3. Case Study A 60 year-old woman was recently diagnosed with chronic myelogenou

Question

3. Case Study A 60 year-old woman was recently diagnosed with chronic myelogenous leukemia (CML). Her physician requested a t(9:22) translocation test. Cytogenetic analysis of 20 metaphases derived from culture of her peripheral blood was reported as 46, XX, t(9:22). Molecular analysis of the t(9:22) translocation was performed by reverse transcriptase-PCR using Forward primers that are complementary to the major and minor breakpoints in the BCR gene on chromosome 22 Reverse primer complementary to the c-abl gene on chromosome 9 Forward and reverse primers complementary to c-abl were included in the reaction mixture to serve as an RNA integrity (amplification) control o o o After reverse transcriptase -PCR, the products were separated by gel electrophoresis, and bands were visualized by ethidium bromide staining. PCR product was seen for: The c-abl amplification control in the lane containing patient specimen PCR product was seen in the patient lane, the size consistent with a product derived from a t(9:22) translocation utilizing the b3a2 breakpoints a) What is the interpretation of these results? b) Why do we use an internal RNA integrity control? c) Are these results consistent? d) Is additional laboratory testing warranted to confirm the diagnosis of CML?

Explanation / Answer

a) Chronic myeloid leukemia (CML) is a blood cancer which is involved to have a translocation between 9 and 22 chromosome denoted as t(9;22) (q34;q11). In all CML patients the breakpoint occurs as major (M-bcr). This breakpoint within M-bcr which in e13a2 ( b2a2) or e14a2 (b3a2) determines two main types of the fused BCR-ABL m RNA which is defined as b2a2 and b3a2 transcripts which differs by 75 nucleotides.

Interpretation of result

It is a BCR-ABL transcripts e14a2 (b3a2). When compared to the e13a2 (b2a2) transcripts the patient with e14a2 (b3a2) transcript will response to drug better and deeper response at earlier stage showing a longer event-free survival.

Note: Here t: indicates translocation and q: is q arm of the chromosome (lower portion) and e: is the exon in the chromosome and a: indicates ABL gene b: BCR gene.

b)RNA is used as internal control to confirm the quality and integrity of the RNA because RNA is instable nucleic acid, which is a key element for testing the BCR-ABL fusion in CML. It is a assurance to confirm the RNA integrity and the reliability of the reaction or test.

c) No, these result are not consistent. After treatment there will an improvement or no improvement in the result (difference in the transcript levels which is based on the patient response towards the given drug).

d)BCR/ABL transcripts should be measured quantitatively using Real-time PCR method for follow-up of the treatment. No other additional test is required to confirm the diagnosis of CML.

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