Translocation RQ-PCR Assays
Chronic Myeloid Leukaemia (CML) accounts for 15-20% of adult leukaemias. CML is a clonal haematopoietic stem cell malignancy that results in the formation of a BCR-ABL cytoplasmic fusion oncoprotein as the result of the reciprocal translocation between the long arms of chromosome 9 and chromosome 22 t(9;22) The disease is characterized by excess proliferation of myeloid progenitors that retain the capacity for differentiation during the stable or chronic phase of the disease. In addition the BCR-ABL fusion is also seen in ALL albeit more rarely. In this disease the BCR-ABL fusion is the result of a slightly different break point and results in the p190 BCR-ABL fusion protein.
A quantitative PCR (Q-PCR) utilizing the Applied Biosytems 7900 Taqman platform is used for the highly sensitive quantitative detection of BCR-ABL fusion transcripts (p210 and p230) in order to monitor disease progression and residual disease. The procedure used at KCH is harmonized to the Europe Against Cancer (EAC) guidelines to ensure that we provide highly accurate and comparable BCR-ABL / ABL transcript ratios. In addition we are one of a small group of laboratories in the UK currently engaged with the regional genetics laboratory to obtain an international conversion factor. This will enable our results can be directly compared with those of any other participating laboratory across the world. This provides even greater consistency which is desirable particularly in multi center drug trails. Our conversion factor exercise will be completed in early 2010. A separate Q-PCR assay is used to detect the p190 variant.
Persistent Q-PCR positivity after treatment is indicative of possible drug resistance and predictive of clinical relapse. A molecularly negative result is associated with disease free survival. Patients who convert to Q-PCR positivity after a negative result are at high risk of relapse. These patients may be candidates for 2nd and 3rd generation tyrosine kinase inhibitors. Failure to respond optimally to Imatinib can be caused by mutation in the p-loop domain of the translocated ABL kinase gene. Our repertoire of tests within the LMH also includes Abl kinase p-loop mutation screening which can be carried out on samples previously sent to the laboratory for BCR-ABL monitoring by request
Presence of heparin anticoagulant will inhibit PCR applications. Clotted samples are unsuitable for DNA analysis.
Samples must be clearly labelled with the patient's first name, surname, D.O.B, hospital number and the date the sample was taken.
The Rayne Institute
King's College Hospital
123 Coldharbour Lane
London SE5 9NU
Blood Sciences Laboratory
Ground Floor Bessemer Wing
King’s College Hospital
Denmark Hill
London SE5 9RS
Mon-Fri, 9.00am-5.30pm
Radich 2009 Blood 114: 3376-3381
Emig M, Saussele S, Wittor H, Weisser A, Reiter A, Willer A, Berger U, Hehlmann R, Cross N, Hochhaus A. Accurate and rapid analysis of residual disease in patients with CML using specific fluorescent hybridization probes for real time quantitative RT-PCR. 1999 Leukaemia, 13, 1825-1832
Kreuzer K, Lass U, Bohn A, Landt O, Schmidt C. LightCycler technology for the quantitation of bcr/abl fusion transcripts. 1999 Cancer Research 59, 3171-3174
2. RUNXI-RUNXTI t(8;21)
The t(8;21) translocation is detected in approximately 7% of de novo AML (20-40% in the M2 subtype). The fusion gene encodes a chimeric protein, which blocks myeloid differentiation and may be important in myeloid leukemogenesis. AML patients with t(8;21) have a relatively good prognosis.
This PCR reaction is designed to detect the fusion transcript AML1-MTG8(ETO) in t(8:21) positive AML patients at presentation and for accurate monitoring of residual disease level to identify patients ate high risk of relapse.
Tobal K, Yin JA. Monitoring of minimal residual disease by quantitative reverse transcriptase-polymerase chain reaction for AML1-MTG8 transcripts in AML-M2 with t(8; 21). Blood. 1996 Nov 15;88(10):3704-9.
Tobal K., et al (2000) Molecular quantitation of minimal residual disease in acute myeloid leukemia with t(8;21) can identify patients in durable remission and predict clinical relapse. Blood. 2000 Feb 1;95(3):815-9.
Van Donegan J.J et al 1999 Standardized RT-PCR analysis of fusion gene transcripts from chromosome aberrations in acute leukemia for detection of minimal residual disease in acute leukemia. Report of the BIOMED-1 Concerted Action: investigation of minimal residual disease in acute leukemia. Leukemia 13 1901-1928
3. PMLRARA; t(15;17)
This PCR reaction is designed to detect the fusion transcript PMLRARA in patients with APML at presentation and for monitoring the disease.
The t(15;17) chromosomal translocation is a specific characteristic associated with acute promyelocytic leukaemia (APL, AML –M3). Two chimeric genes PML-RARA and RARA-PML are detected in 100% and 40% of AML patients respectively. The PML breakpoints cluster in three regions intron 6 BCR-1 (L-long-form), exon 6 BCR-2 (V-variable-form) or intron 3 BCR-3 (S-short-form) that fuse with the 3 prime portion of RARA intron 2. The isoforms occur in 55-60%, 35-40% and 8% of APL patients respectively. Several groups have reported that molecular monitoring of PML-RARA to evaluate MRD is clinically valuable in APL.
RT-PCR positivity after treatment is predictive of clinical relapse and a negative result is associated with disease free survival. Patients who convert to PCR positivity after a negative result are at high risk of relapse.
Tobal et al (2001). Monitoring minimal residual disease and predicting relapse in APL by quantitating PML-RARalpha transcripts with a sensitive competitive RT-PCR method.Leukemia.,15(7):1060-5.
Lo Coco F et al 1992 Molecular evaluation of residual disease as a predictor of relapse in acute promyelocytic leukemia. Lancet 340 1437-1438
Gallagher RE et al. 2003. Quantitative real-time RT-PCR analysis of PML-RAR alpha mRNA in acute promyelocytic leukemia: assessment of prognostic significance in adult patients from intergroup protocol 0129. Blood;101(7):2521-8.
Grimwade 2002. The significance of minimal residual disease in patients with t(15;17).
Best Pract Res Clin Haematol;15(1):137-58. Review.
4. CBFB-MYH11 INV(16)
This PCR reaction is designed to detect this fusion transcript in patients with AML at presentation and for monitoring the disease.
The CBFB gene encodes a Beta subunit of translocation factor, CBFB, and alterations of CBFB are closely associated with AML. The MYH11 gene encodes the smooth muscle myosin heavy chain. The chimeric gene contains the 5 prime region of CBFB gene fused to the major region of MYH11 gene. The fusion protein impairs neutrophil development and alterations in the CBFB gene can contribute to the genesis of myelodysplasia.
The inversion of chromosome 16 (inv 16) and the translocation t(16;16)is a characteristic chromosomal aberration found in the M4Eo subtype of acute myeloid leukaemia (AML). This abnormality is detected in 7-10% of de novo AML (90% of M4Eo subtype) but is not limited to this subtype. The alternative splicings of the genes result in different transcripts. The majority of individuals with AML have type A, type D and E is found in a minority.
Shurtleff S.A. et al 1995 Heterogeneity in CBF beta/MYH11 fusion messages encoded by the inv(16)(p13;22) and the t(16;16)(p13;22) in acute myelogenous leukemia. Blood 85 3695-3703
Liu P et al 1993 Fusion between transcription factor CBF beat/PEBP2 beta and myosin heavy chain in acute myeloid leukemia. Science 261 1041-1044
Tobal et al 1995. Detection of CBFB/MYH11 transcripts in patients with inversion and other abnormalities of chromosome 16 at presentation and remission. Br J Haematol;91(1):104-8.
Van Donegan J.J. et al 1999 standardised RT-PCR analysis of fusion gene transcripts from chromosome aberrations in acute leukemia for detection of minimal residual disease in acute leukemia. Report of the BIOMED-1 Concerted Action: investigation of minimal residual disease in acute leukemia. Leukemia 13 1901-1928
Kogan S.C. et al 1998 The PEBP2-beta-MYH11 fusion created by inv(16)(p13:22) in myeloid leukemia impairs neutrophil maturation and contributes to granulocytic dysplasia. Proc. Natl. Acad. Sci 95 11863-1186
5. IgHBCL2 t(14;18)
This PCR reaction is designed to detect the fusion transcript IgH-BCL2 in patients with t(14:18) positive Leukemia at presentation and for monitoring the disease.
The t(14;18) translocation involves the bcl-2 oncogene on chromosome 18 and the immunoglobulin heavy chain locus on chromosome 14. Two different regions of the bcl-2 gene on chromosome 18 are known. One of the Major breakpoint region (mbr) is located in a 200bp segment of the 3’ untranslated region of exon 3 of the bcl-2 gene. 70% of the breakpoints on chromosome 18 are clustered in this region. The second breakpoint the minor breakpoint region (mcr) is located about 20kb downstream of the mbr within an 800bp segment. The breakpoint on chromosome 14 is at the 5’ end of the six JH segments of the human IgH locus.
Kluin-Nelemans H et al 1991 A new Non-Hodgkins B cell line (DOHH2) with chromosomal translocation t (14; 18)(q32; q21) Leukemia 5 221-224
Lopez-Guillermo A. et al 1999 Correlation of Bcl-2 rearrangement with clinical characteristics and outcome in indolent follicular lymphoma. Blood 93 3081-3087
Hosler G et al 199 Development and validation of a quantitative polymerase chain reaction assay to evaluate minimal residual disease for T cell acute lymphoblastic leukemia and follicular lymphoma. Am. J. Pathol. 154 (4) 1023-1035
Moos M et al 1998 The remission status before and the PCR status after high dose therapy with peripheral blood stem cell support are prognostic factors for relapse free survival in patients with follicular non Hodgkins lymphoma. Leukemia 12 1971-1978
Dolken L et al 1998 Quantitative detection of t (14; 18) positive cells by real time quantitative PCR using fluorogenic probes. Biotechniques 25 1058-1064
Dolken L et al 1996 Bcl-2/JH rearrangements in circulating B cells of healthy donors and patients with nonmalignant diseases. J. Clin. Oncol 14 1333-1344
Johnson P et al 1996 Polymerase Chain reaction for detection of the t (14; 18) translocation in lymphomas from Methods in Molecular Diagnostics of Cancer ed F.E. Cotter Humana Press Inc NJ p63-74
Ji W. et al 1995 Frequent detection of bcl-2/JH translocations in human blood and organ by a quantitative polymerase chain reaction. Cancer Res 55 2876-2882
Bohling S et al 1998 Rapid simultaneous amplification and detection of the mbr1/JH chromosomal translocation by fluorescence melting curve analysis. Am J. Pathol 154 97-103
6. FIP1L1-PDGFRA
For determining the presence or absence of the FIP1L1-PDGFRA fusion transcript produced by an interstitial deletion at chromosome 4q12 that is seen in some cases of hypereosinophilic syndrome and systemic mast cell disease. This test can be used for diagnosis. In some patients these diseases are associated with the FIP1L1-PDGFRa fusion, and therefore respond well to treatment with imatinib (Gleevec®). Although HES is relatively rare, accurate diagnosis can identify patients who may be treated effectively. This analysis is referred to the Leukaemia Research Group, Wessex Regional Genetics Laboratory, Salisbury District General Hospital - please note TAT for FIP1L1-PDGFRa analysis will be up to 1 month accordingly.
FIP1L1-PDGFRA fusion: prevalence and clinicopathologic correlates in 89 consecutive patients with moderate to severe eosinophilia Pardanani et al Blood 2004 vol. 104 no.10 pp3038-3045
Last updated: 07/08/2015
