Liquid biopsies, which analyze cell-free DNA in blood, can be used to study cancer evolution and drug resistance over time. By tracking genetic mutations in plasma samples taken at different time points, researchers can create a temporal map of how a tumor changes with and without treatment. This approach has identified biomarkers of drug resistance and shown that tumors with higher levels of circulating tumor DNA have worse outcomes and are less responsive to some therapies. While liquid biopsies hold promise for early cancer detection and precision medicine, more large clinical studies are still needed to validate their clinical utility.
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Using liquid biopsies to study cancer dynamics and drug resistance
1. Using liquid biopsies
to study cancer
dynamics and drug
resistance
Alessandro Romanel, PhD
Laboratory of Bioinformatics and Computational Genomics
Centre for Integrative Biology (CIBIO), University of Trento
Speck&Tech, 27 February 2018
2. What is the human genome?
Complete set of nucleic acid sequences for humans
Encoded as DNA
8. What is a DNA variation?
Less than 1% of the human genome
Single Nucleotide Variations (SNVs)
Copy Number Variations (CNVs)
Germline variations
Polymorphism when fraction >1%
Mutation when <1%
Somatic variations
Bob: ACGTGGCATACCAATACCTGGTGTAAGTTTA
Alice: ACGTGGCATACCGATACCTGGTGTAAGTTTA
Bob:
Alice:
9. What is cancer?
Disease where abnormal cells divide without control
Most cancers start due to somatic variations
Gene functions are altered (e.g. oncogenes)
Primary
carcinoma
lymphoma
leukemia
sarcoma
Metastatis
spread
10. Cancer is heterogeneus
Patient 1
Patient 2
Patient 3
Primary Metastasis BMetastasis A
Mutation 2
Mutation 1
Mutation 4
Mutation 3
Intra-patientInter-patient
11. Clonality of mutations
Clonal mutation
Mutation 2
Mutation 1
Mutation 4
Mutation 3
Primary or metastasis Subclonal mutation
12. Cancer evolution and drug
resistance
Prostate cancer (PCa)
>1M cases worldwide, strongly heritable (57%)
respond to
castration
(ADT)
metastatic
PCa
Tumorvolumeandactivity
CRPC
13. How to study cancer
evolution and resistance?
Collect and evaluate sequential samples over disease
Collection of repeated tumor tissue biopsies is challenging and
may not reflect real heterogeneity
18. Targeted sequencing panel
Select genomic regions of interest
Genes involved in the disease
Exonic/Coding regions
Informative SNPs
Other non-coding regions
Include genes that are frequently
aberrant in the disease
19. Targeted sequencing panel
exons
DNA
Standard textual formats (BED files) to store this information
ACGGGTCGGAAATGTGCGATGTCCGATGTCGATGTGGCCCCGATGTCCGATGTCGATGTCCGATGTCGATGTG
Panel design
21. Generate/preprocess data
FASTQ files
Quality Control
Alignment to human reference genome
Removal of duplicates
INDEL realignment
Recalibration
QC and data cleaning
BAM files
Library preparation
23. What is tumor DNA fraction?
Romanel A*, Gasi Tandefelt D*, et al, Science Transl Med 2015
Normal DNA
Tumor DNA
24. Learning from control samples
99.63%
99.63%
0.020.0150.01
Noise estimated from control samples
Somatic SNVs detection Somatic CNVs detection
The local total coverage is >= 100
The alternative base is supported by at least 5 reads
The allelic fraction (AF) is > 0.02 (estimated from control samples)
Exclusion of all genomic positions close to amplicon edges
Exclusion of all positions not satisfying strand bias criteria
The allelic fraction of the position for the control samples is <0.01
25. What is tumor DNA fraction?
Romanel A*, Gasi Tandefelt D*, et al, Science Transl Med 2015
TOTAL DNA ~ 90 ng/ml
TUMOR FRACTION 4-fold higher
Normal DNA
Tumor DNA
27. Patient Time
point
Observed allelic
fraction of TP53
mutation
Tumor DNA
fraction
Percentage of
tumor
molecules
harboring
TP53
mutation
Rossi 1 10% 1 10%
Rossi 2 10% 0.2 50%
Rossi 3 5% 0.5 100%
Verdi 1 50% 0.7 71%
Any time we need to compare multiple plasma samples,
either longitudinal or cross-sectional
Why is important?
28. Normal cell
Tumor cell 1
Maternal
Paternal
Maternal
Paternal
Informative SNPs
reference
alternative base
Allelic Fraction
Proportion of reads
supporting the
reference base
SNP1 SNP2 SNP3
Allelic fraction property
29. gene A gene B
Baca S et al, Cell 2013
Prandi D et al, Genome Biology 2014
Tumor fraction estimation
Clonal Subclonal
30. Is an informative biomarker
Romanel A*, Gasi Tandefelt D*, et al, Science Transl Med 2015
Tumor fraction is a biomarker
Advanced patients with high tumor DNA fraction live less and
some treatments are less effective
35. Create a temporal map
Carreira S*, Romanel A*, et al, Science Transl Med 2014
Identify mechanisms or biomarkers of drug resistance
36. Mutations with temporal relationship with progression
Carreira S*, Romanel A*, et al, Science Transl Med 2014
Romanel A*, Gasi Tandefelt D*, et al, Science Transl Med 2015
Drug resistance biomarkers
AR
41. Current limitations
Many tumors lack well-establish biomarkers
Heterogeneity adds a level of complexity in
liquid biopsy test establishment
Biomarkers for different tumors at different stages
DNA in blood is truly representative of all
tumors?
Can liquid biopsies improve cancer survival?
Large studies and clinical trial needed
Precision/recall of aberration identification
Advances in technology
Improvement of computational methods
42. Laboratory of Functional and
Computational Oncology
Francesca Demichelis
POST-DOC positions
Contact: f.demichelis@unitn.it
OPEN POSITIONS at CIBIO