Prima

NGS Based IRMA

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Prima

Chronic Myeloid Leukemia and Imatinib

Chronic Myeloid Leukemia (CML) accounts for 15%–20% of all adult leukemia. The classical Philadelphia chromosome t(9;22)(q34;q11) which is a diagnostic marker for CML, results in the fusion of N-terminal region of BCR gene with the C-terminal kinase domain of ABL1 gene that produces a constitutively active chimeric protein kinase responsible for leukemogenesis in CML.Chronic Myeloid Leukemia (CML) accounts for 15%–20% of all adult leukemia. The classical Philadelphia chromosome t(9;22)(q34;q11) which is a diagnostic marker for CML, results in the fusion of N-terminal region of BCR gene with the C-terminal kinase domain of ABL1 gene that produces a constitutively active chimeric protein kinase responsible for leukemogenesis in CML.

Imatinib, a targeted tyrosine kinase inhibitor, is the current therapy of first choice for patients suffering from CML. However, many patients who initially respond to Imatinib have been shown to relapse after a period due to the development of resistance. The most common mechanism of resistance to Imatinib is due to mutations in the ABL1 kinase domain that affect the ability of Imatinib to bind to the active site.

Why is testing for mutations causing Imatinib resistance important?

Over 130 mutations have been reported in Imatinib resistant patients, of which most frequent variations attributed to the resistance include, M237I, M244V, L248V, G250E, Q252H, Y253F/H, E255K/V, D276G, E279K, V299L, F311I/L/V, T315I, F317L, M351T, E355G, F359V/C, V379I, L384M, M388L, H396R/P, S417Y/T, E450G, E453K/V/D, E459L/K/G, F486S [1-4]. The T315I mutation appears to confer resistance to multiple targeted tyrosine kinase inhibitors, while other mutations may be more responsive to other therapies [1]. Identifying the mutation status of ABL1 kinase domain is highly valuable in determining the therapeutic strategy for each patient, by increasing Imatinib dose, switching to second generation inhibitors as Dasatinib or Nilotinib or alternate treatment approaches.

Why is NGS based IRMA (Imatinib Resistance Mutation Analysis) important

Why is NGS based IRMA (Imatinib Resistance Mutation Analysis) important
  • Complete characterization of the spectrum of minor (<20%) mutated variants
  • The ability to follow the dynamics of resistant mutations over time
  • The ability to follow the dynamics of resistant mutations over time
  • Reconstruction of the clonal architecture of mutated populations in the case of multiple mutations occurring within the same amplicon
  • Early detection with high sensitivity
  • Reliable detection of emerging BCR-ABL1 mutations
  • The ability to detect all resistant mutations and not just hotspots
Assay characteristics NGS Sanger sequencing Real-time PCR(RT-PCR)
Throughput High Low Medium
Sensitivity of mutation detection (LOD) >1% >20% >5%
Ability to detect novel mutations Yes Yes No
Identification of InDels Yes Yes Yes
Distinguish between compound mutations Yes No No
Identification of Polyclonal mutation Yes No No
Quantification of mutation burden Yes No No

Test Details

Test Code: MGM198: Imatinib Resistance Mutation Testing by NGS

Specimen requirement:
  • EDTA anticoagulated peripheral blood or 1μg of RNA
  • Minimum 3ml of peripheral blood is required

It is critical that the transportation temperature of peripheral blood is maintained at 2-8° C. For RNA samples, temperature should be maintained at -40° C for the dry ice shipment. Samples should reach the laboratory within 48 hours of collection.

Turnaround Time (TAT)

The test results are provided to the clinician within 10 working days of sample receipt

Validation Results

Sample type Mutation type Sensitivity Specificity Accuracy Limit of detection
Peripheral blood RNA SNV / Short-Indels 100% 100% 100% ≥1%

References

  1. 1. Soverini, Simona, et al. "Bcr-Abl kinase domain mutation analysis in chronic myeloid leukemia patients treated with tyrosine kinase inihibitors: recommendations from an expert panel on behalf of European LeukemiaNet." Blood (2011): blood-2010
  2. 2. Gorre, Mercedes E., et al. "Clinical resistance to STI-571 cancer therapy caused by BCR-ABL gene mutation or amplification." Science 293.5531 (2001): 876-880.
  3. 3. Chaitanya, Puligundla Krishna, et al. "The role of mutation testing in patients with chronic myeloid leukemia in chronic phase after imatinib failure and their outcomes after treatment modification: Single-institutional experience over 13 years." Indian journal of medical and paediatric oncology: official journal of Indian Society of Medical & Paediatric Oncology 38.3 (2017): 328.
  4. 4. Nardi, Valentina, Mohammad Azam, and George Q. Daley. "Mechanisms and implications of imatinib resistance mutations in BCR-ABL." Current opinion in hematology 11.1 (2004): 35-43.
  5. 5. Soverini, Simona, et al. "Implications of BCR-ABL1 kinase domain-mediated resistance in chronic myeloid leukemia." Leukemia research 38.1 (2014): 10-20
  6. 6. Soverini, Simona, et al. "Unraveling the complexity of tyrosine kinase inhibitor-resistant populations by ultra-deep sequencing of the BCR-ABL kinase domain." Blood (2013): blood-2013
  7. 7. Cavelier, Lucia, et al. "Clonal distribution of BCR-ABL1 mutations and splice isoforms by single-molecule long-read RNA sequencing." BMC cancer 15.1 (2015): 45
  8. 8. Polakova, Katerina Machova, et al. "Next-generation deep sequencing improves detection of BCR-ABL1 kinase domain mutations emerging under tyrosine kinase inhibitor treatment of chronic myeloid leukemia patients in chronic phase." Journal of cancer research and clinical oncology 141.5 (2015): 887

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