Minimal residual disease

Minimal residual disease (MRD) is the name given, to small numbers of leukaemic cells that remain in the patient during treatment, or after treatment when the patient is in remission (no symptoms or signs of disease). It is the major cause of relapse in cancer and leukaemia. Up until a decade ago none of the tests used to assess/detect cancer, were sensitive enough to detect MRD. Now, however, very sensitive molecular biology tests are available - based on DNA, RNA or Proteins - and these can measure minute levels of cancer cells in tissue samples, sometimes as low as 1 cancer cell in million normal cells.

In cancer treatment, particularly leukaemia, MRD testing has several important roles: determining whether treatment has eradicated the cancer or whether traces remain, comparing the efficacy of different treatments, monitoring patient remission status and recurrence of the leukaemia or cancer and choosing the treatment that will best meet those needs (personalization of treatment)

The tests are not simple, are often part of research or trials, and some have been accepted for routine clinical use.

Background: the problem of minimal residual disease (MRD)

Most research on MRD has been done on leukaemia. Mainly two types: adult chronic myeloid leukaemia, and childhood acute lymphoblastic leukaemia (the commonest childhood cancer). What follows, is a very simple discussion, of how leukaemia arises, how MRD arises, and why MRD is important.

Leukaemia is a cancer of white blood cells. The tissue mainly affected is bone marrow.

For most human leukaemias, the cause is not known. Risk factors can include chemicals, X-rays, but for most cases, it's not possible to identify the cause.

The disease is due to a defect in genes.

Leukaemia starts when one cell in the body acquires such a change in a key gene. That cell starts to grow and divide.

The body normally has a range of different types of blood cells, in standard proportions. In leukaemia, one type starts growing and dividing without stopping, and eventually large numbers of these cells accumulate. If the cells affected were gut cells or skin, a solid tumor would result. Leukaemic cells grow and divide without regulation, and crowd out the normal variety of blood cells.

It is only when the disease is advanced that people notice symptoms. By the time someone recognises symptoms of leukaemia, they have very many leukaemic cells in their body - 1 kg or 1,000,000,000,000 leukaemic cells.

If doctors take a sample of bone marrow , they can see these leukaemic cells under a microscope. Leukaemic cells look like normal immature blood cells, and healthy marrow is often 1-2% immature ('blast') cells. However, in leukaemia, there are abnormally high numbers of immature cells, making up 40-90% of marrow. Additional examination of the bone marrow by tests including flow cytometry and FISH are necessary to diagnose the specific malignancy.

The initial 5 weeks of treatment kill most leukaemic cells, and the marrow begins to recover. Immature white blood cells maybe be present in the patient, but these blasts are not always malignant cells.

In most cases a few leukaemic cells survive this treatment, and persist in the marrow for months or years. Approximately 0.001% of those present at the start survive cancer treatment. There is no way to identify them by looking at the marrow using only a microscope. DNA based tests and immunology tests are needed to identify the cells.

About 30 years ago leukaemia was universally fatal. The first treatments treated patients for a few weeks (not months or years as at present), producing remission, but nearly all patients relapsed after a few weeks or months. It is now known that minimal residual disease can regrow once treatment was stopped. Genetic tests can confirm the leukaemic cells at relapse are descendants of those present when the disease first appeared.

Relapse is a problem in leukaemia, and the doctors aim to prevent relapse rather than treat it. Currently, most children do not relapse - the disease is "cured" at first attempt. If the disease relapses, it is usually more resistant to treatment than when first diagnosed. If one gives the same drugs again, the disease may not respond as well. Patients who have relapsed once are at high risk of relapse in the future.

Tests which uncover minimal residual disease (one cancerous cell in a population of one million normal cells) are helpful for directing treatment and preventing relapse. A single remaining leukaemic cell can be fatal, as malignant cells divide without control. Conditioning regiments can continue as long as the patient is healthy enough to sustain damage by cytotoxic treatments.

Most research on MRD was done on leukaemia and lymphomas. Researchers hope that the discoveries made can be applied to understand and treat other cancers.

Techniques for measuring minimal residual disease in leukaemia

DNA based tests

These are based on detecting a leukaemic specific DNA sequence. Generally this is achieved through the use of the polymerase chain reaction, a highly sensitive technique that underpins much of molecular biology. The DNA sequence chosen, may contribute to the genesis of the leukaemia, or may simply be linked to it.

The markers used for DNA based testing are often chromosomal translocations such as t(14;18) involving BCL2 and t(11;14) involving BCL1 (CCND1). Other genes utilized for MRD detection include microsatellites, immunoglobulin and T cell receptor.

RNA based tests

These are based on detecting a leukemic specific RNA sequence. Generally this is achieved through the use of reverse transcription of the RNA followed by polymerase chain reaction. RNA based tests are normally utilized when a DNA test is impractical. For example, the t(9;22) BCR-ABL translocation may occur over a large length of the chromosome which makes DNA based testing difficult and inefficient. However, RNA is a much less stable target for diagnostics than DNA and requires careful handling and processing.

The markers used for RNA based testing are almost exclusively chromosomal translocations such as t(9;22) BCR-ABL, t(15;17) PML-RARA and t(12;21) ETV6-RUNX1 (TEL-AML1).

Patient specific testing

Patient specific MRD detection using immunoglobulin (IG) or T cell receptors (TCR) is gaining popularity as a way of measuring MRD in leukemias that do not contain a chromosomal translocation or other leukemic specific marker. In this case the leukemic specific IG or TCR clone is amplified using PCR and the variable region of the IG or TCR is sequenced. From this sequence PCR primers are designed that will only amplify the specific leukemic clone from the patient.

Both the DNA and RNA based tests require that a pathologist examine the bone marrow to determine which leukaemic specific sequence to target. Once the target is determined, a samples of blood or bone marrow is obtained, nucleic acid is extracted, and the sample analyzed for the leukaemic sequence. These tests are very specific, and detect leukaemic cells at levels down to 1 cell in a million, though the limit typically achieved is 1 in 10,000 to 1 in 100,000 cells. For comparison, the limit of what one can detect using traditional morphologic examinations using a microscope is about 1 cell in 100.

Immunological tests

Immunological based testing of leukaemias utilizes proteins on the surface of the cells. White blood cells (WBC) can show a variety of proteins on the surface depending upon the type of WBC. Leukaemic cells often show quite unusual and unique combinations (leukemic phenotype) of these cell surface proteins. These proteins can be stained with fluorescent dye labeled antibodies and detected using flow cytometry. The limit of detection of immunological tests is generally about 1 in 10,000 cells and cannot be used on leukaemias that don’t have an identifiable and stable leukaemic phenotype.

Use of and common targets in minimal residual disease detection in different leukaemias, lymphomas and solid tumors

Acute lymphoblastic leukaemia (ALL)

Targets: t(9;22) BCR-ABL, t(12;21) ETV6-RUNX1 (TEL-AML1), Patient specific assays for immunoglobulin and T cell receptor genes

Uses: Chromosomal translocation MRD detection is widely used as a standard clinical practice. Patient specific assays are gaining acceptance but are still generally only used in research protocols.

Acute myeloid leukaemia (AML)

Targets: t(15;17) PML-RARA, t(8;21) AML1-RUNX1T1 (AML-ETO), inv(16)

Uses: Chromosomal translocation MRD detection widely used as a standard clinical practice.

Chronic lymphocytic leukaemia

Targets: Cell surface proteins, Patient specific assays for immunoglobulin and T cell receptor genes

Uses: Immunological methods are gaining wider use as more advanced flow cytometers are utilized for clinical testing. Patient specific assays are still generally only used in research protocols.

Chronic myelogenous leukemia

Target: t(9;22) BCR-ABL

Uses: MRD detection of the t(9;22) is considered standard of care for all patients with CML and is extremely valuable for patients being treated with imatinib mesylate (Gleevec/Glivec).

Follicular lymphoma

Targets: t(14;18) IgH/BCL2, Patient specific assays for immunoglobulin and T cell receptor genes.

Uses: The t(14;18) is regularly used for MRD detection. Patient specific assays are still generally only used in research protocols.

Mantle cell lymphoma

Targets: t(11;14) IgH/CCND1 (IgH/BCL1), Patient specific assays for immunoglobulin and T cell receptor genes

Uses: The t(11;14) is regularly used for MRD detection, but the assay can only reliably detect 40-60% of the t(11;14) translocations. Patient specific assays are still generally only used in research protocols.

Multiple myeloma

Targets: M-protein levels in blood, Patient specific assays for immunoglobulin and T cell receptor genes (high levels of somatic hypermutation often prevent this assay from reliably working).

Uses: M-protein level in the blood is standard of care and is used for almost all patients with multiple myeloma. Patient specific assays are still generally only used in research protocols.

Solid tumors

Research into MRD detection of several solid tumors such as breast cancer and neuroblastoma has been performed. These assays have been used to sample lymph nodes and blood for residual or metastatic tumor cells. Applicable targets for MRD detection have been more difficult to determine in solid tumors and the use of MRD in solid tumors is much less advanced than the use in leukemia and lymphoma.

Animal species other than humans

Leukaemias and lymphomas can be monitored in the same way in non-human animals, but we are not aware of any veterinary applications to date.

Significance of minimal residual disease.

Level of MRD is a guide to prognosis or relapse risk

In some cases, the level of MRD at a certain time in treatment, is a useful guide, to the patient's prognosis. For instance in childhood leukaemia, doctors traditionally take a bone marrow sample after 5 weeks, and assess the level of leukaemia in that. Even with microscope, they were able to identify a few patients whose disease had not cleared, and those patients received different treatment. MRD tests also make use of this time, but the tests are much more sensitive.

When past patients were studied: Patients with high levels at this stage - here "high" means often leukaemia more than 1 cell in 1000, were at risk of relapse. Patients with levels below 1 in 100,000 were very unlikely to relapse. Those in between, some relapsed. This led to the idea that MRD testing could predict outcome, and this has now been shown. The next step is whether, having identified a patient whom standard treatment leaves at high risk - there are different treatments they could be offered, to lower that risk. Several clinical trials are investigating this.

Other research groups have looked at other times in treatment -e.g. 3 months, 6 months, 1 year, or end of current treatment (2 years) and these can predict outcome also.

There are also a few scientific studies, showing that level of MRD after bone marrow transplant, shows the risk of relapsing.

Monitoring people for early signs of recurring leukaemia

Another possible use is to identify if or when someone starts to relapse, early, before symptoms come back. This would mean regular blood or marrow samples. This is being explored mainly in chronic myeloid leukaemia (CML), where one can study the leukaemia in blood, which is easier to sample regularly than bone marrow. The molecular tests can show tumour levels starting to rise, very early, possibly months before symptoms recur. Starting treatment early , might be useful: the patient will be healthier; fewer leukaemic cells to deal with; the cells may be amenable to treatment, since at clinical relapse they have often become more resistant to drugs used.

Individualization of treatment

This whole area, comes under individualization of treatment, or if one prefers, identification of risk factors. Currently most patient receive the same treatment: but leukaemia is a very variable disease, and different patients probably have widely different treatment needs, to eradicate the disease.

For instance, the initial 5 week induction treatment, might rapidly clear disease for some patients. For others, the same treatment might leave significant amounts of disease . Measuring MRD level, helps doctors decide which patients need what. In other words, it identifies patients individual risk of relapse, and can theoretically allow them to receive just enough treatment to prevent it.

Without MRD information, doctors can do nothing but give the same treatment to all patients. They know that this will be inadequate for some and excessive for others, but there is little else they can do, as it is not possible to tell who needs what. Identification of risk factors, to help individualise treatment, is a big field in medicine.

Treatment for MRD

Generally the approach is to bring a cancer into remission first (absence of symptoms) and then try to eradicate the remaining cells (MRD) Often the treatments needed to eradicate MRD, differ from those used initially. This is an area of much research.

It seems a sensible idea to aim to reduce or eradicate MRD. What is needed, is evidence on which is the best method, and how well it works. This is emerging. Treatments which specifically target MRD can include.

(a) intensive conventional treatment with more drugs, or a different combination of drugs (b) stem cell transplant, e.g. marrow transplant. This allows more intensive chemotherapy to be given, and in addition the transplanted bone marrow may help eradicate the minimal residual disease (c) immunotherapy (d) monitor the patient carefully for early signs of relapse. This is an area of active research in several countries. (e) treatment with monoclonal antibodies which target cancer cells (f) cancer vaccines

Areas of current research and controversies

Clinical usefulness of MRD tests

It's important that doctors interpreting tests, base what they say, on scientific evidence. If you visit hospital and get tested for something - e.g. a blood count - most of the tests are used often, and have been done thousands or millions of times before, on many different people. The doctors reading the test results, have a large body of evidence, to interpret what the results mean. By contrast, MRD tests are new, and the diseases are uncommon. The tests have been done on relatively few people. Consequently there is less evidence available, to guide doctors, in interpreting the tests, or basing treatment decisions on them. In plain English, this means the doctors are likely to be very cautious, and rely more on other tests which they know and trust, than these. At least at present, while evidence is accumulating. -

Method for testing, and when to test

There are controversies about the best times to test, and the best test method to use. There are national and international approaches to standardize these. In childhood leukaemia and chronic myeloid leukaemia, there appears to be consensus emerging.

Is there such a thing as a safe level of MRD

There is also controversy about whether MRD is always bad inevitably causing relapse - or whether sometimes low levels are 'safe' and do not regrow. It is usually assumed, cancer cells inevitably grow and that if they are present disease usually develops. But there is some evidence from animal studies, that leukaemic cells can lie dormant for years in the body and do not regrow. For this reason, i may be that the goal of treating MRD may be to reduce it to a "safe" level - not to eradicate it completely.

Is MRD testing useful for all patients?

Some types of leukaemia are difficult to treat. In these, it is not clear how MRD testing would help. The patients may not do well on current treatment, but sometimes it is not clear what other treatment, if anything, might be better. There is thus an argument that as the test is not necessary: it might involve an additional procedure for the patient; it will contribute no useful information on treatment, it is not necessary.

MRD testing by hospital and other labs

Where done

MRD testing is not yet a routine test, nor is it carried out in all places.

Currently most MRD testing - in leukaemia research - is done during clinical trials, and would be funded as part of that trial, for patients enrolled on the trial. The tests are specialised, so samples are usually sent to a central reference laboratory in each region or country. The tests are not done in most routine diagnostic labs, as they tend to be complex, and also would be used relatively infrequently.

Cost

MRD testing is technically demanding and time consuming; the tests are expensive, so are usually available only through specialist centres, as part of clinical trials.

Availability of MRD testing.

At the time of writing (Jan 2008) MRD testing is available in some clinical trials in the UK, Europe, Australia and the US.

Interpretation of MRD test results.

Most clinical tests used to guide treatment - e.g. even a simple blood count - have been done millions of times, and doctors can interpret the results confidently, based on this extensive previous knowledge. By contrast, MRD tests are new and have been carried out on relatively few people (a few thousand at most). Researchers and doctors are still building the extensive database of knowledge needed, to show what MRD tests mean.

The consequence: unless a patient is enrolled on a trial which requires the test - clinicians tend to be somewhat cautious about requesting it, and cautious about interpreting the results. This is likely to change in future, as tests become more routine.

Resources

General

• Leukaemia Research Fund (United Kingdom)
• Children with Leukaemia (United Kingdom)
• Janica Lane; Stephen H., PhD. Wolinsky; Schneider, Stephen Henry (2005). The patient from hell: how I worked with my doctors to get the best of modern medicine, and how you can too. Cambridge, MA: Da Capo Books. ISBN 0-7382-1025-0. 

Textbooks

• Frei E, Kufe DW, Holland JF, (2003). "Part VII Pediatric Oncology, Section 37: Pediatric Oncology: Childhood Acute Lymphoblastic Leukemia". Cancer medicine 6. Hamilton, Ont: BC Decker. pp. 141a. ISBN 1-55009-213-8. http://www.ncbi.nlm.nih.gov/books/bv.fcgi?highlight=minimal%20residual%20disease&rid=cmed6.section.38145. 
• Frei E, Kufe DW, Holland JF, (2003). "Part VII Pediatric Oncology, Section 37: Pediatric Oncology: Neuroblastoma: The Concept of Risk-Related Therapy". Cancer medicine 6. Hamilton, Ont: BC Decker. pp. 142c. ISBN 1-55009-213-8. http://www.ncbi.nlm.nih.gov/books/bv.fcgi?highlight=minimal%20residual%20disease&rid=cmed6.section.39141#39150. 
• Frei E, Kufe DW, Holland JF, (2003). "Section 34: Hematopoietic System: Adult Acute Lymphocytic Leukemia: Evaluation of Minimal Residual Disease". Cancer medicine 6. Hamilton, Ont: BC Decker. pp. 129. ISBN 1-55009-213-8. http://www.ncbi.nlm.nih.gov/books/bv.fcgi?highlight=minimal%20residual%20disease&rid=cmed6.section.34862. 

Research papers

• Haferlach T, Bacher U, Kern W, Schnittger S, Haferlach C (Jan 2008). "The diagnosis of BCR/ABL-negative chronic myeloproliferative diseases (CMPD): a comprehensive approach based on morphology, cytogenetics, and molecular markers". Ann Hematol. 87 (1): 1–10. doi:10.1007/s00277-007-0403-6. PMC 2082654. PMID 17938925. http://www.pubmedcentral.nih.gov/articlerender.fcgi?tool=pmcentrez&artid=2082654. 
• Schmitt C, Balogh B, Grundt A, et al. (Jun 2006). "The bcl-2/IgH rearrangement in a population of 204 healthy individuals: occurrence, age and gender distribution, breakpoints, and detection method validity". Leuk Res. 30 (6): 745–50. doi:10.1016/j.leukres.2005.10.001. PMID 16297448. 
• Cavé H, van der Werff ten Bosch J, Suciu S, et al. (Aug 1998). "Clinical significance of minimal residual disease in childhood acute lymphoblastic leukemia. European Organization for Research and Treatment of Cancer--Childhood Leukemia Cooperative Group". N Engl J Med. 339 (9): 591–8. doi:10.1056/NEJM199808273390904. PMID 9718378. http://content.nejm.org/cgi/pmidlookup?view=short&pmid=9718378&promo=ONFLNS19. 
• Brisco MJ, Condon J, Hughes E, et al. (Jan 1994). "Outcome prediction in childhood acute lymphoblastic leukaemia by molecular quantification of residual disease at the end of induction". Lancet 343 (8891): 196–200. doi:10.1016/S0140-6736(94)90988-1. PMID 7904666.