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BCR/ABL

Quantitative p210 BCR/ABL

Qualitative p190 BCR/ABL

References

Quantitative p210 BCR/ABL

Background

The Philadelphia (Ph) chromosome is the result of a reciprocal translocation between chromosomes 9 (ABL gene) and 22 (BCR gene) resulting in deregulated ABL tyrosine kinase (TK) activity.  The breakpoints in the BCR gene are predominately in the major breakpoint cluster region (M-BCR) of chromosome 22 (exon 2 or exon 3), but can also occur at a second upstream breakpoint in the BCR gene (exon 1), which occurs almost exclusively in Philadelphia positive acute lymphoblastic leukemia (Ph+ALL).  Transcripts from the M-BCR encode a BCR/ABL protein of 210kDA, called p210 BCR/ABL.  Patients can have transcripts from one or multiple breakpoints. 

Clinical Significance

Diagnosis: The presence of the p210 BCR/ABL mRNA is consistent with either chronic myeloid leukemia (CML) or acute lymphoblastic leukemia (ALL).  These two diseases are also distinguishable by other clinical and pathological findings.

Transplant setting:  The presence of p210 BCR/ABL mRNA in the 6-12 months post-transplant is associated with an increased risk of relapse1.  The quantitative assay defines the risk of relapse:  an approximately 1 log increase between two time points is considered clinically significant for an increased risk of relapse2, 3.

Non-transplant setting: BCR/ABL levels are correlated with response in CML.  In the IRIS trial, the level of BCR/ABL at 12 months post initiation of treatment with a Tyrosine Kinase Inhibitor (TKI) was associated with outcome4.  In this trial, a 3-log reduction from baseline was defined as a major molecular response (MMR).  CML chronic phase patients who had an MMR had a >95% progression free survival rate. In addition, an increase in BCR/ABL levels while receiving TKI therapy was associated with the subsequent occurrence of a TKI resistant mutation5.  Thus, the recommendations of the National Comprehensive Cancer Network (NCCN) is to obtain peripheral blood BCR/ABL testing every 3 months for all patients on TKI therapy after achievement of complete cytogenetic response (CCR)6.

 

Method

The Cepheid GeneXpertTM combines RNA extraction, nucleic acid amplification and detection of the BCR/ABL target using real-time reverse transcription polymerase chain reaction (RT-PCR).  The BCR/ABL test is a nested, multiplex assay capable of detecting BCR/ABL p210 translocation (exon 2 or exon 3) and the ABL endogenous control.  Quantitation of BCR/ABL is determined by a ratio of BCR/ABL to ABL.  The GeneXpertTM is able to detect a singe BCR/ABL copy within 100,000 normal cells.  To assure accuracy, patient samples are run in duplicate.

Method before 02/01/2007

RT-PCR is used to detect BCR/ABL RNA transcripts.  White blood cells (WBCs)  are separated from the bone marrow aspirate or peripheral blood sample by red blood cell lysis and subsequent washing.  Total RNA is extracted from the WBCs using the TRIzolTM method.  A RT-PCT reaction is followed by a nested PCR amplification of the target sequence.  Presence or absence of the amplified sequence is visualized using agarose gel electrophoresis.  Patient samples are run in duplicate with appropriate positive and negative controls.  The RNA quality is assessed by RT-PCR of Beta-2 microglobulin mRNA.  This highly sensitive technique can identify a single BCR/ABL transcript in a dilution of 100,000 to 1,000,000 normal cells.

Qualitative p190 BCR/ABL

Background

The Philadelphia (Ph) chromosome is the result of a reciprocal translocation between chromosomes 9 (ABL gene) and 22 (BCR gene) resulting in deregulated ABL tyrosine kinase (TK) activity.  The breakpoints in the BCR gene are predominately in the major breakpoint cluster region (M-BCR or p210) region of chromosome 22 (exon 2 or exon 3) but can also occur at a second upstream breakpoint in the BCR gene (exon 1), which occurs almost exclusively in Philadelphia positive acute lymphoblastic leukemia (Ph+ALL).  Transcripts from the minor breakpoint region (m-BCR) encode a BCR/ABL protein of 190kDA, called p190 BCR/ABL.  Patients can have transcripts from one or multiple breakpoints.  P190 (m-BCR) analysis is QUALITATIVE ONLY at this time and is performed on all Ph+ALL patient samples and on CML samples if specifically requested.

Clinical Significance

Diagnosis: The presence of the BCR/ABL mRNA is consistent with either chronic myeloid leukemia (CML) or acute lymphoblastic leukemia (ALL).  The p190 breakpoint (exon 1) is found predominately in patients with ALL and rarely in patients with CML.  The presence of the p210 breakpoint (exons 2 and 3) is consistent with either CML or ALL.  These two diseases are also distinguishable by other clinical and pathological findings.  Presence of any of the three breakpoints in ALL is associated with aggressive disease and has been shown to be a poor prognostic factor.

Transplant setting:  The presence of BCR/ABL mRNA in the 6-12 months post-transplant is associated with an increased risk of relapse and the risk to patients with the p190 variant may be higher.

Methods

RT-PCR is used to detect BCR/ABL RNA transcripts.  White blood cells (WBCs) are separated from the bone marrow aspirate or peripheral blood sample by red blood cell lysis and subsequent washing.  Total RNA is extracted from the WBCs using the TRIzolTM method.  A RT-PCR reaction is followed by a nested PCR amplification of the target sequence.  Presence or absence of the amplified sequence is visualized using agarose gel electrophoresis.  Patient samples are run in duplicate with appropriate positive and negative controls.  The RNA quality is assessed by RT-PCR of Beta-2 microglobulin mRNA.  This highly sensitive technique can identify a single BCR/ABL transcript in a dilution of 100,000 to 1,000,000 normal cells.


References


1. Blood (1995);85:2632-2638

http://bloodjournal.hematologylibrary.org/cgi/content/abstract/85/9/2632

2. Blood (2001);98:1701-1707 http://bloodjournal.hematologylibrary.org/cgi/content/abstract/98/6/1701

3. Blood (2001);97:1560-1565 http://bloodjournal.hematologylibrary.org/cgi/content/abstract/97/6/1560

4. NEJM (2003);349:1423-1432
http://nejm.highwire.org/cgi/content/abstract/349/15/1423

5. Blood (2004);104:2926-2932 http://bloodjournal.hematologylibrary.org/cgi/content/abstract/104/9/2926

6. J Natl Compr Canc Netw (2005);3:732-755

7. Cepheid GeneXpert Dx System.  v.1.6

8. Clinical Chemistry (2007); 53: 1593-1600
http://www.clinchem.org/cgi/content/abstract/53/9/1593

9. Blood (2006);108:28-37
http://bloodjournal.hematologylibrary.org/cgi/content/abstract/108/1/28

10. Blood (1997);89:2602-2609 http://bloodjournal.hematologylibrary.org/cgi/content/abstract/89/7/2602

11. Biology Blood and Marrow Transplant (2003);9:206-212
http://www.bbmt.org/article/S1083-8791(03)70011-1/abstract

Questions pertaining to testing may be addressed to the Molecular Oncology Laboratory at 206-667-2592