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"Measurement of residual leukemia during remission in childhood acute lymphoblastic leukemia." (21)
This 1997 paper by Roberts and colleagues comes out of the University of Texas M.D. Anderson cancer Center. Although they only studied 25 patients, their methodology is, in the opinion of your editor, quite elegant. Their comments in the discussion section are also worth sharing. Plus, their results support a controversial concept (more on this later).
Roberts and colleagues amplified the IgH gene from bone marrow samples obtained at diagnosis and remission and isolated the PCR band from the gel. They then sequenced this DNA to confirm that it was leukemic DNA and also that each subsequent test for MRD showed up the same clone. For each patient, they designed patient-specific primers for use in the PCR reactions. The amount of residual leukemia was determined with a limiting-dilution method. (22) (This reference is a technical report in the journal Leukemia which explains in detail the methods they used; note that the same authors are on both papers.) They claim a detection limit of 5x10-6.
They only studied 24 patients. 7 patients relapsed: 5 in the bone marrow, two in CNS and bone marrow. For the 17 patients who remained in complete remission the median follow-up time was 45 months.
In the seven patients who relapsed, levels of MRD increased noticeably over a period of time prior to relapse, as evidenced by twelve and twenty fold increases in MRD levels between successive determinations. These increases preceded the clinical diagnosis of relapse by four to nine months. 15 of the 17 patients who remained in remission had detectable MRD. They double checked this MRD result in 12 of these15 patients using the blast colony assay; the assay was positive for 7 of the 12 patients.
In the patients who remained in remission, the PCR assay values generally declined to the lowest levels during the second year of treatment. However, only half the samples during this period tested negative by the PCR assay. They found evidence of residual leukemia in more patients than previous studies, which they attribute to the use of fresh, viable, purified bone marrow cells and the fact that the sensitivity of their PCR was consistently greater than the ratio of leukemia cell DNA to normal bone marrow cell DNA than reported in the other studies, since they used replicate PCR amplifications that used 10 micrograms of DNA each (equivalent to 2 million cells). They took extensive precautions to avoid false positive PCR results -- all PCR products were sequenced for confirmation and they correlated the results with a clonogenic blast colony assay.
Direct quotes: "Our results indicate that cure and the absence of leukemia cells may not be synonymous. . . our results challenge the dogma about the nature of cure, which is based on animal models of leukemia. Our data imply that more than 10,000 leukemia cells may persist in a patient who remains in long-term remission and that the cure of ALL may not require the elimination of all leukemia cells. ... We speculate that chemotherapy may cure patients with some forms of acute leukemia by effects other than those directly related to chemotherapy. Our data support those of Gale and Butturini (26), who suggest that since maintenance chemotherapy does not eliminate all remaining leukemia cells, other processes could control the accumulation of these cells. These may include modifications to the program controlling the growth of leukemia cells, altered immune surveillance, or the interaction of the malignant cells with the microenvironment of normal bone marrow. The identification of HLA-unrestricted cytotoxic T cell clones specific for lymphoblasts suggests that the immune system can destroy ALL cells."
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