Quantitative Reverse Transcriptase Polymerase Chain Reaction (qRT-PCR)

What the test measures and its relationship to other tests.

At diagnosis, virtually every white cell in a blood or marrow sample will be leukaemic (Ph+) so the result should, in theory, be 100% Ph+. However, because there are higher levels of Ph+ cells present at diagnosis, q-PCR testing is not accurate, which is why Ph positivity varies between 50% and 100%. This test may be used to establish a baseline value of Ph+ cells at diagnosis.

After the start of therapy q-PCR is used at specific time points after cytogenetic/FISH tests. Once tests show that the Ph+ cell population has reduced to less than 10%, q-PCR testing can more accurately quantify the amount of residual disease left in the marrow.

The goal of TKI therapy is to reduce the abnormal BCR-ABL1 gene to a deep molecular level, preferably to at least 0.1% (MMR/MR3).
During the first 3, 6, 9 and 12 months of therapy Ph+ cells should reduce significantly. When the level of Ph+ cells falls below 1% q-PCR testing is extremely accurate and will be used to monitor the stability of a molecular response. Under ideal conditions, this test can detect
1 Ph+ cell in every 100,000 cells, although more commonly it detects 1 Ph+ cell in every 10,000.

The BCR-ABL1 gene and its mRNA, the Protein Tyrosine Kinase Bcr-Abl1

Chromosomes are found in a cell’s nucleus and are made up of tightly wound stretches of DNA, the genetic code essential for the life of the cell and therefore the life of the individual.

The nucleus is a protected environment, nothing can get inside it and neither can DNA move outside it. In order to deliver instructions (as code) for a myriad of cell processes, short stretches of DNA are duplicated as molecules known as RNAs, of which there are several forms. The RNA we are interested in here is messenger RNA or mRNA.

RNA travels outside the nucleus into the cell cytoplasm where protein tyrosine kinases are formed. Proteins express signals, setting in motion a variety of cell processes including division, proliferation and cell death (apoptosis).

In CML the BCR-ABL1 gene duplicates its coded instruction as a messenger RNA (mRNA). In this form the DNA code moves outside of the nucleus into the cytoplasm where the protein Bcr-Abl1 signals the Ph+ cells to divide in a deregulated (leukaemic) manner.

Tyrosine kinase inhibitors (TKIs) target the abnormal protein Bcr-Abl1, effectively blocking the signal for continuous Ph+ cell division. This reduces the abnormal Ph+ cell population to very low levels along with the clinical manifestations of CML.

Continued TKI therapy is highly effective over the longer term allowing the majority of patients to live out their normal life-span.