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MRD Part I Counting Marbles: The Hunt for Residual Disease

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By Susan Leclair

In the late 1960s and 1970s, a series of articles were published to describe the numbers of cells in various malignant populations.  Discussions of this have been ongoing since that time with changes in definition, staging, and choices of therapy.

Think for the moment of a 2-gallon jug. Fill it with white marbles (normal cells) and put it to one side. Take another jug and fill it with about 75% black marbles (malignant cells) and 25% white marbles (normal cells). As in most baking recipes, make sure that you stir thoroughly before you set aside each jug. Take another jug – yes, this will go on for a little while. Have patience. Into this jug, put about 50% black and 50% white marbles. And in another jug, you guessed it, 25% black marbles and 75% white marbles.  Finally (yea!) put 20 individual black marbles into the last jug and fill to the top with white ones.  Now let’s look at them.

Jug number 2 (75% malignant and 25% normal)

There are studies that have tried to assess marrow stress at various times in disease. That second jug is symbolic of how your marrow probably looked at the time of diagnosis. You had so many malignant cells that they were overwhelming your normal cells, hence the anemia, the thrombocytopenia, the lack of correct healing, etc.  The object of therapy is to get this number (the tumor burden) to as low as it can be. Yes, the target is zero, but that might be harder to achieve than one might think. That first step in traditional chemotherapy is called induction remission.

Jug number 3 (50% malignant cells and 50% normal cells)

The first point of therapeutic success is when the individual begins to feel better, although for lot of traditional chemotherapy with their lists of significant side effects, that is harder to determine than one might think. In the jug of marbles scenario, if you close your eyes and reach into the jug to pull out one marble, it is a fairly even shot that you will find a white one.  Don’t think less of this as the resurgence of your normal cells is a great accomplishment. For the laboratory, it is the time at which the peripheral blood looks acceptable – that is, the white cells are no longer abnormal, the anemia has recovered as has the platelet count. Typically, physicians notice that the lymph nodes are smaller, fever may have disappeared, and imaging studies show a return to more normal structures. When those overt signs and symptoms are gone, it is called a clinical remission. But, as the marbles tell you, the black ones are still present, putting your sense of well-being at odds with the cellular reality, hence the traditional use of a second and typically different medication for “consolidation”.

This scenario (illustrated by jug number 3 above) set up a conundrum for the mid-twentieth century laboratory. We needed more sensitive tests when the commonly available ones were no longer useful. Enter chromosome analysis.

Jug number 4 (25% malignant cells and 75% normal cells)

Chromosome counting and analysis showed that, in many situations, there could be no signs or symptoms yet there was the presence of specific chromosomal damage linked to certain diseases. One good example of this was during the investigation of the Philadelphia chromosome and chronic myelogenous leukemia. In order to determine if the chromosome happened frequently or just to people with CML, a lot of healthy patients agreed to allow their bone marrow be examined. It turned out that among the hundreds of normal people, there were a very few who had the Philadelphia chromosome yet had no diagnosis, no signs or symptoms and no trace of CML. Following these people for a few years showed that each one of them eventually became symptomatic and were diagnosed with CML.

The problem with simple karyotyping was that the damage had to be extensive, such as the loss of a chromosome or an arm of a chromosome before it could be seen. It could not show if a piece of a chromosome had been turned upside down or moved to another site. So, a modification of the karyotype which allowed the chromosome to be highlighted or dimmed was used to more closely pinpoint what parts of the chromosome were disturbed. If these tests were negative, one could say that a person might be in a chromosomally negative remission. One caution here is that these tests are performed using a standard light microscope, so smaller alterations or structures were often missed.

In the 1980’s, the use of flow cytometry came into common use. Flow cytometry is like a computer. It is not so much of a test as it a technique, just like in cooking where you can broil or sauté or bake a piece of meat. It gets cooked but there are different tastes and textures. First used to identify markers, proteins and other compounds on the outside of the cell membrane, the application of flow cytometry to identify subsets of diseases such as B-cell CLL and the rest gave a much more nuanced way to evaluate the different cells in any given sample. But it still had problems with numbers.

Jug number 5 (less than 1% malignant cells and 99+% normal cells)

The last jug as of today!  In the late 1990s, it became possible to take a sample of cells, extract the DNA from the cells and then by judicious stimulation, cause the specific DNA you were interested in to undergo thousands of replications. Then, if you used the sensitivity of flow cytometry, you could get a sense of the presence of a very small number of specific gene mutations, reliably down to 1 cell in 10,000. At present, with the most sensitive of the new genetic tests you can be reasonably sure that a person has less than 1 malignant cell in 100,000 or even some cases, 1 in a million cells. But notice there is a problem in that it appears Jug number 5 has no black marbles at all.  There are so many white marbles that they totally overwhelm the black. So, how do you report this out – logically and truthfully. You can’t report negative because, well, you do not know that. You only know that there is the possibility of cells beyond the test’s ability to find them. Now we get the uMRD – or undetected minimal residual disease which basically means that no matter how we tried, we didn’t find any malignant cells, but the test is not sensitive enough to find every last cell.


Susan Leclair is Chancellor Professor Emerita at the University of Massachusetts Dartmouth; Senior Scientist, at Forensic DNA Associates; and Moderator and Speaker, PatientPower.info – an electronic resource for patients and health care providers.

Originally published in The CLL Society Tribune MRD Special Edition.