Liquid Biopsies - ctDNA

Question 1

What is a Biopsy?

Answer 1

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

Question 2

What are the limitations of taking primary biopsies in cancer patients?

Answer 2

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

Question 3

What is a Liquid Biopsy?

Answer 3

• 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

Question 4

What is the potential clinical impact of the liquid biopsy in cancer care?

Answer 4

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

Question 5

What is the origin of the traces of tumour DNA in the blood steam?

Answer 5

• 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

1. Passive: by necrotic cancer cells
2. Active: actively released by tumour to condition target cellular niches at distant locations throughout the body

Question 6

Why do cancer patients have a higher quantity of cfDNA in their curculation?

Answer 6

• 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

Question 7

What factors affect the rate of shedding of cfDNA by a tumour?

Answer 7

Rate of shedding affected by the tumour;

1. location
2. size
3. vascularity

Leads to variability in levels across patients

Question 8

What other non-cancer causes can lead to increased cfDNA in the plasma?

Answer 8

infectious and autoimmune diseases

stroke

infarction

trauma

Pregnancy

Question 9

What are the properties ctDNA?

Answer 9

• Very low concentrations
• Highly fragmented
  
  • Vast majority if fragments <150bp
• Tumour-specific methylation markers
• half-life of less than two hours

Question 10

What information can be gained from assessing the fragmentation of cfDNA?

Answer 10

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

Question 11

What is the clinical utility of methylation profiling of ctDNA?

Answer 11

• 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%

Question 12

What are the applications of liquid biopsies?

Answer 12

• 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)

Question 13

What technologies underpin liquid biopsy?

Answer 13

1. PCR based – Low cost and sensitivity
2. Digital PCR – Detects known point mutations in ctDNA at low allele fractions
3. NGS – Plasma DNA analysis, high throughput, detects chromosomal rearrangements and CNVs, though requires high coverage
4. CellSearch system, FDA approved kit for enumeration of CTCs
5. BEAMing

Question 14

What is the concordance between mutations detected in the tumour and via ctDNA analysis?

Answer 14

• 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

Question 15

What challenges associated with liquid biopsies?

Answer 15

• 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

Question 16

Early challenges of liquid biopsy?

Answer 16

Low quantity of ctDNA was not reliably detectibly with early techniques limiting clinical impact.

Question 17

What is the standard clinical practice for WGS of tumours and what are the liitations to this method?

Answer 17

• 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

Question 18

What is the most likely explanation for discordance between tumour WGS and ctDNA WGS?

Answer 18

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

Question 19

How does ctDNA compare to CTC as an analyt for liquid biopsy?

Answer 19

• 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%

Question 20

How does ctDNA compare to protein biomarkers e.g. cancer antigens?

Answer 20

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

Question 21

How can ctDNA facilitate personalised cancer treatment?

Answer 21

• 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

Question 22

Can ctDNa be used a marker for Prognosis and Relapse risk?

Answer 22

• 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

Question 23

Can ctDNa be used a marker for resistance?

Answer 23

• 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

Question 24

What alternative specimens can be used for ctDNA analysis?

Answer 24

• 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.

Question 25

Are there any FDA approved liquid biopsy testscurrently available?

Answer 25

• 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

Question 26

Give examples of other non-FDA approved liquid biopsy tests

Answer 26

• 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

Question 27

What are the key limitations of liquid biopsy?

Answer 27

• 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

Question 28

What are the major benefits to patient care delivered by ctDNA?

Answer 28

• 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 .

Question 29

What future developments are likely to come in ctDNA diagnostics?

Answer 29

• 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