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Embed code for: Next Generation Sequencing in Chronic Myeloid Leukemia
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Improving therapy for Chronic Myeloid Leukemia
The impact of Next-Generation Sequencing
Chronic myeloid leukemia
15-20% of all leukemias
Average age of onset – 50-60 years
Unregulated and increased growth of myeloid cells in the bone marrow
Philadelphia chromosome - t(9;22)(q34;q11)
Proto-oncogene ABL1 (chromosome 9)
Breakpoint cluster region BCR gene (chromosome 22)
3 main BCR-ABL1 fusion gene hybrids encode BCR-ABL1 protein isoforms p210, p190, and p230
Imatinib – binds to the TK through ATP binding domain
Stabilizes the inactive, non-ATP binding form of BCR-ABL, thereby preventing tyrosine autophosphorylation
High degree of specificity for the ATP binding site
Chronic Myeloid Leukemia from a Historical Perspective
Rous Sarcoma Virus (1911)
Protein Phosphorylation (1933)
Tyrosine kinases (1979)
in CML (1960)
Current challenges in CML research
Choice of therapy – mutations that lead to drug resistance
Minimal Residual Disease( MRD) and discontinuation of therapy
The future of monitoring patients:
The development of efficient methods which facilitate fast, inexpensive and sensitive BCR-ABL1 quantization in emerging economic regions
Quantization of low levels of BCR-ABL1 to assist detection and monitoring of MRD
Sensitive and accurate detection of mutations that drive resistance to TKI therapy
The discovery of molecular markers that predict response to therapy or disease progression
Challenges in treatment choice
Standard therapy – Tyrosine Kinase Inhibitors (TKI)
20%-40% of patients present intolerance or resistance to first-line treatment, imatinib
Second line TKI –nilotinib, dasatinib
Third line TKI – ponatinib (most effective in patients with T351I mutation)
Compound mutations -two or more mutations present in the same molecule
Polyclonal mutations refer to mutations present in different molecules
Low-level mutations are detected only by sequencing of individual PCR products, not by Sanger Sequencing (SS)
Multiple mutations detected by SS – 11.4%
NGS – detects more compound mutations; detects low level mutations
Sanger Sequencing and its limitations
It cannot robustly identify mutated populations
10% to 15%, it provides only rough estimates of mutated clone abundance
It cannot discriminate between polyclonal and compound mutations
The information it provides may not be sufficient to predict treatment response
What can be achieved by Next Generation Sequencing?
NGS is one of the most promising technologies in detecting resistance mutations.
Ability to distinguish compound mutations from PCR-artifacts and polyclonal mutations and specific detection of true compound mutations.
Ultra-deep sequencing (UDS) = sequencing of a nucleotide position multiple times, achieving high sensitivity, which allows for
(1) full characterization of minor mutated variants;
(2) the ability to follow the dynamics of resistant mutations over time;
(3) reconstruction of the clonal architecture of mutated populations in the case of multiple mutations occurring within the same amplicon.
The switch from a TKI to another determined the dominant mutation population.
Evolution of BCR-ABL–positive cells is mainly shaped by:
The “absolute” fitness
The “relative” fitness
We found that conventional Sanger sequencing had misclassified or underestimated BCR-ABL mutation status in 55% of the samples, where mutations with 1% to 15% abundance were detected.
NGS identified more mutations
A substantial proportion of cases was found not to harbor any mutation
Other mechanisms of molecular disease persistence might be involved
A long- range NGS approach permitting massively parallel sequencing of the entire TKD length of 933bp in a singe read
LR-NGS technique detection limit is identical to that reported for the more laborious and costly four-fragment NGS approach
Permits reliable and more economic assignment of mutations
Potential method of choice for mutational screening
In 43% patients in Group 1, the 2GTKI-resistant mutations that triggered relapse were already detectable at low levels in the switchover sample.
All the low level dasatinib-or nilotinib-resistant mutations detected by NGS at switchover expanded whenever the patient happened to receive the 2GTKI to whom they were insensitive.
BCR-ABL1 KD mutation status may be much more complex than SS shows
NGS at the time of imatinib failure reliably identifies clinically relevant mutations
An NGS-based mutation screening -> better therapeutic decisions
The setting in which the NGS could be most useful: Ph+ leukemia patient has to be shifted to second-line therapy
Using WGS novel BCR-ABL1 fusion gene with breakpoints in the BCR intron 14 and the ABL1 intron 2
Fusion protein – compromised SH3 domain altered drug response and unique clinical manifestations
Is NGS the path for improving patient management and therapy choice?
NGS can achieve much more than conventional approach
The identification of certain mutations has an impact on treatment choice
It is an emerging technology for overcoming treatment resistance
However, the cost-efficiency has to be further investigated
Current Developments in Molecular Monitoring in Chronic Myeloid Leukemia, Therapeutic Advances in Hematology. 2016
A novel BCR-ABL1 fusion gene identified by next-generation sequencing in chronic myeloid leukemia, Molecular Cytogenetics, 2016
The Philadelphia chromosome in leukemogenesis, Chinese Journal of Cancer, 2016
What challenges remain in chronic myeloid leukemia research?
Tyrosine Kinase Inhibitors for the Treatment of Chronic-Phase Myeloid Leukemia: Long-Term Patient Care and Management, J Adv Pract Oncology, 2016
BCR-ABL Mutations in Chronic Myeloid Leukemia Patients with Failure and Warning to First and Second-Line Tyrosine Kinase Inhibitor Therapy: What Is the Advantage of Next-Generation Sequencing over Conventional Sequencing?, Blood Journal, 2015
Next-generation sequencing for senzitive detection of BCR-ABL1 mutations relevant to tyrosine kinase inhibitor choice in imatinib-resistant patients, Oncotarget, 2016
Unraveling the complexity of tyrosine kinase inhibitor-resistant populations by ultra-deep sequencing of the BCR-ABL kinase domain, Bloog Journal, 2014
http://www.bloodjournal.org/content/122/21/384?sso-checked=truef minor mutated variants;
LR-NGS technique detection limit is identi