Treatment overview >  Minimum Residual Disease (MRD)

with PCR ("molecular photocopying") of blood or marrow samples

Last update: 03/21/2018

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Minimal residual disease (MRD) refers to cancer cells that may remain in the body of a person with lymphoid cancer after treatment. These cells are present at levels undetectable by traditional microscopic examination of blood, bone marrow or a lymph node biopsy. Very low levels of MRD can be reliably detected only by using sensitive molecular technologies.

One goal of testing for Minimal Residual Disease (MRD) is to judge the quality of the response to treatment, which might predict the durability of the remission - who is at greatest risk of relapse.  Most likely the test will prove to be most useful for assessing response when the goal of therapy is to induce a durable remission or cure.

A validated MRD test would help to identify patients who may benefit from more therapy or a change of therapy; or who may benefit by avoiding unnecessary additional treatment to limit side effects and risks, including financial.  In clinical trials, a validated MRD test would help to pick the winner of new treatments under evaluation with much greater efficiency.

Polymerase Chain Reaction (PCR) techniques are used to identify trace amounts of tumor-specific fragments (one or more portions of DNA or RNA)  in the blood or marrow after treatment.   The bone marrow may be the more reliable sample to evaluate, depending on the type of lymphoma.

The PCR test is sometimes called "molecular photocopying."  The first step in the real-time PCR process is to detect a fragment of interest (with a binder).  The bound fragment is then copied (amplified) and tagged with light so that it can be visualized.  The earlier the fragment is detected in the copy process the more fragments exist in the sample (quantification).  

Animated explanation: 
Real-Time Polymerase Chain Reaction (PCR) - Multi-Lingual Captions
showing how a DNA or RNA fragment is detected, amplified and tagged, and quantified.

MRD Analogy: A standard bone marrow test is like taking a sample from a swimming pool. The test is only conclusive if leaves are found in the bucket, because there may be leaves floating elsewhere in the larger pool.  MRD testing is like examining the same sample of water in the bucket for tiny fragments of substances that exist only in leaves.  Thus, a negative finding with MRD testing provides much greater confidence that the blood or bone marrow is clear of the abnormal cells.

The tools and science of PCR testing are evolving rapidly.  The strongest evidence for the utility of MRD is for ALL, where MRD testing after induction is part of NCCN guidelines.

PCR is the basis of the molecular methods for MRD detection, but there are several different types of PCR assays:

There is one type of assay in which the cancer cells all harbor a particular mutation or translocation that can be assessed with PCR. Unfortunately, this type of testing does not apply to all patients as not all have a known or common mutation/translocation.

Another type of PCR-based MRD testing is known as as allele-specific oligonucleotide PCR (ASO-PCR; also sometimes just referred to as real-time quantitative PCR or RT-qPCR). In this method, a personalized primer set and assay is developed for each patient to amplify the immune receptor gene harbored by the clonal cancer cells (a slow, laborious and expensive process, and one that is not widely available – it's done mostly in academic centers). This assay is then used to sensitively and quantitatively detect cancer cells based on their characteristic immune receptor sequence.

A third kind of PCR-based molecular MRD detection method, however, is the one used in Sequenta's ClonoSIGHT test. In this case, a universal set of PCR primers amplifies the immune receptor gene from every B cell (and/or T cell depending on the disease) in a diagnostic sample and then uses next-generation sequencing to identify and quantify each of these sequences. The cancer sequence is identified by virtue of its high frequency compared to all other sequences. This initial testing is done on a diagnostic sample, but once identified, the specific lymphoma B or T cell receptor sequence can be tracked in follow–up samples (this is the MRD testing). This method is relatively new and is therefore not really discussed in most reviews about MRD testing.

Recent studies have found a correlation between the duration of response to treatment and MRD status for Mantle Cell lymphoma (below).   Indeed, at ASH 2013 there were a high number of reports on the potential utility of MRD testing following treatment for lymphoma and also for surveillance for relapse:

* ASH Paper, 2013: Minimal Residual Disease (MRD) Predicts PFS In Mantle Cell Lymphoma: CALGB 50403 (Alliance) http://bit.ly/18Bzj6q

* ASH Paper, 2013:  Improved Igh-Based MRD Detection By Using Droplet Digital PCR: a Comparison With Real Time Quantitative PCR In MCL and MM http://bit.ly/ID5ifU

ASH Paper: Comparative Analysis Of Next-Gen Sequencing and Real-Time Quantitative PCR For MRD in Follicular Lymphomas http://bit.ly/1jiAvUe

NGS represents a feasible tool for IGH-based MRD monitoring that allows analysis of a larger group of FL pts.

(IGH: immunoglobulin heavy chain variable region - a portion of the b-cell receptor that is expressed clonally on the tumor and is tumor-specific)

Our results show that the two methods have a high level of correlation.  Lymphoma infiltration of dx samples and somatic mutation of IGH is a critical point for identification of the tumor-specific clonotypes by NGS, therefore different MRD methods should complement each other to allow MRD assessment for the majority of pts. Furthermore IGH NGS sequencing has the potential to detect and track IGH evolution in FL.

* ASH Paper, 2013: Minimal Residual Disease Measurement By Deep Sequencing Reflects Changes In Disease Load During Therapy In Diffuse Large B Cell Lymphoma Patients http://bit.ly/19fCOzP

* ASH Paper, 2013: Treatment Of Early Stage Follicular Lymphoma With Involved Field Radiotherapy and Rituximab. Role Of Bcl-2 Molecular Monitoring http://bit.ly/1cXfObg

* ASH Paper, 2013: DNA Sequencing-Based Monitoring Of Serum Predicts Clinical Relapse Before CT Imaging in Diffuse Large B-Cell Lymphoma http://bit.ly/1b20z3X

* ASH Paper, 2013: Detection Of Classical Hodgkin Lymphoma In Peripheral Blood Using High-Throughput Sequencing Assay http://bit.ly/1k81yiG

* ASH Paper, 2013: Comparison Of Deep Sequencing and Allele-Specific Oligonucleotide PCR Methods For MRD Quantitation In Acute Lymphoblastic Leukemia and Mantle Cell Lymphoma: CALGB 10403 and CALGB 59909 (Alliance) http://bit.ly/1ix5dev

The clinical significance of being MRD negative (also called a molecular remission) is still being studied.  PCR tests are generally given after a complete response to treatment has been determined using CT and a bone marrow biopsy, mainly in clinical trials.   The tests must be validated prospectively in clinical trials.

Limitations and uncertainty: The analysis of the blood sample by PCR ("molecular photocopying") is limited to the compartment that's tested (blood, bone marrow) and its value may depend on the elements of the malignant cells it is set up to detect.  It may be that the validity of the test will vary and depend on the type of treatment, type of lymphoma, the compartment tested (blood/marrow), or how the test is scheduled over time.
 

An advocate's perspective:

There's an urgent need to validate biomarkers for response to treatment to reduce the risk of unproductive toxicity – the patients experiencing only the side effects of the treatment with minimal or no benefit – that can narrowing the patient’s future treatment options.  Further, the  uncertainty and the fear of unproductive toxicity adds significantly to the burden of living with lymphoma. 

Tests that better determine the response to treatment can help to reduce toxicity or identifying patients who may benefit from additional therapy.

The quantitative assessment of MRD months has been explored as a potential surrogate for PFS, but needs validation. http://bloodjournal.hematologylibrary.org/content/95/8/2651.full 

We propose that the study of maintenance/consolidation protocols following the initial therapy of indolent lymphoma should be based on emerging and novel biomarkers for efficacy in order to limit the risk of over treating patients.  For example, patients might be eligible to participate in the randomized phase of study based on an incomplete response to induction therapy (MRD positive) as measured by PCR tests of the blood.  This would be similar to the approach for Hodgkins lymphoma using PET imaging to select only patients with an incomplete response to induction therapy (PET positive) for additional or more intensive therapy. 

Advantage to a validated MRD test: 

It would help to limit exposure to ionizing radiation from repeated CT and PET imaging.  The ease of giving the test could lend itself to repeating the test with shorter intervals - such as every 15 or 30 days as a way to detect relapse earlier and with more precision than with imaging, which is typically given every 2 or 3 months in clinical trials.

Validating MDR as a surrogate for PFS would be major advance. It could decrease the time and expense of carrying out subsequent studies, and also provide important benefits to patients in regular practice – helping to guide clinical decisions; such as if the patient has received enough therapy, needs to continue therapy, or needs a different therapy.

It's important to note that it was the use of viral load (a similar concept) as a validated surrogate for clinical benefit that lead to the rapid development and comparison of drugs to treat HIV.  

List Clinical Trials evaluating biomarkers

Lymphoma or CLL
Find Trials that apply MRD testing

In the News

FL - circulating DNA testing for lymphoma We have high hopes for this approach to monitoring -- that it can be more sensitive than imaging and less burdensome (a blood sample), and one day it can be used as a way to monitor disease status and duration of response to treatment - helping to assess and compare protocols with better accuracy. * Dynamic monitoring of circulating tumor DNA in non-Hodgkin lymphoma. - PubMed - NCBI http://1.usa.gov/1NfE53H abstract

* Oncology Times 2014:
For CLL and Myeloma Patients, MRD Remains Uncertain Goal http://bit.ly/1aLFPic

Kay, MD:  “Right now, we conventionally use clinical trial data for progression-free survival and overall survival estimates, but you'd have to be immortal to use those in routine trials. But if you could substitute MRD as a surrogate for progression-free or overall survival you could shorten clinical trials, test the promising new drugs more quickly, and then if they are effective, bring them to the patient in a reasonable time frame.”

Furman, MD:  The new tyrosine kinase inhibitors cause preferential lymphocytosis by interfering with adhesion molecules that hold the cells in the lymph nodes and in the different niches, ... “So I believe that MRD status will have lost its predictability and its importance when we deal with these tyrosine kinase inhibitors.”

* ASH Paper, 2013: Minimal Residual Disease (MRD) Predicts PFS In Mantle Cell Lymphoma: CALGB 50403 (Alliance) http://bit.ly/18Bzj6q

* ASH Paper, 2013:  Improved Igh-Based MRD Detection By Using Droplet Digital PCR: a Comparison With Real Time Quantitative PCR In MCL and MM http://bit.ly/ID5ifU

ASH Paper: Comparative Analysis Of Next-Gen Sequencing and Real-Time Quantitative PCR For MRD in Follicular Lymphomas http://bit.ly/1jiAvUe

* ASH Paper, 2013: Minimal Residual Disease Measurement By Deep Sequencing Reflects Changes In Disease Load During Therapy In Diffuse Large B Cell Lymphoma Patients http://bit.ly/19fCOzP

* ASH Paper, 2013: Treatment Of Early Stage Follicular Lymphoma With Involved Field Radiotherapy and Rituximab. Role Of Bcl-2 Molecular Monitoring http://bit.ly/1cXfObg

* ASH Paper, 2013: DNA Sequencing-Based Monitoring Of Serum Predicts Clinical Relapse Before CT Imaging in Diffuse Large B-Cell Lymphoma http://bit.ly/1b20z3X

* ASH Paper, 2013: Detection Of Classical Hodgkin Lymphoma In Peripheral Blood Using High-Throughput Sequencing Assay http://bit.ly/1k81yiG

* ASH Paper, 2013: Comparison Of Deep Sequencing and Allele-Specific Oligonucleotide PCR Methods For MRD Quantitation In Acute Lymphoblastic Leukemia and Mantle Cell Lymphoma: CALGB 10403 and CALGB 59909 (Alliance) http://bit.ly/1ix5dev

Resources and Background Reports

	Ther Adv Hematol. 2013 June; 4(3): 189–198.   Clinical implications and prognostic role of minimal residual disease detection in follicular lymphoma http://1.usa.gov/12H6NDw MRD detection is more informative on bone marrow (BM) cells than on peripheral blood (PB) cells [Gribben et al. 1994] as the tumor infiltration is higher. Moreover in the rituximab era [Ghielmini et al. 2004], the extremely effective clearance of FL cells from the PB ensured by rituximab further suggest that BM might represent a more reliable tissue for MRD analysis in FL.
	Commercially available test for MRD: http://www.sequenta.com/ * Sequenta's ClonoSIGHT MRD Test Can Detect Relapse of Diffuse Large B-Cell Lymphoma More Than Seven Months Before CT Scan http://bit.ly/1gfDTQ3 Note:  PAL does not have an opinion regarding the validation of this test for regular clinical use.
	Book on topic: Monitoring Lymphoma with PCR  http://bit.ly/f9wGad
	Molecular monitoring of low grade non-Hodgkin's lymphoma by gene amplification, 1991, full text http://www.ncbi.nlm.nih.gov/pmc/articles/PMC1977878/  | Related articles Abstract: Molecular monitoring by the polymerase chain reaction (PCR) was used to detect and follow minimal disease in working formulation category B and C on non-Hodgkin's lymphoma. Rearrangement of the bcl-2 gene served as the target for gene amplification. Thirty patients were studied. Bone marrow histology was compared to PCR analysis of bone marrow aspirate and blood. PCR upstaged disease status in approximately 50% of patients. Results are shown from a patient whose disease was followed with PCR during chemotherapy from initial remission to relapse. We conclude that PCR of bone marrow and blood can be used to upstage disease status in low grade lymphoma and PCR of blood may be used to monitor response to treatment with obvious patient benefit. The general approach of molecular monitoring provides a means for appraising therapies in the setting of subclinical disease.
	All advanced stage non-Hodgkin's lymphomas with a polymerase chain reaction amplifiable breakpoint of bcl-2 have residual cells containing the bcl-2 rearrangement at evaluation and after treatment. 1991 full text: http://bloodjournal.hematologylibrary.org/content/78/12/3275.long
	* Blood: Immunoglobulin heavy-chain consensus probes for real-time PCR quantification of residual disease in acute lymphoblastic leukemia  http://bloodjournal.hematologylibrary.org/content/95/8/2651.full Tumor-related immunoglobulin heavy-chain (IgH) rearrangements are markers for polymerase chain reaction (PCR) detection of minimal residual disease (MRD) in B-cell malignancies. Nested PCR with patient IgH allele-specific oligonucleotide primers can detect 1 tumor cell in 104 to 106 normal cells. In childhood acute lymphoblastic leukemia (ALL), persistence of PCR-detectable disease is associated with increased risk of relapse. The clinical significance of qualitative PCR data can be limited, however, because patients can harbor detectable MRD for prolonged periods without relapse. Recent studies indicate that a quantitative rise in tumor burden identifies patients who are at high risk for relapse. Therefore, an efficient and reliable PCR method for MRD quantification is needed for ALL patients. We have developed a real-time PCR method to quantify MRD with IgH VH gene family consensus fluorogenically labeled probes. With this method, a small number of probes can be used to quantify MRD in a large number of different patients. The assay was found to be both accurate and reproducible over a wide range and capable of detecting approximately 1 tumor cell in 5 × 104 normal cells. We demonstrate that this methodology can discriminate between patients with persistence of MRD who relapse and those who do not. This technique is generally applicable to B-cell malignancies and is currently being used to quantify MRD in a number of prospective clinical studies at our institution.
	AACR: technical Biomarkers for Cancer Risk, Early Detection, and Prognosis: The Validation Conundrum http://bit.ly/1hNQ58K
	Biomarkers PAL