Demystifying PCR-Based Molecular Monitoring in CML

When imatinib, the first tyrosine kinase inhibitor (TKI), was approved for the treatment of chronic myeloid leukemia (CML) in 2001, the disease’s natural history and treatment were transformed. Before interferon was first used as a CML therapy in the 1980s, long-term survival for those diagnosed with CML was rare, with fewer than 15% of patients surviving to 8 years after diagnosis. By the 1980s and 1990s, with the introduction of combination interferon and chemotherapy such as cytarabine, 5-year survival improved to about 50%. Since 2001, after imatinib’s approval, the 5-year survival rate rose to 87%.¹ Patients on imatinib who achieve major molecular remission following 2 years of therapy can now expect to live as long as their healthy counterparts.²

Imatinib was revolutionary, representing the first so-called “molecularly targeted therapy” for cancer. Unlike less narrowly targeted therapies, imatinib binds to and blocks the activity of the ABL1 tyrosine kinase, which is responsible for the pathogenesis of CML.

Traditionally, patients’ bone marrow samples were used to perform metaphase cytogenetics analysis, to identify whether the Philadelphia chromosome was present and monitor response to treatment. Following treatment, if 20 or more metaphases contain no Philadelphia chromosome, the patient was considered to have a complete cytogenic response. Beginning in the 1990s, a larger number of chromosomes could be tested with fluorescent in situ hybridization (FISH) probes for the BCR-ABL1 fusion, but still only a few hundred cells could be analyzed.

Now, with the advent of polymerase chain reaction (PCR)–based molecular assays, “response to TKI therapy should always be monitored by the degree of molecular response, looking for the presence of BCR-ABL1 messenger RNA levels,” said Michael Deininger, MD, PhD, professor and Chief of Hematology and Hematologic Malignancies in the Department of Internal Medicine at the University of Utah and the Huntsman Cancer Institute. “This newer test has superseded the cytogenetics test for monitoring patients with CML on therapy.” This is in part because potent TKI therapy results in deep responses, and a molecular analysis is a much more sensitive way to detect any measurable residual disease compared with a cytogenetic test, he explained.

“In the first large trial of imatinib, which compared the TKI with interferon therapy, our lab and two other laboratories performed the molecular BCR-ABL1 assessment,” said Jerald Radich, MD, who specializes in the molecular genetics of leukemia at the Fred Hutchinson Cancer Research Center in Seattle. The test was then validated in other clinical labs and became the standard way to monitor CML. ASH Clinical News spoke with Drs. Deininger and Radich about the development of PCR molecular-based testing to monitor patients with CML, how it is used in CML, and how the testing technology is evolving.

The Advent of CML Molecular Monitoring

Back in the 1980s, Nora Heisterkamp, PhD, now a professor at the City of Hope department of systems biology in California, and her colleagues at the US National Cancer Institute discovered that the “Philadelphia chromosome,” which was present in almost all CML patients, was the result of a fusion of two genes, BCR and ABL1. Typically, these two genes are located on different chromosomes.³

Separately, Owen Witte, MD, professor at the University of California, Los Angeles in the departments of immunology, microbiology, and molecular genetics, together with colleagues at several institutions showed that the fusion BCR-ABL protein translated from the Philadelphia chromosome directly leads to CML.⁴

Because the level of BCR-ABL1 messenger RNA correlates with BCR-ABL1 protein and with the burden of disease, researchers subsequently developed a PCR-based molecular assay to monitor the levels of the BCR-ABL1 mRNA using patients’ peripheral blood samples. The test provides a unique way to monitor both the presence of disease and quantitate a patient’s disease burden.

The current method for molecular monitoring of BCR-ABL1 mRNA is quantitative reverse transcriptase (qRT)-PCR. Typically, the quantitative assay can detect just a few copies of BCR-ABL1 mRNA in a background pool of 100,000 total mRNA transcripts.⁵ This is then normalized to expression of a reference gene.

To perform qRT-PCR, total RNA is extracted from a patient’s blood or marrow sample. The single-stranded mRNA template is reverse transcribed to generate synthetic, double-stranded complementary DNA (cDNA), followed by real-time quantitative coamplification of the BCR-ABL cDNA and an internal control cDNA gene. In quantitative molecular assays, standard curves are constructed by serial dilutions of known amounts of cloned plasmid, an extrachromosomal DNA molecule that can replicate independently from chromosomal DNA, that contains the fusion DNA. These materials are part of the standard commercial kits now widely used to perform the assay. Results are usually available in 4-10 days.

“There is a way to use DNA to detect the BCR-ABL1 gene fusion, but because the break point where the BCR gene fuses to ABL1 spans a few thousand kilobases and differs from patient to patient, a PCR-based test based on that would require designing unique primers for the PCR test for each patient, which is why this method has not caught on,” explained Dr. Radich.

Which Kits Are Available?

There are currently three FDA-approved qRT-PCR tests to detect BCR-ABL:

• Asuragen’s QuantideX qPCR BCR-ABL IS Kit
• Bio-Rad’s QDx digital PCR kit
• Cepheid’s Xpert BCR-ABL Ultra test

Each test uses a different methodology to quantify BCR-ABL transcripts. The QuantideX qPCR BCR-ABL IS Kit can be used on a patient’s whole blood sample.

The QDx digital PCR kit employs so-called “digital” PCR, which offers a way to increase the signal-to-noise ratio for low-abundance transcripts. In the standard “analog” RT-qPCR method, technicians generate an absolute standard curve using an RNA sample with known amounts of BCR-ABL1 in parallel with a patient sample to extrapolate the previously unknown value of BCR-ABL1 transcripts in the patient’s sample.

With digital PCR testing, the patient’s sample is divided into thousands of nanoliter droplets that are amplified either in individual tiny wells or in emulsified “bubbles,” depending on the platform, resulting in a higher reaction efficiency than standard PCR testing. Each well or droplet optimally contains a single template molecule. If there is amplification, it is read as “positive” and assigned a digital code of 1; if there is no amplification, it is read as negative and assigned a score of zero. Because this digital scoring allows determination of the absolute quantity of molecules, generating a standard curve is unnecessary. This method produces tens of thousands of data points from a single sample and, according to one study, increases the limit of detection of the BCR-ABL1 mRNA by more than 1-log, compared with conventional qRT-PCR.⁶

The third test, Xpert BCR-ABL Ultra, uses an automated cartridge system that includes all the ingredients needed to quantitate the transcripts in a patient’s blood sample. This “automated” PCR molecular testing has the advantage of requiring fewer steps from a technician, providing results more efficiently and quickly and minimizing room for error.⁷

“[The PCR–based molecular assay] has superseded the cytogenetics test for monitoring patients with CML on therapy.”

—Michael Deininger, MD, PhD

“These three technologies provide, essentially, the same limits of detection,” said Dr. Radich. “Digital droplet PCR may be a little bit more sensitive, but essentially all provide a way to quantitative BCR-ABL transcript values over a 4- or 5-log range.”

“As patients achieve deep responses, the question is whether higher-sensitivity monitoring would be able to identify patients with particularly deep responses versus those with not-so-good responses who may continue to have active CML,” said Dr. Deininger.

Dr. Radich also highlighted another CML testing method in development: The use of dried blood spots that can be sent to a centralized lab and could be used in clinics located in limited-resource areas. This method uses less blood, and thus cannot be as sensitive as conventional methods, but can be stored and shipped over a period of weeks.

Testing, Standardized

According to the National Comprehensive Cancer Network and the European LeukemiaNet guidelines for CML, the RT-qPCR test is recommended to confirm a diagnosis.^8,9 Then, after a patient begins TKI therapy – either with imatinib or with one of the next-generation BCR-ABL TKI inhibitors (dasatinib, nilotinib, bosutinib, or ponatinib) – the guidelines suggest monitoring BCR-ABL transcript levels using RT-qPCR every 3 months.

“The values at 3 months, 6 months, and 12 months have been shown to be predictive of subsequent outcomes, which helps us categorize patients’ risk of subsequent therapy failure,” Dr. Deininger explained.

RT-qPCR is recommended every 3 months until BCR-ABL1 transcripts are less than 0.1%, then every 3 to 6 months.

Patients are categorized according to their risk of disease relapse or progression: “Optimal response” patients who can continue their same treatment; those who, after follow-up testing, need to immediately change their therapy; and ”warning cases” who are at risk and should be closely monitored to understand the best time to potentially change their therapy.

In the International Randomized Study of Interferon versus STI571 (IRIS) trial, imatinib-treated patients had at least a 3-log reduction in BCR-ABL1 transcript levels compared with the standardized baseline, which translated to a negligible risk of disease progression over a 12-month period.¹⁰ Based on these results, a major molecular response (MMR) is now defined as a 3-log reduction or a BCR-ABL1 transcript level of 0.1%. CML researchers led by Timothy Hughes, MD, at the University of Adelaide and the South Australian Health and Medical Institute then devised the international scale (IS), a molecular monitoring system that standardizes RT-qPCR results across laboratories worldwide using one of three control genes (BCR, ABL1, or GUSB). The IS has become the gold standard for expressing patients’ PCR values.

“The advantage of the international scale is that each lab can use its own assay, then can use a normalization factor to calculate the BCR-ABL transcript value on a relative scale,” said Dr. Deininger. “This way, all the results from different labs can be compared with each other. This is helpful because, with PCR in general, there is quite a lot of variation. The lower the BCR-ABL1 expression, the greater the relative variation becomes.”

However, according to both Drs. Radich and Deininger, physicians might not adhere to the RT-qPCR BCR-ABL1 testing guidelines for several reasons. First, since TKIs are effective, CML is often perceived as a relatively indolent form of cancer that does not pose an immediate risk to the patient’s life. Second, “CML is not that common compared with other tumor types,” said Dr. Deininger. “Many smaller oncology practices see only a few patients with CML and may not be that familiar with the guidelines.”

Dr. Radich also questioned whether the suggested frequency of monitoring is optimal. “We started doing the PCR monitoring every 3 months in the TKI clinical trials because that’s how we had previously done it in the setting of allogeneic transplant,” he said. “This practice became established in every clinical trial and, in clinical practice, it kind of just stuck.”

He suggested that, “if a patient has a deep response, we can decrease the frequency from every 3 months to less frequent monitoring after a few years. In practice, patients are not monitored as closely [as in clinical trials] and less frequent monitoring does not seem to be having a huge effect on their outcomes.”

The Rise of Resistance

While decreases in a patient’s q-RT-PCR BCR-ABL1 result signal a response to treatment, lack of reduction of transcript levels by at least 1-log (10%) 3 months or more after therapy can signal a suboptimal outcome. In addition, loss of a previously achieved response is worrisome. “If a patient’s disease was demonstrating a response, but then their BCR-ABL1 transcript numbers start to climb, that means they are either not taking their drug as prescribed or their clinician should be looking for a potential resistance mutation and switching them to a different TKI,” said Dr. Radich.

If resistance is suspected, clinicians need to identify a novel resistance mutation swiftly, so that the patient can switch to a different TKI therapy that could inhibit the ABL protein harboring the mutation – potentially thwarting the emerging resistant clone. Next-generation sequencing is typically performed to identify the presence of a mutation within ABL.

Changes to Come?

For Dr. Radich, it is possible for a RT-qPCR test to be too sensitive. “The PCR techniques are getting more sensitive and, while more sensitive intuitively sounds better, at some point, we will begin to pick up cases of minute residual disease that would never have relapsed,” he said. “So, the question is, ‘How deep do we need to go?’”

Another question on the horizon is whether researchers can use PCR-based molecular monitoring to identify patients who have achieved a deep long-term response and could discontinue their TKI, without raising their risk of relapse.

Finally, researchers have begun to study whether patients can take responsibility for their own results, Dr. Deininger told ASH Clinical News. He cited an ongoing study in Europe in which patients with CML have access to their RT-qPCR results. “This is emerging as a useful way to improve compliance for our patients,” he said. “They have to take destiny into their own hands.” —By Anna Azvolinsky