Making a Difference with Microdeletions

Acute lymphoblastic leukemia (ALL) is the most common cancer of childhood – and despite its five-year survival rate of over 85 percent (1), it also remains the most common cause of cancer-related death in children. What factors influence a child’s likelihood of survival after a diagnosis of ALL? Age, subtype, white blood cell count at diagnosis, and spread of disease (for instance, into the central nervous system fluid) can all have a significant impact – but genetic abnormalities are a key contributor. Alterations to the chromosomes can take many forms, including translocations, deletions and aneuploidies. All of these can occur in ALL – and when they do, it’s likely that they can affect a patient’s treatment response, relapse risk, and ultimate likelihood of survival.

Minimal residual disease (MRD) – the continued presence of residual cancer cells in the patient’s bone marrow after treatment – is a vital factor when it comes to predicting the potential relapse and overall disease prognosis of ALL patients. Even a single cancer cell can reproduce and seed a disease recurrence that may be much more difficult to treat than the initial presentation. Fortunately, sensitive molecular techniques can detect residual disease based on unique gene rearrangements in B- and T-cell lineages and on recurrent genetic abnormalities characteristic of leukemias. Using this information in conjunction with other patient data, we can reasonably approximate the risk that a given patient will relapse, allowing us to plan treatment in accordance with that risk.

Identifying risk

High-risk patients are defined by either adverse genetics or poor response to therapy. The genetic features that define high-risk ALL include Philadelphia chromosome (BCR-ABL1 fusion gene) positivity or the mixed lineage leukemia gene rearrangement now known as KMT2A. We define poor response to therapy by blast percentage count following the first seven days of treatment (poor prednisolone response at day 8) or high MRD taking into account levels on days 33 and 79 of therapy. In contrast, standard-risk patients are defined by a lack of high-risk features, a lack of central nervous system involvement, and excellent MRD response (with PCR negativity when tested with a highly sensitive marker at days 33 and 79). Medium-risk patients have intermediate MRD response (with lower, but still detectable, disease).

In comparison with other patients, those at high risk receive more intensive chemotherapy after the initial induction and consolidation blocks, and may proceed to bone marrow transplant. In a clinical trial context, standard-risk patients would be randomized to receive less intensive therapy to reduce toxicity, whereas medium-risk patients would be randomized to slightly more intensive treatment to reduce the risk of relapse.

MRD testing allows us to create new measures of disease status in ALL. Clinical remission is still defined as a reduction in lymphoblasts to less than five percent in a bone marrow aspirate; an increase in the blast count to greater than five percent is a clinical relapse. “Molecular remission” is defined as undetectable MRD (PCR-negative), and “molecular relapse” as a reappearance of measurable levels of MRD in the bone marrow. There is growing evidence that molecular relapse often occurs months before the re-emergence of symptoms and clinical relapse.

MRD is used in two different ways to predict clinical relapse and guide treatment. First, if a patient achieves molecular remission rapidly (that is, MRD negativity by day 33), their risk of relapse is low relative to patients with higher levels of MRD, who usually still achieve molecular remission, but require a longer treatment period to do so. Second, MRD can be used to detect molecular relapse, which is used in selected higher-risk patients after treatment (for instance, bone marrow transplant) is complete.  In practice, detecting molecular relapse requires more MRD tests for the patient, so most research focuses on improving our initial estimates for risk by MRD and other tests.

The current reliance on early MRD response to predict which patients are most likely to relapse is better than older systems (for instance, the National Cancer Institute’s Rome criteria). But even our current methods fall short of being a perfect predictor, and some patients relapse even when MRD response indicate that it’s unlikely, probably because the underlying genetics of the leukemia cells also have an impact. One option is to apply different MRD thresholds to different ALL genetic subtypes using a new algorithm (2). Another is to devise a multifactorial risk score that includes high-risk microdeletions to improve relapse prediction – and that’s what we opted to do in our paper (3).

Making use of microdeletions

We already knew microdeletions were an important consideration in ALL; several characteristic recurrent microdeletions had been already discovered by others using microarray technology or comparative genomic hybridization (CGH). The most common of those microdeletions could be detected by a diagnostic technique known as multiplex ligation probe amplification (MLPA). Along with several other groups, particularly in the UK and Europe, we tested large numbers of patients for microdeletions using MLPA and established their potential prognostic value.

Most microdeletions in ALL cause a partial loss of either a tumor suppressor (such as RB1) or a gene that promotes normal differentiation of lymphocytes (such as IKZF1). Our study showed both RB1 and IKZF1 to be associated with poor prognosis. In one specific case, the P2RY8-CRLF2 fusion, a microdeletion upstream of the CRLF2 gene causes increased expression of a gene that promotes cell growth through tyrosine kinase pathways. Regardless of the way in which they affect disease, however, it’s clear that microdeletions have a significant effect on relapse risk and overall prognosis – and that means we can use them to our advantage when stratifying patients.

Pathology laboratories that already use MLPA kits or CGH arrays to diagnose genetic conditions could readily incorporate testing for microdeletions found in B-cell precursor ALL. Alternatively, our lab has developed PCR assays for the most common IKZF1 deletions and for the P2RY8-CRLF2 fusion. In the short term, we anticipate that microdeletion evaluation will be part of the standard workup for pediatric ALL patients enrolling on the AIEOP-BFM ALL 2017 trial (Combination Chemotherapy Based on Risk of Relapse in Treating Young Patients With ALL) due to open for enrolment in 2018 in several European countries and Australia.

Knowing the score

Along with our colleagues, we have incorporated microdeletion testing into a new risk score for pre-B-cell ALL – a refinement on current risk stratification schemes for non-high risk pediatric patients (3). The risk score allows the identification of a new group of patients, previously hidden in the standard- or medium-risk groups, who face a 50 percent chance of relapse. It combines clinical features at diagnosis (age, white cell count, and the NCI Rome criteria), treatment response as measured by MRD detection, and the presence of specific microdeletions (IKZF1 and the P2RY8-CRLF2 fusion) in the ALL blasts. MRD testing techniques based on real-time quantitative PCR or quantitative flow cytometry allow the sensitive detection of ALL cells down to a sensitivity of one cell per 10⁴ or 10⁵ normal bone marrow cells (0.01–0.001 percent). In contrast, microscopic examination of a bone marrow aspirate has a sensitivity of approximately one cell per 100 normal bone marrow cells (one percent). The use of either PCR-MRD or flow-MRD techniques to stratify ALL patients into risk groups is the established standard of care in developed countries, based on numerous studies showing that rapid clearance of ALL in early treatment can identify patients with the highest chance of cure and the smallest risk of relapse. Our risk model scores patients using a combination of MRD threshold (>5x10^-5 at day 33, the end of the induction phase), high NCI risk (either high white cell count or age >10 years), and the presence of IKZF1 and CRLF2 deletions. The resultant risk score is a significant improvement over our original standard- and medium-risk stratification, and we validated it in an independent cohort of ALL patients.

By using more precise risk stratification, we can reduce the number of ALL relapses in the future. The best chance of long-term cure from ALL is with initial treatment – it’s easier to cure ALL at first diagnosis than after a relapse. We have shown in a prior publication that the intensification of treatment for high-risk ALL patients improves survival by reducing the risk of relapse (4). The risk score in our current paper (3) could be used to selectively identify ALL patients who would previously have been classified as medium-risk, but who in fact have a high risk of relapse. Such patients could then be offered intensified, high-risk ALL therapy. The risk score would mean that fewer patients at higher risk of relapse would be treated with less intensive, less effective therapy. The key to the score, though, lies in the identification of microdeletions – so pathology and laboratory medicine will continue to play a central role in the diagnosis and risk classification of ALL.