Template for Reporting Results of Monitoring Tests for Patients With Chronic Myelogenous Leukemia (BCR-ABL1⁺)

Completion of the template is the responsibility of the laboratory performing the biomarker testing and/or providing the interpretation. When both testing and interpretation are performed elsewhere (eg, a reference laboratory), synoptic reporting of the results by the laboratory submitting the tissue for testing is also encouraged to ensure that all information is included in the patient's medical record and thus readily available to the treating clinical team.

Select a single response unless otherwise indicated.

Note: Use of this template is optional.*

*Reporting on the data elements in this template is not required.

• ___ Peripheral blood

• ___ Bone marrow

• ___ Other (specify): _______________________________________________

• ___ No abnormalities detected

• ___ t(9;22)(q34;q11.2); BCR-ABL1 (Philadelphia chromo‐ some [Ph]) detected

• Total number of metaphases examined: _______________________________________________

• Number of Ph⁺ metaphases:

• ___ Other abnormalities detected (specify): _______________________________________________

• ___ No BCR-ABL1 fusions detected

• ___ BCR-ABL1 fusions detected

• Total number of cells examined:

• Number of BCR-ABL1⁺ cells:

• ___ BCR-ABL1 amplification (duplication) detected:   _______ copies per cell

• ___ No BCR-ABL1 transcripts detected

• ___ BCR-ABL1, p210 type (e13/14a2) transcripts detected

• Normalized copy number (e13/14a2 transcripts per reference gene): _______________________________________________

• Percent BCR-ABL1 (International Scale [IS]): _______%

• ___ BCR-ABL1, p190 type (e1a2) transcripts detected

• Normalized copy number (e1a2 transcripts per reference gene): _______________________________________________

• ___ Other BCR-ABL1 transcripts detected (ie, e19a2; p230 type) (specify): _______________________________________________

• Normalized copy number (other BCR-ABL1 transcripts per reference gene): _______________________________________________

• ___ No mutation detected

• ___ Mutation(s) detected

• ___ p.T315I mutation

• ___ Other (specify): _______________________________________________

• Significance of mutation:

•   ___ Reported to confer resistance to tyrosine kinase inhibitors (TKIs)

•   ___ Unknown resistance profile

•   ___ Normal sequence variant and/or not associated with resistance

• Date of most recent cytogenetic study: _______________________________________________

• Most recent cytogenetic results:

• ___ No abnormalities detected

• ___ t(9;22)(q34;q11.2); BCR-ABL1 (Ph) detected

•   Total number of metaphases examined: _______________________________________________

•   Number of Ph⁺ metaphases: _______________________________________________

• ___ Other abnormalities detected (specify): _______________________________________________

• Date of most recent FISH study: _______________________________________________

• Most recent FISH results:

• ___ No BCR-ABL1 fusions detected

• ___ BCR-ABL1 fusions detected

• Total number of cells examined: _______________________________________________

• Number of BCR-ABL1⁺ cells: _______________________________________________

• ___ BCR-ABL1 amplification (duplication) detected: _______ copies per cell

• Date of most recent BCR-ABL1 quantitative RT-PCR (qRT-   PCR) study: _______________________________________________

• Most recent BCR-ABL1 qRT-PCR results:

• ___ No BCR-ABL1 transcripts detected

• ___ BCR-ABL1 (e13/14a2; p210 type) transcripts detected

• e13/14a2 normalized copy number: _______________________________________________

• Percent BCR-ABL1 (IS): ________%

• ___ BCR-ABL1 (e1a2; p190 type) transcripts detected

• e1a2 normalized copy number: _______________________________________________

• ___ Other BCR-ABL1 transcripts detected (specify type): _______________________________________________

•   Normalized copy number: _______________________________________________

• BCR-ABL1 RT-PCR assay sensitivity: _______________________________________________

• Fusion transcripts covered:

•   ___ e13/14a2 (p210)

•   ___ e1a2 (p190)

•   ___ Other (specify): _______________________________________________

• BCR-ABL1 mutation analysis assay sensitivity: _______________________________________________

• BCR-ABL1 mutation analysis assay coverage:

•   ABL1 codons _____ through _____ or list: _______________________________________________

• BCR-ABL1 mutation analysis method:

•   ___ Sanger sequencing

•   ___ Pyrosequencing

•   ___ Allele-specific PCR

•   ___ Denaturing high-performance liquid chromatography

•   ___ Next-generation (massively parallel) sequencing

•   ___ Other (specify): _______________________________________________

• BCR-ABL1 reference sequence accession number (if applicable): _______________________________________________

Chronic myelogenous leukemia (CML) is characterized by the presence of an abnormal clonal myeloid population harboring t(9;22)(q34;q11.2) (known as Ph), resulting in the presence of BCR-ABL1 mRNA transcripts and an abnormal fusion protein with constitutive ABL1 tyrosine kinase activity. Detection and monitoring of t(9;22)(q34;q11.2) or BCR-ABL1 fusion transcripts by a variety of laboratory methods, including classical cytogenetic karyotyping, FISH, and qRT-PCR, provides an effective way to assess the response to TKI therapy. Furthermore, these techniques provide a mechanism for the early detection of emerging TKI resistance and for identifying newly acquired genetic abnormalities that may be associated with transformation to a more aggressive phase of disease or with resistance to particular TKIs. Clear, concise, and accurate reporting of results is extremely important for effective clinical management. The National Comprehensive Cancer Network (NCCN) publishes extensive clinical guidelines for appropriate laboratory monitoring of CML patients to ensure accurate characterization of the hematologic, cytogenetic, and molecular response to therapy.¹  The NCCN guidelines continue to evolve and should be consulted for the most up-to-date recommendations.

Cytogenetic analysis is typically performed at diagnosis and at certain intervals during treatment with TKIs, particularly if there is evidence of a suboptimal therapeutic response or evidence of emerging resistance. Although karyotyping is the least sensitive method for detecting t(9;22)(q34;q11.2), it is essential for establishing the depth of the cytogenetic response to therapy and for assessing whether or not important therapeutic milestones have been met. Cytogenetic analysis is also critical for the detection of additional abnormalities that are commonly present at disease progression, such as trisomy 8 (+8) or isochromosome 17q (i(17q)), among others. Reporting of the cytogenetic results should include both the total number of metaphases examined and the number of Ph⁺ metaphases as well as any additional abnormalities that are identified. For BCR-ABL1 fusions, FISH is often used as an adjunct to karyotyping because of the increased sensitivity of the technique. It may also allow for the detection of rare cryptic translocations that are otherwise undetectable by karyotyping. It is important to report FISH results with the total number of cells analyzed along with the number of BCR-ABL1⁺ cells. FISH is also important for detecting genomic duplication or amplification of the BCR-ABL1 locus, which may contribute to TKI resistance in a subset of CML patients.

BCR-ABL1 qRT-PCR testing is the most sensitive method for the detection and monitoring of the abnormal fusion transcripts and may be performed on peripheral blood or bone marrow samples. Unless otherwise clinically indicated, it is not necessary to obtain bone marrow specifically for molecular testing. In an effort to promote the standardization of qRT-PCR reporting and the interlaboratory comparison of test results, a standardized reporting scale, known as the IS, was introduced and has been widely adopted by laboratories worldwide.²  Serial testing of patients by qRT-PCR during TKI therapy allows for the accurate assessment of important molecular treatment milestones. Importantly, both the depth and the kinetics of the response are critical for the evaluation of therapeutic efficacy and for the assignment of overall prognosis.³  A major molecular response is defined as BCR-ABL1 qRT-PCR values of 0.1% or less for IS, a 3-log reduction from the standardized baseline. A complete molecular response is defined as undetectable BCR-ABL1 levels using a test with 4.5-log sensitivity. The definition of complete molecular response highlights the importance of test performance characteristics, such as sensitivity. To evaluate the response kinetics it is necessary to place current results in the appropriate clinical context using the clinical history and the results of prior testing. For simplicity, this reporting template includes space for only a single prior test result, but this issue may be revisited in future template updates.

Patients with CML who are undergoing treatment with TKIs may manifest signs of therapeutic resistance in a variety of ways, including progression to accelerated or blast phase, failure to achieve timely cytogenetic or molecular milestones, or signs of the loss of a previously achieved response. A subset of patients may acquire resistance to TKI therapy due to substitution mutations in the translocated ABL1 kinase domain. Specific mutations may impart resistance to certain kinase inhibitors, but not others. Because the choice of subsequent TKI therapy depends on the identity of the mutation detected, it is important to report this information clearly in terms of the amino acid change (ie, p.F359V). In most current studies, the most commonly detected mutation in resistant CML patients is p.T315I,⁴  an abnormality that promotes resistance to all but one of the currently approved TKIs (as of January 2014). A number of germ line polymorphisms also occur in the ABL1 kinase domain and should not be confused with true resistance mutations.⁵  Insertion-type and deletion-type mutations occur as well but have uncertain clinical significance.⁶  Rare mutations are identified in signaling domains in the translocated ABL1 sequence called the Src homology-2 (SH2) and Src homology-3 (SH3) domains. Most current BCR-ABL1 mutation tests are focused on the kinase domain and do not provide information on potential SH2/SH3 mutations, but certain rare mutations in these domains have also been reported to confer resistance to TKI therapy.⁷