monitoring of therapy response, early detection of resistance, and knowledge of tumor recurrence even months before clinical relapse .
29
What future developments are likely to come in ctDNA diagnostics?
* Researchers are examining circulating RNA as a potentially improved cancer profile
* DNA methylation for its ability to signal other types of tissue damage such as the destruction of pancreatic islet cells in type 1 diabetes mellitus or oligodendrocytes in relapsing multiple sclerosis
* New applications for liquid biopsy may be the detection of circulating extracellular vesicles or exosomes secreted by cancer cells
* Continued improvement in performance and the design of trials based on new driver mutations
