Despite progress, ‘huge gap’ remains in understanding, application of minimal residual disease
The detection of minimal residual disease in patients with hematologic malignancies has altered the definition of clinical remission.
Minimal residual disease (MRD) assays — capable of finding cancerous cells that otherwise go undetected — already offer prognostic information for childhood acute lymphoblastic leukemia and acute myeloid leukemia. They also can play a meaningful role in treatment for chronic myeloid leukemia, chronic lymphocytic leukemia and multiple myeloma.
The widespread use of flow cytometry, polymerase chain reaction and next-generation sequencing has allowed clinicians to more accurately determine the depth of clinical complete remission. However, because benchmarks to distinguish MRD vary greatly by disease, standardization of assessments has proved challenging.
Oncologists continue to investigate the best methods used to test MRD, how often those tests should be administered, and whether they should dictate treatment length and intensity. Further, it is unclear if widespread use of MRD assays will become standard or accelerate drug development.
“We are still in a transition period — an uncomfortable zone in which some assays that are clinically available haven’t been fully vetted for clinical use,” Jonathan M. Gerber, MD, director of the leukemia division at Levine Cancer Institute at Carolinas HealthCare System and a HemOnc Today Editorial Board member, told HemOnc Today. “We are beginning to redefine what remission really is. Certainly, it is disconcerting if there is still MRD detectable. Persistent MRD after the completion of treatment almost always portends a poorer prognosis.”
HemOnc Today spoke with hematologic oncologists about the benefits of and challenges associated with MRD tests, the potential risks and rewards of tailoring treatment to achieve MRD negativity, and the increasing difficulty in determining when a patient truly is disease free.
Prior to the introduction of MRD detection, patients with leukemia, myeloma and lymphoma were considered in morphological complete remission when — under microscopic analysis — they had normal neutrophil and platelet counts and less than 5% of cancerous cells present in bone marrow.
Flow cytometry can detect one leukemic cell among more than 10,000 normal cells (0.01% level) and polymerase chain reaction (PCR) can detect one cancer cell in more than 100,000 normal cells (0.001%). These became effective prognostic tools in the early 1990s — especially when measured against traditional morphologic examination by light microscopy, which is capable of detecting one leukemic cell in every 20 examined (5%).
However, until the past 10 or 15 years, both methods were relegated to the research arena.
“Some people call it ‘measurable’ residual disease, and that is probably the more appropriate term because it is highly dependent on the tools and techniques used to detect it,” Farhad Ravandi, MD, professor in the department of leukemia within the division of cancer medicine at The University of Texas MD Anderson Cancer Center, told HemOnc Today. “When you get better, higher-quality remissions, it becomes more of interest to see if you can detect minimal disease. The problem with MRD is what to do about it.”
When PCR technology came into clinic, oncologists began to realize that young adults with ALL who for years were in clinical complete remission were found to have minute numbers of demonstrable leukemic cells.
“Psychologically, to [these patients], it was some kind of a shock,” Kanti R. Rai, MD, professor of molecular medicine at Hofstra North Shore-LIJ School of Medicine and a HemOnc Today Editorial Board member, said in an interview.
Today, flow cytometry and PCR — along with highly sensitive next-generation gene sequencing — have gained “solid ground” in clinical practice and have become the “gold standard” for assessing molecular remission, Rai said.
“There is evidence that MRD ought to be a very serious clinical consideration before we solidify a diagnosis of complete remission,” Rai said. “That is where it is most exciting. In clinical remission, bone marrow shows no evidence of leukemic or lymphoma cells. But with PCR or flow cytometry, lo and behold, there are significant or nonsignificant numbers of cancer cells. Rarely are there zero cancer cells.”
MRD levels above or below 0.01% — as measured by flow cytometry or PCR — dictate whether a patient is MRD positive or negative.
Studies have highlighted how these levels can predict outcomes for a range of hematologic malignancies.
In a study presented at last year’s ASH Annual Meeting and Exposition, Gupta and colleagues showed that children with ALL in morphologic clinical remission who were MRD negative had a significantly higher 5-year EFS rate than those who were MRD positive (87.1% vs. 59.1%; P < .0001). Based on these data, “MRD should replace morphology in defining remission in subjects with ALL,” researchers concluded.
In CLL, a study by Böttcher and colleagues — published in 2012 in Journal of Clinical Oncology — showed that, compared with patients with low levels of MRD, risks for disease progression increased for patients with intermediate (HR = 2.49) and high (HR = 14.7; P < .0001 for both) MRD levels. Median OS was 48.4 months among patients with high MRD but not reached for patients with lower levels.
A study by Buckley and colleagues, published earlier this year in Haematologica, showed MRD positivity prior to allogeneic hematopoietic cell transplant was associated with worse leukemia-free survival (HR = 2.76; 95% CI, 1.9-4), OS (HR = 2.36; 95% CI, 1.73-3.22) and cumulative incidence of relapse (HR = 3.65; 95% CI, 2.53-5.27) among patients with AML. However, researchers observed no association with nonrelapse mortality (HR = 1.12; 95% CI, 0.81-1.55).
And in multiple myeloma, a study by Martinez-Lopez and colleagues — published in 2014 in Blood — showed patients with MRD–negative disease by next-generation sequencing had a significantly longer time to tumor progression (median, 80 months vs. 30 months; P < .0001) and OS (median, not reached vs. 81 months; P = .02) than MRD–positive patients.
The National Cancer Research Institute of London continues to assess the role of MRD in disease surveillance in the AML17 and AML19 trials, the results of which could affect FDA guidelines.
“We all agree that MRD–directed treatment is going to be coming,” Sergio A. Giralt, MD, chief hematologic oncologist at Memorial Sloan Kettering Cancer Center and a HemOnc Today Editorial Board member, said in an interview. “The debate is: What is the best test, what is the best timing and what is the best threshold?”
Flow cytometry, developed in the late 1960s, was the first method used to detect MRD in bone marrow. At that time, it provided detailed characteristics of single cells as they passed through light. Within the past decade, the development of 10-color flow panels enabled the detection of more aberrant cells.
A single flow cytometer can range from $30,000 to more than $100,000, and a test can cost $300 to $1,000 per patient, depending on the institution and reimbursement.
Other than cost, one of the biggest drawbacks of flow cytometry is the limited number of medical centers with experts who can read the flow panels. University of Washington and Johns Hopkins Medicine are considered leaders in the field of flow cytometry, but many smaller centers mail blood samples to larger centers for analysis.
This creates two challenges, Gerber said.
“One is that some samples actually degrade during the transport process, so you’re not sure if you have a quality sample to analyze,” he said. “The second is that you have to take multiple samples and more volume from the patient, and some of these are diluted by peripheral blood flowing in through the marrow hole.”
Once bone marrow samples are ready for analysis, there are different modalities with which they are read, Gerber said. Experts can look for markers from the original leukemia and hunt for the same phenotype during clinical remission, or they can compare normal bone marrows and search for aberrant characteristics in the leukemic sample. If there is an abnormal combination — either extra or missing markers on the cells — that sample would be deemed a prognostic marker for relapse.
“The other limitation is that the leukemia can evolve over time,” Gerber said. “Either different clones can arise, or the original clone can change its markers after treatment or at different phases of its evolution. It definitely is a tricky process. It takes an experienced team to perform the assay and particularly to analyze the results.”
Another issue with flow cytometry is the challenge of making it standardized across all labs.
“It cannot be that my MRD test works at Memorial Sloan Kettering and it does not work any place else,” Giralt said. “It has to be easily reproducible and translatable. It makes it a lot harder for smaller institutions, because they probably won’t be able to have an expert flow cytometrist. So, now they’re thinking about sending it out, but there are many issues with send-out. It has to be practical.”
PCR, next-generation sequencing
PCR testing is comparable in cost to flow cytometry but is more sensitive and has the potential to be more easily standardized “because it’s a machine,” Giralt said.
Introduced in the early 1990s, PCR is a highly sensitive form of molecular testing with the potential to produce millions to billions of gene samplings for sequencing, cloning and analysis.
PCR assays can routinely examine cells to the 0.01% or even 0.001% levels.
However, with more than 100 billion cells, “there’s still a lot that you’re missing and there still can be a substantial burden of leukemia that even our most sensitive assays now miss,” Gerber said.
“The kicker for all of these is that you have to know what you’re looking for,” he added. “If you don’t have a probe on a PCR set to detect a certain region or to find a mutation — and for flow cytometry, if you don’t have the right combination of antibodies to probe with for markers on the cell surface — you may miss something that was truly there.”
Like PCR, next-generation sequencing also is molecular based. It is the newest tool in detecting MRD, permitting sequencing of large numbers of antigen receptor gene arrangements to provide a picture of residual leukemia and normal blood cells.
Perhaps the biggest obstacle facing next-generation sequencing is that it is not always reimbursed by insurance companies, Gerber said.
A next-generation sequencing panel usually costs $3,000 to $10,000, depending on the institution.
In the largest study to date on the benefits of MRD detection, Avet-Loiseau and colleagues showed that next-generation sequencing is highly predictive of 3-year PFS in patients with myeloma in complete remission before maintenance (MRD negative vs. positive, 87% vs. 63%) and after maintenance (92% vs. 64%).
Some clonal hematopoietic mutations — such as FLT3 and NPM1 — have been well studied and have shown to be associated with a higher likelihood of relapse, Ravandi said. However, the prognostic relevance of other mutations, such as DNMT3A, may be less clear.
For the added layer of identification, Gerber said he prefers the use of both flow cytometry and molecular testing to detect MRD.
“Next-generation sequencing is great at covering a large panel at diagnosis and finding abnormalities that you can track later,” Gerber said. “The beauty of combining the molecular and the flow cytometric assays is that they are independent of one another. PCR and next-generation sequencing both look for a potential molecular marker — flow cytometry looks for cell surface markers, and you’re potentially getting independent confirmation of an abnormality if it’s positive on both lines of testing.”
If there is an aberrant phenotype on flow cytometry of unclear clinical significance, but molecular sequencing also identifies an abnormality, that should provide more confidence in treatment decisions.
“On the flip side, if there are conflicting results, you would be much more hesitant to act — especially if it’s a mutation that is of uncertain significance,” Gerber said. “Right now, it is hard to say which one is better than the other. It is a very reassuring thing to have two completely independent forms of testing that might agree with one another.”
‘Threshold of relevance’
Regardless of method, oncologists agree MRD testing should take place at three points during the induction phase in all patients, as well as once at the end of consolidation in high-risk patients who have positive results on their first MRD test.
Despite this consensus, the MRD level that confirms remission remains less clear.
“We need to identify the threshold of relevance,” Giralt said. “Negative can mean a lot of things. People are still detecting disease at very low levels, which may not have clinical significance. The threshold is different for each disease. The threshold of MRD after allogeneic transplant may not be the same for a nontransplant patient.”
In CLL, for example, if markers such as CD5, CD19 and CD23 are negative, clinical remission is declared. However, under the examination of flow cytometry, those cells may be as high as 1 x 10-2 or as low as 1 x 10-6.
“Some evidence is emerging that those complete remission patients with MRD positivity are more likely to relapse in 2 or 3 years than patients in MRD–negative complete remission,” Rai said. “In CLL, we are at that stage of the game and everybody is now quietly, without announcing it in the protocols, declaring complete remission by clinical criteria, but for research we would like to declare complete remission by MRD negativity.”
The argument is whether MRD negative is the 1 x 10-4 level or 1 x 10-2 level, Rai said.
Further, finding treatments directed at eradicating MRD has been difficult for hematologic oncologists — a challenge they hope will be addressed in ongoing clinical trials.
“That’s been a frustrating topic because most therapies kill the bulk leukemia cells, but they do not necessarily impact MRD,” Gerber said. “In ALL, MRD assays have been in clinical use for quite some time — especially in the pediatric populations — and some of the agents that they’ve used have had more success at achieving an MRD–free state. On the AML side, there have been a few trials that are now trying to target leukemia stem cells, which appear to be more resistant to treatment and thus represent a disproportionate share of the MRD.”
Approval for MRD–specific drugs is needed, Giralt said.
“I definitely think drug development is needed because MRD is here to stay,” he said. “People are going to be wondering what to do with this information. We need more studies.”
Due to its proven prognostic value, MRD has been increasingly suggested as a surrogate endpoint for survival in clinical trials, potentially accelerating drug development.
In a statement provided to HemOnc Today, the FDA acknowledged the use of MRD to influence clinical decision-making in the management of several hematologic malignancies, including risk-adjusted therapies and the timing of HSCT.
“The FDA recognizes the markedly improved sensitivity and specificity provided by several methods currently employed in the assessment of MRD, including next-generation sequencing, compared with standard morphological assessment of bone marrow and peripheral blood to assess response to therapy,” the statement said. “The FDA recognizes that several sponsors have interest in utilizing MRD as [a] response biomarker and intermediate endpoint in clinical trials, and the agency is currently developing a draft guidance on the use of MRD as a drug development tool in the development of new drugs for hematological malignancies.”
The FDA declined to comment on pending drug applications, but it noted blinatumomab (Blincyto, Amgen) has been approved for relapsed and refractory ALL in adults and children and has been demonstrated in several studies to have an effect on MRD.
Impact on treatments
The ultimate goal of MRD assays is to guide therapeutic decisions. Patients with more than one leukemic cell per 1,000 are considered at high risk for relapse, and those with levels below one cancerous cell per 100,000 are considered very unlikely to relapse.
Although clinicians agree that patients who test positive for MRD have a greater risk for relapse, there seems to be disagreement about whether being clinically in remission but MRD positive warrants new treatment.
“Not yet,” Rai said. “This is a tough nut to crack because we really don’t know that all these MRD–positive patients will relapse. There are some people who say we should start treatment right away, not realizing that any treatment brings toxicities. These patients are ostensibly in good quality of life. Some, but not all, will be willing to take that gamble.”
Although there are studies that show patients who achieve MRD negativity have better outcomes, “this is likely prognostic versus predictive,” Sagar Lonial, MD, FACP, chair and professor of the department of hematology and medical oncology at Winship Cancer Institute at Emory University School of Medicine and a HemOnc Today board member, said in an interview.
“These may be people who were going to do better no matter what treatment they received due to good biology,” he said. “What is unknown is if you take someone who is MRD positive, switch treatment and convert them to MRD negative, do you actually change their outcomes? Those studies are ongoing.”
Some research suggests patients with persistent MRD–positive disease can benefit from augmented therapy.
In acute leukemias, MRD can “absolutely” be used to intensify treatment, Ravandi said. It also has been utilized in pediatric leukemias, such as ALL and AML.
“In younger patients, it’s possible to intensify treatment,” he said. “The problem is that if you look at all patients, it’s a bit difficult to intensify treatments [in adults] because they can’t tolerate high doses of chemotherapy.”
A study by Vora and colleagues, published in 2014 in The Lancet Oncology, evaluated whether children and young adults with clinical standard- and intermediate-risk ALL who had persistent 0.01% or greater MRD at the end of induction therapy benefitted from augmented postremission therapy with an additional eight doses of pegylated asparaginase, 18 doses of vincristine and escalated-dose IV methotrexate.
After a median follow-up of 70 months, more patients with MRD who received augmented therapy compared with standard therapy achieved 5-year EFS (89.6% vs. 82.8%; OR = 0.61; 95% CI, 0.39-0.98). Five-year OS also was higher with augmented therapy, but it did not reach statistical significance (92.9% vs. 88.9%; OR = 0.67; 95% CI, 0.38-1.17).
However, more adverse events occurred in the augmented group, including treatment-related hypersensitivity (6.7% vs. 0.8%), pancreatitis (3% vs. 0.4%), mucositis (4.1% vs. 1.1%) and stomatitis (18% vs. 4.5%).
“Almost every time, in every disease, MRD positivity is a bad thing,” Gerber said. “Treating the disease earlier seems to do better than when there’s a much more substantial burden.”
On the flip side of augmenting treatment, MRD also can be used to determine which patients can stop therapy or avoid transplantation.
In the EURO-SKI trial, conducted by Mahon and colleagues and presented at last year’s ASH Annual Meeting and Exposition, patients with CML with a deep molecular response — or BCR-ABL less than 0.01% — were safely able to stop tyrosine kinase inhibitor therapy, demonstrating a molecular RFS of 61% (95% CI, 58-65) at 6 months, 55% (95% CI, 51-58) at 12 months, 50% (95% CI, 47-54) at 24 months, and 47% (95% CI, 43-51) at 36 months.
Most patients were able to regain their deep molecular response, and duration of imatinib therapy of at least 5.8 years prior to stopping therapy increased likelihood of molecular RFS.
“One of the things being talked about now is if somebody is MRD negative, can you stop therapy?” Lonial said. “In myeloma, we tend to use a lot of continuous therapy and chronic maintenance. My hesitation in adopting that concept is that we do not know the right time points to say a patient can stop. In other words, if a patient is MRD negative after 6 or 8 weeks of initial induction therapy, is that the same as being MRD negative 2 years later? Probably not.”
Even patients who achieve MRD negativity carry a risk for relapse, Lonial added.
“MRD negative is not a surrogate for cure,” he said. “If you know it’s not a surrogate for cure, why would you really want to argue to stop?”
MRD assessment also may redefine the role of HSCT for several malignancies.
For instance, blinatumomab has been effective for taking patients with ALL who are MRD positive and making them MRD negative, Ravandi said.
“One could argue that if you become MRD negative with blinatumomab, you may do actually really well, even without transplant,” he said. “So, why subject the patient to a risky procedure? This debate is going to become more intense in the future.”
In patients with acute promyelocytic leukemia (APL), a subtype of AML, Ravandi said it’s “relatively easy” to achieve complete molecular remission with arsenic trioxide–based induction, also eliminating the need for transplant.
“We now have data for 10 years that a vast majority of people with APL who become MRD negative don’t relapse,” Ravandi said. “They have an outstanding survival so, clearly, we don’t need to transplant people with APL. This is a situation that hopefully will evolve with other subtypes of AML and ALL.”
Fifteen years ago, patients with Philadelphia chromosome–positive ALL were candidates for immediate transplant after first remission, Ravandi said. With the introduction of nontoxic TKIs — such as imatinib, dasatinib, blinatumomab and ponatinib (Iclusig, Ariad) — the proportion of patients who become MRD negative has increased, and the role of allogeneic stem cell transplant has become a subject of debate.
“With more absolute MRD negativity, hopefully studies will show that if you have achieved an excellent MRD–negative status you can do away with transplant,” he said.
Future of MRD
With next-generation sequencing allowing doctors to examine the depth of response to 1 x 10-6, results from long-term studies — such as the IFM/DFCI trial, and the AML17 and AML19 trials — could lead to standardization of MRD testing in some leukemias and myelomas, Lonial said.
“The challenge in trying to interpret those datasets — which are likely to be available in the next few years — is that some of it depends on things like genetic risks,” Lonial said. “A patient with indolent, low-risk myeloma who is MRD positive probably has a different outcome than a patient with 17p–deleted myeloma who is MRD positive. What does that benchmark mean for your type of biology? That’s a much more complicated analysis and trial.”
Although most hematologic oncologists acknowledge there is a place for MRD testing in clinical practice, there is no consensus about whether it should be a secondary layer of analysis or a primary prognosticator for treatment.
“I am a compromiser,” Rai said when asked his position on MRD. “I want to maintain a patient’s good quality of life. By 2022, I predict and I expect that the MRD concept will become a reality. People like myself will have joined the bandwagon. But as of 2017, it is an iffy question which we all feel is correctly in the domain of researchers.”
Hematologic cancers may be too complex to be treated universally, and they may require a collaboration of prognostic tests and treatments to help deepen and extend the levels of remission, Gerber said.
“These diseases are rare enough that it’s hard to do MRD testing as a single institution,” he said. “We’ve become complacent. Some people think we have already solved the MRD issue and, clearly, we haven’t. Very frequently we have a patient who is in complete remission and MRD negative, and the next follow-up 3 months later, they are MRD positive, followed soon after by relapse. So, there is still a huge gap.
“You’re talking about so many cells that, even when you get to fractions of a percent, it’s still a lot of cells,” Gerber added. “Until our assays are able capable of detection in those small ranges, both with great sensitivity and specificity, it’s going to take multiple modalities to solve this.”
While awaiting more absolute recommendations from the FDA, hematologic oncologists will continue trying to determine how to treat what is considered today’s “new remission.”
“We’re all humbled by MRD,” Gerber said. “The best way to think of MRD is as the gap between remission and cure, as we continue to refine the remission criteria. It is confusing to say someone is in complete remission but MRD positive. If we know that, we arguably shouldn’t call them in remission anymore, provided the MRD is, in fact, clinically relevant.”
A perfect MRD assay would be able to distinguish who is and is not cured, Gerber added.
“One of the most challenging things is that, despite all of the advances and the fact that some of these tests are remarkably sensitive, we haven’t reached that point yet,” he said. – by Chuck Gormley
Avet-Loiseau H, et al. Abstract 191. Presented at: ASH Annual Meeting and Exposition; Dec. 5-8, 2015; Orlando, Fla.
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Buckley SA, et al. Haematol. 2017;doi:10.3324/haematol.2016.159343.
Gupta S, et al. Abstract 758. Presented at: ASH Annual Meeting and Exposition; Dec. 3-6, 2016; San Diego.
Mahon F-X, et al. Abstract 787. Presented at: ASH Annual Meeting and Exposition; Dec. 3-6, 2016; San Diego.
Martinez-Lopez J, et al. Blood. 2014;doi:10.1182/blood-2014-01-550020.
Vora A, et al. Lancet Oncol. 2014;doi:10.1016/S1470-2045(14)70243-8.
For more information:
Jonathan M. Gerber, MD, can be reached at email@example.com.
Sergio A. Giralt, MD, can be reached at firstname.lastname@example.org.
Sagar Lonial, MD, FACP, can be reached at email@example.com.
Kanti R. Rai, MD, can be reached at firstname.lastname@example.org.
Farhad Ravandi, MD, can be reached at email@example.com.
Disclosure: Gerber, Giralt, Lonial, Rai and Ravandi report no relevant financial disclosures.