Development of molecular genetic methods for monitoring myeloid malignancies

The malignant diagnosis of a haematological disorder can in most cases be made by clinical signs and routine microscopic examination. However, it has become necessary to characterize the malignant clone with various markers, which give either knowledge of the prognosis of the disease or give tools for the laboratory follow up of the patient. In lymphatic diseases there are excellent markers of clonality. On the contrary in myeloid malignancies the few well characterized markers are mostly helpful in the clinical management of rare myeloid subgroups. The aim of our project has been to develop methods for laboratory monitoring of myeloid diseases by two major approaches 1) detection of methylation alterations in the short arm of chromosome 11 and 2) novel approaches for sensitive point mutation detection. The short arm of chromosome 11 has areas where the DNA becomes hypermethylated in acute leukemias and lymphomas. In this chromosomal area the calcitonin gene serves as a good marker for methylation alterations due to several CpG sites in the 5'area of the gene. Even if the gene is normally methylated in most cases of chronic myeloid leukemia (CML), we have found that the hypermethylation of the calcitonin gene marks progression of CML and precedes any other signs of acceleration with several months. The point mutations of certain proto-oncogenes, such as the N-ras gene, are attractive markers for detecting residual diseases after chemotherapy of high malignant haematological disorders. However, conventional methods for detecting point mutations have been both insensitive and cumbersome, and thus unsuitable for clinical routine laboratories. With the solid-phase minisequencing we can technically easily and accurately detect small quantities of mutated cells.