BCR/ABL Testing Guidelines for CML

· Sample Processing:

· 5 mL of PB is adequate at diagnosis but a minimum of 10–20 mL is recommended for follow-up samples to achieve adequate sensitivity

· Although there is no rigorous published data to indicate the acceptable time interval between taking the sample and laboratory processing, it is generally accepted that samples should be processed as soon as possible

· In our experience, samples must be processed by the laboratory within, and not later than, 72 h from collection.

· Processing samples older than 72 h should only be considered for pre-treatment samples to establish BCR-ABL1 transcript type

· Even pre-treatment samples that are more than 5 days old should be discarded and a repeat sample requested

· Investigations at diagnosis (CCGM-CML recommendations):

· Baseline bone marrow cytogenetics (karyotype)

· Peripheral blood q-RT-PCR

· Cytogenetic response (minimum 20 metaphases):

· Complete – no Ph-positive metaphases

· Partial – 1-35% Ph-positive metaphases

· Major – complete or partial

· Minor - > 35% Ph-positive metaphases

· Molecular response:

· Complete molecular response – BCR-ABL mRNA undetectable by qPCR

· Major molecular response (MMR) - >= 3-log reduction of BCR-ABL mRNA

· A 3-log reduction corresponds to 1 malignant cell in a background of 1000 normal cells and has been called a ‘‘major molecular response.’’

· “Deep molecular response” – MR4.0; <= 0.01% BCR-ABL1 IS) (NCCN 1.2019)

· Requires a reliable qPCR test with a sensitivity of detection of at least MR4.5 (<=0.0032% IS

· (MR4, MR4.5, MR5) [Cross et al Leukemia (2015) 29, 999–1003]

· “Stable molecular response” – Deep MR for >= 2 years as documented by at least 4 tests (NCCN 1.2019)

· One of the criteria for TKI discontinuation

· “Complete molecular response (CMR) – variably described, and is best defined by the assay’s level of sensitivity (eg. MR4.5) (NCCN 1.2019)

· Calculating IS from raw data:

· ((sum of BCR-ABL1 copies)/(sum of reference gene copies)) × CF × 100 [Cross et al Leukemia (2015) 29, 999–1003]

· CF is the lab-specific conversion factor, determined by sample sharing with a lab that already has an established CF

· Note that undetectable result should have a minimum estimated control gene copy number (see Cross et al.)

· BCR-ABL1 ratios are expressed as a percentage (%) as follows: BCR-ABL1 molecules divided by the total number of ABL1 molecules and multiplied by 100; For example: BCR-ABL1: 4500 molecules ABL1: 23 000 molecules; Ratio = 4500 divided by 23 000 (= 0Æ195) and multiplied by 100 (= 19Æ5%). (Foroni et al)

· If ABL1 copy number is >=10 000, results should be both reliable and sensitive and can be issued as ‘positive’ or ‘undetectable’.

· If ABL1 copy number is 5000: ‘undetectable BCR-ABL1’ results should not be given (due to the risk of false negative) and one of the following comments should be included in the report: (see the paper)

· Conversion factor. Laboratories with an appropriately validated conversion factor should also consider converting their BCR-ABL1/ABL1 values to the International Scale. However this should be clearly specified in the final report to the clinicians.

· Calculating log reduction from IS:

· 100% BCR-ABLIS corresponds to the International Randomized Study of Interferon and STI571 (IRIS) study standardized baseline [Cross et al Leukemia (2015) 29, 999–1003]

· 0.1% BCR-ABL IS is defined as a major molecular response (MMR or MR3) [Cross et al Leukemia (2015) 29, 999–1003]

· <= 0.01% BCR-ABLIS is MR4

· <= 0.0032% BCR-ABLIS is MR4.5

· <= 0.001% BCR-ABLIS is MR5

· Expression of results on the IS depends on each testing laboratory either having obtained a laboratory-specific conversion factor (CF) by sample exchange with an established reference laboratory or by using kits and reagents that have been calibrated to the World Health Organization International Genetic Reference Panel for quantitation of BCR-ABL1 mRNA

· Indications for cytogenetics and qPCR for BCR-ABL mRNA:

· Diagnosis:

· Cytogenetics on BM

· qPCR on BM

· Bone marrow FISH for deletions in chromosome 9 (relevant for interferon treatment) (CCGM-CML recommendation in 2006)

· if BM not available:

· FISH on PB for BCR-ABL is acceptable

· treatment response:

· BCR-ABL qPCR every 3 months (FISH maybe used until CCR) (CCGM-CML)

· BM cytogenetics annually, if a major cytogenetic response is maintained (CCGM-CML)

· NCCN - BM cytogenetics at 6 months

· 12 months if complete cytogenetic response (CCyR) not seen at 6 months

· 18 months if complete CCyR not seen at 12 months

· CCyR (NCCN):

· BCR-ABL transcript levels every 3-6 months

· BM cytogenetics if clinically indicated

· CCGM-CML milestones for imatinib therapy:

· CHR at 3 months

· Major cytogenetic response at 12 months

· CCR at 18 months

· MMR at 24 months

· CCGM-CML definition of disease progression:

· Transformation from CP to AP or BC

· Cytogenetic (clonal) evolution in Ph-positive cells

· Loss of CCR

· Confirmed increase of 0.5 log or more (q-RT-PCR) for patients in CCR or better

· Detection of ABL mutations with loss of response

· CCGM-CML:

· If a >= 0.5 log increase occurs, the test should be repeated within 4 weeks; mutational analysis is recommended (ABL-kinase sequencing for mutations)

· NCCN (updated 2018):

· qPCR at diagnosis, then q3 mo, then q3-6 mo. after MMR for 2 years

· Monthly molecular monitoring for 1 year, then every 6 weeks for the 2^nd year, and every 12 weeks thereafter (indefinitely) is recommended after discontinuation of TKI therapy.

· If resumption of TKI required, test monthly until MMR is established, then every 12 weeks indefinitely for those who have reinitiated TKI therapy after a loss of MMR

· Rising level (1 log increase) of BCR-ABL transcripts:

· If major molecular response (MMR) retained, repeat in 1-3 mo.

· If no MMR, obtain BM cytogenetics

· Consider mutation testing

· BCR-ABL1 KD mutations:

· Indications for ABL kinase domain (KD) mutation analysis:

· Chronic phase:

· Inadequate initial response

1. Failure to achieve complete hematologic response at 3 months

2. Minimal cytogenetic response at 6 months

3. Major cytogenetic response at 12 months

4. >10% BCR-ABL1 (IS) at 3 months

· Any sign of loss of response

1. Hematologic relapse

2. Cytogenetic relapse

3. 1 log increase in BCR-ABL transcript ratio AND loss of MMR (NCCN)

4. CML progresses to advanced stages of disease

5. MMR not re-established after a trial discontinuation and resumption of therapy

· Recommendations from the ELN suggest direct mutation testing when primary treatment with imatinib fails, when the increase in BCR-ABL1 transcripts leads to loss of MMR, or whenever there is a suboptimal response (eg, lack of MMR after 18 months of imatinib)

· ELN and NCCN guidelines specifically recommend a switch to certain TKI agents when particular mutations are detected (?no improved outcomes have been demonstrated however)

· In particular, the presence of the common T315I mutation suggests that ponatinib, and no other TKI, may be effective.

· In addition, NCCN and ELN guidelines suggest a switch to nilotinib (not dasatinib) for patients with the V299L, T315A, or F317L/V/I/C mutations; and a switch to dasatinib (not nilotinib) for patients with the Y253H, E255K/V, or F359V/C/I mutations

·

References:

· Foroni L et al. Guidelines for the measurement of BCR-ABL1 transcripts in chronic myeloid leukemia. British Journal of Haematology 2011;153:179-190.

· Cross NCP et al. Laboratory recommendations for scoring deep molecular responses following treatment for chronic myeloid leukemia. Leukemia 2015;29:999-1003.

· NCCN Guidelines Version 2.2011 for Chronic Myelogenous Leukemia (updated with version 1.2019)

· Laneuville P et al. Recommendations of the Canadian Consensus Group on the Management of Chronic Myeloid Leukemia. Current Oncology 2006;13(6):201-221.

· Press RD, Kamel-Reid S, Ang D. BCR-ABL1 RT-qPCR for Monitoring the Molecular Response to Tyrosine Kinase Inhibitors in Chronic Myeloid Leukemia. J Molecular Diagnostics2013;15(5):565-576.