Tests In Support of Diagnosis

Currently there are several tests done in support of a JMML diagnosis. When JMML is suspected, tests are done to contribute to this diagnosis, along with other diag nostic criteria. New, faster tests are currently under development, but not yet clinically available.

CFU-GM (Colony-Forming Units Granulocyte-Macrophage) Hypersensitivity Assay
A key feature of JMML is the hypersensitivity of myeloid progenitor cells (immature white blood cells) to a substance called GM-CSF (granulocyte-macrophage colony-stimulating factor). A patient’s bone marrow or peripheral bloods cells are sent to a specialized lab (formerly in Alabama) and the cells are kept alive in several tissue-culture plates with culture medium (methylcellulose) and increasing concentrations of GM-CSF. Normal cells will not grow in low concentrations of GM-CSF while JMML cells will grow in low concentrations of GM-CSF. Individual cells grow to form colonies (a cluster of cells) – thus if the report is given that there is “spontaneous or hypersensitive colony growth indicative of JMML,” it means that the JMML cells grew in low or no GM-CSF on the methylcellulose. This test takes several weeks to complete and is only done by specialized labs, so there is often a significant wait for the results.

Genetic Tests for Specific JMML Mutations
Several specific genetic mutations are known to be associated with JMML. A mutation is a “mistake” in the patient’s DNA that can result in a change in normal cellular function. The coding sequence of portions of the patient’s DNA can be examined in the laboratory to look for the known JMML mutations. Certain mutations in the neurofibromatosis-1 (NF-1) gene, RAS genes (only in leukemia cells), or PTPN11 gene (only in leukemia cells) will confirm a diagnosis of JMML. Approximately 20 to 25% of JMML cases are not associated with any known genetic mutation.

Tests to Determine Donor Engraftment Post-transplant

To determine % donor cells vs. % recipient cells for a post-BMT patient, various tests can be used such as FISH, VNTR, or RFLP. There are some newer tests, such as Taq-MAMA and SNP, which are being validated in the lab and may eventually be used by clinical diagnostic labs. Which test your child has done depends on how different their DNA is from the donor and which tests your lab does. The best possible score is different for each test and may range from 97% donor to 100% donor, depending on the nature and inherent accuracy of the test and the ability to use a large enough sample of cells for statistical accuracy. A 100% on one test is not better than a 98% on another if each of those results is the best possible for the test used.

FISH (Florescence In-Situ Hybridization) If donor and recipient are a different sex or have differences that are visible at the level of chromosomal appearance, the FISH test can be done. In a FISH test, patient bone marrow cells are prepared in such a way that whole chromosomes remain intact and can be spread on slides and identified under the microscope. Florescent "tags" can be attached or hybridized with the chromosome preparation and have a bright colored appearance under the microscope. Fluorescent tags that are specific for the recipient DNA will “light up” a chromosome that came from a recipient cell, such as a Y chromosome from a male recipient, when the donor is female.

RFLP (Restriction Fragment Length Polymorphism) Patient and Donor DNA samples from blood or bone marrow are cut with restriction enzymes, which chop the DNA into fragments at specific sites in the DNA. This chopped up DNA is size-separated in the lab so that the restriction fragments can be viewed. Discrete sized DNA fragments will be separated out and then “labeled” by binding a radioactive or fluorescent “tag” known to be different between the donor and recipient. Compared to samples that are from the donor and pre-transplant recipient, the pattern of fragments will be quantified as to % donor or recipient.

VNTR (Variable Number Tandem Repeat) is a test that is done at the level of the DNA to detect differences in characteristic tandem repeated DNA sequences that can distinguish donor and recipient. The VNTR test is based on PCR technology (polymerase chain reaction) to allow amplification of the patient's DNA. As the amount of DNA that can be obtained from a patient's blood or bone marrow is too small to detect, it is necessary to amplify or increase the amount by exact DNA copying, in order to get to a level that is detectable. A fluorescent short piece of DNA (primer) that binds specifically to recipient or to donor DNA is used to amplify the patient’s DNA and quantify the % donor and recipient DNA in a sample.

Taq-MAMA
The TaqMAMA assay uses PCR technology (polymerase chain reaction) to detect the single base-pair changes in the part of the DNA that codes for a specific JMML mutation. As the amount of DNA that can be obtained from a patient's blood or bone marrow is generally too small to see on a gel, it is necessary to increase the amount of DNA by the polymerase chain reaction in order to get to a level that is detectable. This amplification is done in such a way that only DNA containing the JMML mutation (recipient DNA) can be copied and not the "normal" or donor DNA. Some small level of the mutant DNA may be found even in a patient who is 100% donor by other methods. An increase in the level of the mutation detected over time indicates a possible relapse and this increase is typically rapid. This assay, in addition to assessing chimerism, could also be used as a diagnostic tool.

SNP Real-time PCR (Single-Nucleotide Polymorphism)
The SNP assay uses PCR technology (polymerase chain reaction) to detect the single base-pair changes in parts of the DNA that are highly variable between individuals. As the amount of DNA that can be obtained from a patient's blood or bone marrow is generally too small to see on a gel, it is necessary to increase the amount of DNA by the polymerase chain reaction in order to get to a level that is detectable. SNP is similar to TaqMAMA in that it is PCR-based and can detect changes down to a single base-pair change. An important difference is that the single base pair changes called SNPs are not specific to the JMML mutations. While SNP analysis can be used to distinguish donor and recipient DNA, it does not necessarily detect the specific JMML mutation.