Hematopathology Laboratory - Molecular Hematopathology
Molecular Studies:Acceptable specimen types
Pre-transplant and transplant consult studies
Testing for Hematopoietic Malignancies
Common Molecular Abnormality
Current Molecular Hematopathology Assay
|FL / DLBCL||B-cell clonality
|IGH and IGK clonality
BCL2 (MBR, MC7, MC8)
|IGH and IGK clonality
|CLL / MM* / other B-cell NHL||B-cell clonality||IGH clonality, IGK clonality|
|T-cell NHL and T-cell Leukemia||T-cell clonality||TRG Clonality|
|CML / B-cell ALL||
t(9;22) "Philadelphia (Ph) chromosome" BCR/ABL1
|Quantitative p190 and p201
|CML / B-cell ALL||
t(9;22) "Philadelphia (Ph) chromosome" BCR/ABL1
|ABL1 kinase domain mutational analysis**
|PV / PMF / ET / other CMPN / Mastocytosis||JAK2
KITD816V and sequencing
KIT D816V and sequencing
* For MM patients not usually clinically indicated.
** ABL mutational analysis assay is available in making treatment choices in patients with CML who exhibit Imatinib resistance. The ABL mutational analysis assay can be applied to samples which have tested positive for the p210 BCR/ABL transcript.
|DLBCL||Diffuse large B-cell lymphoma|
|MCL||Mantle cell lymphoma|
|CLL||Chronic lymphocytic leukemia|
|CML||Chronic myelogenous leukemia|
|B-ALL||Precursor B-cell Acute lymphoblastic leukemia|
|CMPN||Chronic-phase myeloproliferative neoplasms|
|AML||Acute myeloid leukemia|
Acceptable specimen typesB-cell and T-cell clonality, lymphoma translocation assays: Acceptable specimen types for these assays include fresh blood, fresh bone marrow, or tissue (fresh, frozen or formalin-fixed paraffin embedded [FFPE]). Anticoagulation using EDTA is preferred for blood and marrow. Acid-citrate-dextrose (ACD) and sodium citrate also are acceptable if EDTA is not available. Heparin has been shown to inhibit PCR reactions, so specimens anticoagulated with heparin will only be accepted if no other specimen is available or obtainable. Acid-decalcified bone marrow biopsies are not optimal due to acid-mediated DNA fragmentation. Under certain circumstances (e.g. when fresh, frozen, or FFPE material is not available) testing of samples could be attempted but must be approved in advance by the laboratory director. Tissue or bone marrow fixed/decalcified using B-5, Zenkerís or other mercury-containing fixatives is not acceptable for any molecular testing since mercury-treated DNA in general cannot be amplified by PCR.
JAK2, CALR, MPL, FLT3-ITD, NPM1, CEBPA, KIT D816V BCR/ABL1 and ABL1 Mutational Analysis assays: Fresh blood or bone marrow should be submitted for JAK2, CALR, MPL, FLT3-ITD, NPM1, CEBPA, KIT D816V or ABL1 mutation testing; tissue specimens are generally not appropriate for these tests. Because RNA is labile, specimens for assays requiring RNA, BCR/ABL1 and ABL1 Mutational Analysis, should arrive in the laboratory within 24 hours of collection. EDTA is the required anticoagulant for these RNA based tests; heparinized specimens are not accepted due to inhibition.
SensitivityThe sensitivity of molecular testing for residual disease monitoring varies greatly depending on the particular assay used. Assays to identify translocations may be quite sensitive (down to 0.002% of nucleated cells) owing to the typical absence of such translocations in normal individuals. The sensitivity of the B cell clonality assays (IGH and IGK gene rearrangement) depends on the number of normal B cells present, being relatively insensitive (~2-5% of nucleated cells) in normal untreated marrow and much more sensitive (~0.01% of nucleated cells) during anti-CD20 antibody therapy. Similarly, the T cell clonality assay (TRG gene rearrangement) is generally relatively insensitive given the ubiquitous presence of normal T cells. The FLT3-ITD, NPM1, CEBPA, KIT D816V, JAK2, CALR and MPL mutation assays were designed primarily for the purposes of classification and prognosis in pre-treatment samples. The utility and sensitivity of these assays in the post-treatment setting are under investigation. There is some evidence that the NPM1 mutation assay may be useful in detecting minimal residual disease.
Pre-transplant and transplant consult studiesMolecular studies include a variety of assays, each focused on a specific category or subcategory of hematopoietic disease. As such, these assays should not be regarded as general screening tests, but as targeted assays for the confirmation of diagnoses or to aid in classification. In patients having a known hematopoietic disease, as determined by clinical history, morphology and/or flow cytometry, molecular studies may be indicated to confirm the patientís diagnosis or to assist in disease classification. Molecular studies also may provide important prognostic information, e.g. FLT3-ITD, NPM1 and CEBPA mutation testing in AML patients. Finally, molecular studies may be ordered to monitor minimal residual disease after therapy or transplant.
Post-transplant studiesPost-transplant molecular studies may be routinely used to monitor residual disease. However, as with patients lacking known disease prior to transplant, such studies are only appropriate for patients having an informative molecular abnormality identified prior to transplant (see above). A negative result for an assay known to be uninformative provides no useful information and may represent a false negative result. As in the pre-transplant setting, BCR/ABL1 is the one exception and can be ordered in any patient having disease known to be Ph+ prior to transplant or at relapse.
Non-informative studiesAn important prerequisite for any post-therapeutic molecular monitoring is demonstration that the assay used is informative, i.e. positive when disease is present. The ordering of non-informative assays is generally not appropriate and should be discouraged because such assays will be negative even when disease is present and can lead to false negative results. In particular, molecular studies for the t(11;14) CCND1/IGH translocation associated with mantle cell lymphoma or the t(14;18) IGH/BCL2 translocation associated with follicular lymphoma and diffuse large B cell lymphoma will be informative in only a subset of patients having these lymphomas because these translocations are not always detected by PCR, even if present. When possible, testing of diagnostic material should be performed in all patients to confirm that a given molecular assay is informative prior to (or concurrent with) testing for residual disease. One exception is t(9;22) BCR/ABL1 testing, which is informative in virtually all patients with a diagnosis of chronic myeloid leukemia or t(9;22)-associated (Ph+) acute lymphoblastic leukemia and can be ordered on any patient carrying these diagnoses.
B-cell clonalityMonoclonal populations of B lymphocytes can be identified by testing for immunoglobulin heavy chain (IGH) and/or immunoglobulin kappa (IGK) gene rearrangements using the polymerase chain reaction (PCR). Our method, developed by the collaborative European BIOMED-2 Concerted Action study group, employs three multiplex primer sets for the IGH locus, each recognizing one of three conserved "Framework Regions" (FR1, FR2, or FR3) of IGH variable gene segments (IGHV) and two multiplex primer sets targeting the kappa light chain (IGK) locus. Each multiplex IGH primer set contains six or seven IGHV primers in combination with the appropriate IGHJ region primer. The IGK primers are separated into two tubes: tube A contains IGKV and IGKJ primers and tube B contains IGKV, intronRSS, and KDE primers. Each of the five multiplex tubes contains one primer that is labeled with a fluorescent dye, which allows the amplicons to be analyzed on the ABI 3130 Sequence Detector by capillary electrophoresis.
When the DNA is amplified, the length of the PCR products is determined by the number of random nucleotides added at the time of immunoglobulin gene rearrangement. In a non-neoplastic population of B cells, each PCR product is slightly different in size. When these products are separated by capillary electrophoresis, a roughly Gaussian distribution of PCR product sizes is observed. In B cell lymphomas and other B cell-derived tumors, all of the neoplastic cells have the same heavy or light chain rearrangement and thus produce a PCR product of the same size, resulting in the appearance of a discrete peak on capillary electrophoresis. In a mixed population of B cells, the lymphoma cells must represent at least 2-5% of the total B cells in order to be detectable as clonal peaks in a polyclonal distribution of background peaks. Approximately 90% of B cell lymphomas produce a PCR product using the BIOMED-2 IGH and IGK primer sets. For patients whose lymphoma has been shown to have a detectable clonal B cell population, this assay can be a sensitive detector of minimal residual disease in post treatment, B cell depleted specimens, however B cell clonality testing is generally not recommended for minimal residual disease detection.
BCL1 [also known as t(11;14) or or IGH-CCND1 (cyclin D) translocation]Mantle cell lymphoma is associated with a translocation between chromosomes 11 and 14 resulting in insertion of the cyclin D1 (CCND1) gene into the immunoglobulin heavy chain locus. This t(11;14) is detected using a nested polymerase chain reaction assay. The two successive PCR amplifications use 5' primers directed against the major translocation cluster (MTC) of breakpoints in the CCND1 locus on chromosome 11 coupled with consensus primers directed against IGHJ regions on chromosome 14. The breakpoint occurring in the MTC region allows detection by our PCR method in 40-50% of mantle cell lymphoma cases. If a patientís mantle cell lymphoma cells are positive by this assay, it is a very sensitive detector of minimal residual disease. This assay can detect one neoplastic cell in 104-105 normal cells. However, ~50-60% of mantle cell lymphoma patients will have breakpoints outside the MTC that cannot be detected by these PCR primers; testing of such patients using this MTC assay may lead to false negative results and is not recommended. For this reason we strongly recommend testing a patientís original diagnostic material before the t(11;14) PCR assay is ordered to detect minimal residual disease. Samples with low mutational burden may appear as wild-type.
BCL2 [also known as t(14;18) or IGH-BCL2 translocation]Our BCL2 polymerase chain reaction assay detects the t(14;18), a chromosomal translocation that is the most frequent cytogenetic abnormality in follicular lymphoma and can also be found in a minority of diffuse large B-cell lymphoma cases. Approximately 70% of follicular lymphomas have t(14;18) breakpoints at the major breakpoint region (MBR) that can be detected using the MBR1 primer on chromosome 18 in conjunction with a consensus IGHJ primer recognizing the IGH joining region on chromosome 14. An additional ~5% of follicular lymphomas can be detected using primers for the minor cluster region (MC8 or MC7) on chromosome 18 in conjunction with the consensus IGHJ primer. If a patientís lymphoma is positive for this assay, it is a very sensitive detector of minimal residual disease. However, ~20% of follicular lymphomas will not have breakpoints that can be detected using these PCR primers; testing of such patients using these assays may lead to false negative results and is not recommended. For this reason we strongly recommend testing a patientís original diagnostic material before the t(14;18) PCR assay is ordered to detect minimal residual disease. The t(14;18) PCR assay can detect 1 lymphoma cell in 104-105 normal cells. Samples with low mutational burden may appear as wild-type.
T-cell clonalityMonoclonal populations of T lymphocytes can be identified by testing for T cell receptor ? chain (TRG) gene rearrangements using a multiplex polymerase chain reaction assay. In its germline configuration, the TRG locus consists of six functional TRGV gene segments, eight TRGV pseudogene segments, and five TRGJ segments. There is enough homology among the TRGV region segments and among the TRGJ region segments to allow use of a small number of consensus primers to amplify across the V-J join N region. A polyclonal T cell population will produce a Gaussian distribution of PCR products on capillary electrophoresis, while a clonal process will produce one or two discrete peaks. Approximately 95% of T cell lymphomas produce clonal PCR products using our primer set. A neoplastic clone must represent at least 2-5% of the total T cells in a mixed population for a clonal peak to be detectable in a polyclonal distribution of background peaks.
FLT3-ITDA mutation in the FLT3 gene on chromosome 13 results from internal tandem duplications (ITD) in exons 14 and 15 of the juxtamembrane portion of the gene and causes activation of the FLT3 protein. Approximately 20-30% of patients with acute myeloid leukemia have this mutation, which has been associated with adverse prognosis. In our PCR assay, purified genomic DNA is amplified using primers that flank the ITD region (one forward, HEX-labeled primer and one reverse, NED-labeled primer) and then size fractionated by capillary electrophoresis. The FLT3 ITD to wild-type ratio, known as the allelic ratio, is calculated by dividing the peak height of the ITD product(s) by that of the wild-type product. If more than one FLT3 ITD product is present, the sum of the FLT3 ITD peak heights is divided by the normal peak height. Amplification of normal genomic DNA results in a PCR product of approximately 329 nucleotides (nt), whereas amplification of genomic DNA containing the FLT3 ITD mutation yields the normal 329 nt product as well one or more longer products. This test should be performed on AML patients at diagnosis on EDTA anticoagulated peripheral blood or bone marrow containing neoplastic blasts. FLT3 mutations tend to be unstable and can change with relapse, so this assay is not a reliable method for detecting minimal residual disease in AML. This assay is capable of detecting as few as 5% cells with the mutation. Samples with low mutational burden may appear as wild-type..
NPM1Insertion mutations in exon 12 of the NPM1 gene on chromosome 5 cause abnormal cytoplasmic localization of the NPM1 protein and have been identified in 35-50% of adult acute myeloid leukemia (AML) and in 50-60% of AML cases having normal karyotype (AML-NK). In the absence of FLT3 internal tandem duplication (ITD) mutations, the presence of NPM1 mutations in AML-NK has been associated with better response to induction therapy and favorable overall survival. In our PCR assay for NPM1 insertion mutations, purified genomic DNA is amplified using primers that flank the insertion region (one HEX-labeled, the other unlabeled) and then size-fractionated by capillary electrophoresis. Amplification of normal genomic DNA results in a product of ~189 nucleotides (nt). The vast majority of NPM1 mutations in AML are 4 nt insertions and, because NPM1 mutations are invariably heterozygous, these result in one normal ~189 nt product and one abnormal ~193 nt product. In rare cases, NPM1 insertion mutations of different sizes (i.e. other than 4 nt) have been reported in AML. This test should be performed on AML patients at diagnosis on EDTA anticoagulated peripheral blood or bone marrow containing neoplastic blasts. The literature suggests that NPM1 mutations may be useful as molecular targets for detecting minimal residual disease in AML. This assay is capable of detecting as few as 5% cells with the mutation. Samples with low mutational burden may appear as wild-type.
CEBPAMutations in the transcription factor CCAAT/enhancer binding protein alpha (CEBPA) are found in ~5% to 10% of acute myeloid leukemia (AML) with normal cytogenetics and are associated with a good prognosis. Accordingly, the fourth edition of the WHO Classification of Tumours of the Haematopoietic and Lymphoid Tissues recommends routine mutational screening of CEPBA in addition to FLT3-ITD and NPM1 in new cases of AML. Two classes of CEPBA mutations are most frequent: (1) C-terminal basic leucine zipper (bZIP) region mutations and (2) N-terminal mutations located between the major translational start codon and a second ATG in the same open reading frame. C-terminal bZIP mutations are usually in-frame and may impair DNA binding and/or homodimerization and heterodimerization. N-terminal mutations usually introduce a premature stop of translation of the p42 CEBPA protein while preserving translation of a p30 isoform (p30 expression may act to inhibit the function of the p42 isoform). Recent literature suggests that only patients with double CEBPA mutations have a favorable prognosis. Most patients with CEBPA-mutant AML have double (two) mutations that consist of a C-terminal bZIP mutation and an N-terminal mutation. Rare cases of double bZIP mutations are also reported. AML patients with double CEBPA mutations usually do not have concurrent FLT3-ITD or NPM1 mutations. Limited evidence suggests that FLT3-ITD mutations negate the prognostic benefit of double CEBPA mutations in rare cases of concurrent mutations.
To facilitate cost-effective testing we have adopted a step-wise screening strategy with reflex testing to identify the majority of patients with biallelic CEPBA mutations. The initial test is a PCR capillary electrophoresis sizing assay of the C-terminal bZIP region that detects >95% of patients with double CEBPA mutations. Patients who are positive by the C-terminal sizing assay (~5-10% of all new cases of AML) go on to further testing to confirm an N-terminal mutation. The first reflex test is two overlapping PCR capillary electrophoresis N-terminal sizing assays that will detect an N-terminal mutation in ~80% of cases that are positive by C-terminal sizing. When no N-terminal mutation is detected by sizing assays, complete CEBPA gene sequencing is performed to identify rare point mutations. About 13% of C-terminal mutation positive patients will not have an N-terminal mutation and are reported as have a single (monoallelic) CEBPA mutation. The fragment analysis portion of this assay can detect a heterozygous mutation when it is present in at least ~3% of cells. Sanger sequencing, however, has decreased sensitivity. Samples with low mutational burden may appear as wild-type.
KIT D816V mutationKIT, also referred as to c-kit or CD117, is a tyrosine kinase receptor encoded by the KIT proto-oncogene that belongs to the class III subfamily of tyrosine kinase receptors together with PDGF receptor, CSF-1R, and FLT-3. The ligand for KIT is the stem cell factor (SCF). SCF binding to KIT induces a conformational change, resulting in the dimerization of the receptor, followed by the activation of its intrinsic tyrosine kinase activity. The activated receptor becomes autophosphorylated which serve as docking sites for signal transduction molecules. Multiple downstream signaling pathways can thus be activated, including the Ras/ERK, phosphatidylinositol 3-kinase (PI3-K), Src kinase and JAK/STAT pathways. The activation of these pathways elicits diverse biological responses according to the state and type of the responding cells; however, in general these signaling pathways induce the expression of genes that drive proliferation, maturation, and survival.
Different activating somatic mutations in the KIT gene have been observed in human neoplasms arising from cells that normally express KIT in the adult. These tumors include gastrointestinal stromal tumor (GIST), melanoma, seminomas, acute myeloid leukemia (AML), and systemic mastocytosis. Specifically, the activating D816V mutation in exon 17 has been described in GIST, as a secondary mutation after treatment, systemic mastocytosis, AML, and melanoma. The mutation is the result of an A-to-T transversion at codon 816 that causes a missense mutation with a substitution of aspartic acid for valine. In systemic mastocytosis, D816V mutation is found in >90% of adult cases and constitutes one of the minor diagnostic criteria according to the WHO classification of tumors of hematopoietic and lymphoid tissue.
In this assay, specimens are amplified by two specific primer sets, a control primer set to amplify exon 17 and an allele-specific primer set to amplify part of the exon 17 of the mutant allele. The forward primer from the control primer set has no fluorescent-tag and spans the KIT intron 16 and 17; the reverse primer was labeled with FAM and recognizes intronic sequences downstream of exon 17. The control primer set results in an ~184 base pair (bp) amplicon for both the wild-type and the D816V mutant alleles and serves as a control reaction to ensure that genomic sequences for KIT exon are present in the samples to be analyzed. The allele-specific primer set consists of the same reverse primer as in the control primer set, but the forward primer was specifically designed to only recognize the D816V mutant sequence and was tagged with HEX. This forward primer has a T at the 3í-end instead of the wild-type A to match the point mutation that causes the D816V mutation. In addition, 3 bp from the 3í-end of the forward primer, a mutation that results in a single nucleotide mismatch was added to improve specificity. The mutant allele-specific primer set yields an ~85 bp product when the D816V mutation is present. The analytic sensitivity of this assay is 0.8%. For AML specimens, the UW Genetics lab tests for KIT mutations in exon 8 and 17 by DNA sequencing. Samples with low mutational burden may appear as wild-type.
JAK2 [also known as JAK2 point mutation or JAK2V617FThe somatic point mutation V617F in the JAK2 tyrosine kinase gene at chromosome 9p24.1 (JAK2V617F) has been associated with several chronic myeloproliferative disorders, including polycythemia vera (PV), essential thrombocythemia (ET) and primary myelofibrosis (PMF). In the studies published to date, JAK2V617F mutations have been found in 74-97% of PV patients, 23-57% of ET patients, and 35-57% of PMF patients. Our testing for JAK2V617F is performed by allele-specific multiplex PCR on purified genomic DNA using a HEX-labeled reverse primer for the normal sequence, an unlabeled forward primer for the normal sequence and a FAM-labeled forward primer specific for the mutant sequence, which allows detection of amplification products by capillary electrophoresis. Amplification of normal genomic DNA results in 364 nucleotide (nt) HEX-labeled product and no FAM-labeled product, whereas amplification of genomic DNA that contains the JAK2V617F mutation results in two differentially labeled products: a 364-nt HEX-labeled product in addition to a 203-nt product labeled with both FAM and HEX. This is a sensitive assay, capable of detecting as few as 2.5% cells with the mutation, however the significance of the detection of low levels of cells with a JAK2 mutation is not clear. Samples with low mutational burden may appear as wild-type.
CALR [also known as calreticulin]The somatic insertion/deletion mutation in exon 9 of the CALR gene has been associated with several chronic myeloproliferative disorders, including essential thrombocythemia (ET) and primary myelofibrosis (PMF) and infrequently other myeloid stem cell disorders. In the studies published to date, CALR mutations have been found in ~20 to 25% of ET patients, and 30 to 35 % of PMF patients. Less commonly, this mutation may be found in some low grade myelodysplastic syndromes and other myeloid stem cell disorders. Our testing for CALR exon 9 insertion/deletion mutations is performed by PCR using purified genomic DNA and intron-located primers that flank and amplify exon 9 of CALR (Klampfl et al., NEJM 2013). Amplification of normal genomic DNA results in a 263 nucleotide (nt) FAM-labeled wild-type product, whereas amplification of genomic DNA that contains a CALR mutation results in a different sized product, in addition to the wild-type product. The most common subtypes of CALR mutations reported thus far include the type 1 mutation (52 base pair deletion) and the type 2 mutation (5 base pair insertion). For these mutation subtypes, additional Sanger sequencing will not typically be performed. For other mutation subtypes, specifically non-Type 1 and non-Type 2, confirmatory Sanger sequencing will be performed to confirm the size of the deletion/insertion. This is a sensitive assay, capable of detecting as few as 2% cells with the mutation, however the significance of the detection of low levels of cells with a CALR mutation is not yet clear. Samples with low mutational burden may appear as wild-type.
MPL [also known as leukemia virus MPL proto-oncogene, thrombopoietin receptor]Somatic mutations in the MPL gene have also been infrequently identified in chronic myeloproliferative neoplasms, including JAK2 mutation-negative and CALR mutation-negative essential thrombocythemia (ET) and primary myelofibrosis (PMF). Our testing for MPL mutation is performed by PCR using purified genomic DNA by allele-specific multiplex PCR on purified genomic DNA. Our assay detects the W515L or the W515K mutation, but does not test for the S505N mutation. Amplification of normal genomic DNA results in a 358 nucleotide (nt) wild-type product. By contrast, amplification of genomic DNA that contains a MPL mutation W515 mutation results in differentially labeled products: a 358 nt HEX- or FAM- labeled wild-type product in addition to a 229 nt mutant product labeled with either FAM- (for W515K) or HEX- (for W515L). This is a sensitive assay, capable of detecting as few as 5% cells with the mutation. If indicated, Sanger sequencing confirmation may be performed; however, this is not routinely done. Samples with low mutational burden may appear as wild-type.
Qualitative BCR/ABL RT-PCRThe Qualitative BCR/ABL1 RT-PCR assay will be discontinued as of April 1 2015. Please see Quantitative BCR/ABL for further testing options including evaluation of new diagnoses or minimal residual disease testing.
Quantitative BCR-ABL1The Philadelphia chromosome (Ph) is the product of the reciprocal translocation between the ABL1 gene on chromosome 9, and the BCR gene on chromosome 22. The resulting BCR/ABL1 translocation is found in > 90% of patients with chronic myeloid leukemia (CML), approximately 5% of childhood acute lymphocytic leukemia (ALL) and in 20-50% of adult ALL. Depending on variations in splicing, BCR exon 2 or exon 3 is joined to ABL1 exon 2 to form e14:a2 or e15:a2 BCR/ABL fusion gene producing the p210 fusion protein. Quantitative testing for the p210 BCR/ABL1 translocations (e14:a2 and e15:a2) is performed using the Cepheid GeneXpert system.
The Cepheid GeneXpert combines RNA extraction, nucleic acid amplification and detection of the BCR/ABL1 target using real-time reverse transcription polymerase chain reaction (RT-PCR). A nested, multiplex assay uses primers designed to detect the p210 BCR/ABL1 translocations (e14:a2 and e15:a2) and the ABL1 gene. ABL1 is the reference RNA and evaluates RNA quality, normalizes total RNA, and also may detect sample inhibition. Quantitation of BCR/ABL1 is expressed as a percentage ratio of BCR/ABL1 RNA to ABL1 RNA. This assay is able to detect 1 BCR/ABL1 positive cell within 100,000 normal cells. All BCR/ABL1 testing should be done on EDTA anticoagulated peripheral blood or bone marrow aspirates. Specimens anticoagulated with heparin will not be accepted.
A second BCR/ABL1 translocation occurs when BCR exon 1 joins to ABL1 exon 2, resulting in the p190 (e1:a2) BCR/ABL1 breakpoint. p190 is found almost exclusively in Ph+ALL patients, but can, on occasion, be found in CML patients as an alternative splice variant. p190 is currently not available on the GeneXpert, but a laboratory-developed test using real-time quantitative PCR methodology is available. This assay is able to detect one Ph+ cell in 10,000 normal cells. Samples with low mutational burden may appear as wild-type.
Indications for BCR/ABL1 testing and recommendations for testing:
ABL1 Mutational Analysis (performed by Fred Hutchinson Molecular Oncology Lab)Current therapy for CML and Ph+ALL generally involves one of several tyrosine kinase inhibitors (TKIs). These agents block the ATP binding site in the ABL1 kinase domain, maintaining the protein in an inactive conformation. Point mutations within this region prevent the binding of one or more TKIs, resulting in clinical resistance. Identification of ABL1 kinase mutations can result in early detection of TKI resistance. Mutations in the ABL1 region of the BCR/ABL1 oncogene have differing treatment options, depending upon the specific mutation. By sequencing and identifying mutations in ABL1, this assay will assist physicians in making treatment decisions for patients with CML who exhibit TKI resistance.
Peripheral blood or bone marrow aspirates are extracted to RNA and the BCR/ABL1 transcript is amplified by reverse transcription polymerase chain reaction (RT-PCR). The ABL1 kinase domain is further amplified in a semi-nested PCR reaction and is sequenced using Sanger sequencing. The ABL1 sequence is then compared to a wild-type reference sequence. The detection of ABL1 mutations is confirmed with a second primer. The ABL1 mutational analysis assay can be applied to samples that have tested positive for the p210 BCR/ABL1 transcript.