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Hematopathology Section

Hematopathology Laboratory

Molecular Hematopathology

Testing for Hematopoietic Malignancies

Considered Diagnosis

Common Molecular Abnormality

Current Molecular Hematopathology Assay

FL / DLBCL B-cell clonality
IgH clonality, IgK clonality
BCL2 (MBR, MC7, MC8)
MCL B-cell clonality
IgH clonality, IgK clonality
CLL / MM / other B-cell ALL B-cell clonality IgH clonality, IgK clonality
T-cell NHL T-cell clonality TCRγ
CML / B-cell ALL t(9;22)"Philadelphia (Ph) chromosome" quantitative BCR/ABL
qualitative BCR/ABL
PV/ PMF / ET / other CMPD JAK2 V617F mutation JAK2 point mutation
AML Flt3 Internal Tandem Duplication (IDT)
NPM1 mutation
CEBPA mutaton
NPM mutation
CEBPA mutaton


FL Follicular lymphoma
DLBCL Diffuse large B-cell lymphoma
MCL Mantle cell lymphoma
NHL non-Hodgkin's lymphoma
CLL Chronic lymphocytic leukemia
MM Multiple myeloma
CML Chronic myelogenous leukemia
ALL Acute lymphoblastic leukemia
CMPD chronic-phase myeloproliferative disorders
PV Polycythemia vera
ET Essential thrombocythemia
PMF Primary myelofibrosis
AML Acute myeloid leukemia

B-cell clonality

Monoclonal 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 5 multiplex primer sets: 3 primer sets for detecting clonal populations with rearranged IgH genes and 2 primer sets for detecting clonal populations with rearranged IgK genes. The multiplex primer sets for IgH clonality each recognize 1 of 3 conserved "Framework Regions" (FR1, FR2, or FR3) of IgH variable gene segments (VH). Each multiplex IgH primer set contains 6 or 7 VH primers in combination with a single consensus joining region (JH) primer. IgK testing consists of two multiplex primer sets that bind to conserved regions on either side of the V-J region where programmed genetic rearrangements occur during maturation of B cells. The IgK "A" multiplex primer set contains six kappa variable (VK) region primers paired with two joining (JK) region primers. The IgK "B" multiplex primer set contains the same six kappa variable region primers and an intronic recombination signal sequence (RSS) primer which are paired with a kappa deleting element (KDE) reverse primer. One of the primers in each multiplex primer set 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 rearrangement and joining of the IgH or IgK gene segments. 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 rearrangement and thus produce a PCR product of one 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 1% of the total B-cells in order to be detectable as a clonal peak in a polyclonal distribution of background peaks by the IgH clonality assay. In order to be detectable by the IgK clonality assay, the lymphoma cells must represent at least 5% of the total B cells in a mixed population of B-cells. Approximately 95% of B-cell lymphomas produce a clonal PCR product when both the BIOMED-2 IgH and IgK primer sets are tested. For patients whose lymphoma has been shown to have a detectable clonal B-cell population, these assays can be a sensitive detector of minimal residual disease in post treatment, B-cell depleted specimens.

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BCL1 [also known as t(11;14) or cyclin D1]

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 the J regions of the IgH gene 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 patient's mantle cell lymphoma cells are positive by this assay, it is a very sensitive detector of minimal residual disease. 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.

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BCL2 [also known as t(14;18)]

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) on chromosome 18 that can be detected using an MBR1 primer in conjunction with a consensus JH 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 JH primer. If a patient's 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.

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Mutations in the transcription factor CCAAT/enhancer binding protein (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.

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T-cell clonality

Monoclonal populations of T lymphocytes can be identified by testing for T cell receptor γ chain (TCRγ) gene rearrangements using a polymerase chain reaction assay. In its germline configuration the TCRγ gene consists of 6 functional V gene segments, 8 V pseudogene segments and 5 J segments. There is enough homology among the V region segments and among the J region segments to allow use of small numbers 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 a discrete peak. Approximately 95% of T cell lymphomas produce a PCR product using our primer set. In a mixed population of T lymphocytes, a neoplastic clone must represent about 1% of the total T cells in order to detect a clonal peak in a polyclonal distribution of background peaks.


A 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 flanking primers (one HEX-labeled, the other NED-labeled) and then size fractionated by capillary electrophoresis. The FLT3 ITD length is calculated as the difference in bases between the length of amplified FLT3 ITD and normal allele products. The FLT3 ITD to Normal Ratio is calculated by dividing the peak height of the ITD product by that of the normal allele 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 HEX and NED labeled products of approximately 330 nucleotides (nt), whereas, amplification of genomic DNA containing the FLT3 ITD mutation usually yields the normal 330 nt product as well as one (or rarely more than one) longer HEX and NED labeled product(s). This test should be performed on AML patients at diagnosis on EDTA anticoagulated peripheral blood or bone marrow containing neoplastic blasts.

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Insertion 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 flanking primers (one HEX-labeled, the other unlabeled) and then size-fractionated by capillary electrophoresis. Amplification of normal genomic DNA results in a predominant HEX-labeled 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.

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The 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 2 differentially labeled products: a 364-nt HEX-labeled product in addition to a 203-nt product labeled with both FAM and HEX.

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Qualitative BCR/ABL

A multiplex reverse transcriptase polymerase chain reaction is used to detect the BCR-ABL transcript in Philadelphia chromosome positive (Ph+) Chronic Myelogenous Leukemia (CML) and Acute Lymphoblastic Leukemia (ALL) cells. The BCR-ABL fusion gene is formed by a translocation between chromosomes 9 and 22, which results in the joining of ABL exon 2 and either BCR exon 2 or BCR exon 3 (b2:a2 or b3:a2). The BCR-ABL mRNA formed from this gene codes for a chimeric p210 protein in 90-95% of CML patients. The Philadelphia chromosome can also be found in 30-50% of patients with ALL, and rarely, in Acute Myelogenous Leukemia patients. The majority of Ph+ ALLs have a more 5' BCR breakpoint and result in mRNA, e1:a2, which codes for the chimeric p190 protein. Our assay is a nested multiplex RT-PCR. The initial PCR reaction includes primers for detection of p190 and p210 BCR-ABL transcripts. A second round of amplification with primers internal to those in the first found is performed using 5 µl of the first round product. The second round of PCR is included to insure specificity and increase sensitivity.

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Quantitative BCR-ABL

This test is a reverse transcriptase polymerase chain reaction which is designed to quantify the BCR-ABL transcript in specimens from Philadelphia chromosome positive (Ph+) Chronic Myelogenous Leukemia (CML) and Acute Lymphoblastic Leukemia (ALL) patients. In this assay, RNA is extracted from the patient's leukocytes and reverse-transcribed to produce cDNA. The cDNA is subjected to 2 separate PCR reactions, one each in which BCR-ABL and glucose-6-phosphate dehydrogenase (G6PDH) messages are amplified. The reaction is monitored in real time using fluorescent detection, and the quantity of each RNA is determined based on the PCR cycle at which the fluorescent signal appears. The BCR-ABL reaction is positive if detectable numbers of CML cells are present (approximately 1 CML cell in 10,000 normal cells). The G6PDH reaction is used as a control for the quality of the RNA and also as normalization reference RNA. The result is reported as a ratio (in percent) of the amount of BCR-ABL RNA to G6PDH RNA. By performing this assay on serial blood or bone marrow specimens, the quantitative results can be followed over time to determine whether the number of leukemic cells is increasing or decreasing. The assay is a multiplex PCR which includes forward primers for PCR in BCR-exons e1 and b2 that can both combine with reverse primer in ABL-exon 4. The PCR can detect fusion transcripts resulting from the breakpoints b3a2, b2a2, and e1a2.

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Last updated:11/1/10

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