Myeloid Leukemia

72 Branford and Hughes
The majority of CML patients have breakpoints that result in a fusion mRNA
in which either BCR exon 13 (known as b2 for its location within the major
breakpoint cluster region of the BCR gene) or exon 14 (b3) is fused to ABL
exon a2 to form the b2a2 and b3a2 transcripts. The resultant fusion protein
encoded by BCR-ABL is 210 kd (p210). However, a small number of patients
have breakpoints outside of the standard region and various BCR exons may be
fused to either ABL exon 2 or 3 (reviewed in ref. 20). The resultant atypical
fusion proteins vary in size but maintain an activated tyrosine kinase. Rare
cases of CML involve the e1a2 transcript, which is more commonly seen in
patients with acute lymphoblastic leukemia, and the e19a2 transcript, which
may be associated with chronic neutrophilic leukemia (21,22). Aberrant fusion
transcripts have been described that involve the insertion of intronic sequences
(23–25) and breakpoints within coding regions (26). When using a quantitative
PCR technique to monitor treatment response in CML it is necessary to characterize
the BCR-ABL breakpoint to exclude false-negative results. Our RQ-PCR
technique evaluates the b2a2 and b3a2 transcripts only. Therefore, an investigation
for an atypical transcript is undertaken by a qualitative PCR technique if
a newly diagnosed patient with CML has a negative BCR-ABL value by RQ-
PCR. The PCR technique uses primers that span BCR exon 1 and ABL exon 3.
Amplification products of atypical lengths are sequenced to characterise the
BCR-ABL breakpoint. This procedure is particularly relevant for patients with
Philadelphia chromosome-negative CML. In these cases the presence of a BCR-
ABL transcript must be demonstrated in order for the patient to receive imatinib
therapy.
2. Materials
2.1. RNA Extraction
1. Lysis buffer: 0.144 M NH 4Cl and 0.01 M NH 4HCO 3. Autoclave and store at room
temperature. Stable for 6 mo. The lysis buffer is prepared from stock solutions of
1.44 M NH 4Cl (autoclave) and 1.0 M NH 4HCO 3 (avoid inhaling fumes during
preparation and do not autoclave because the fumes are overpowering if autoclaved).
Stable for 12 mo at room temperature.
2. Sterile 50-mL tubes (Becton Dickinson).
3. Sterile mixing cannula.
4. RNAse- and DNAse-free 1.5-mL and 2.0-mL microcentrifuge tubes (Quantum
Scientific).
5. 18-gauge blunt needles (Becton Dickinson) and 3-mL syringes.
6. Barrier or filter pipet tips.
7. Trizol RNA isolation solution (Invitrogen). Store at 2–8°C. Trizol is used in a
laminar-flow hood. Use gloves and eye protection. If skin contact occurs, apply
glycerol to the site immediately.