An example of the use of clinical molecular biology in dis ease monitoring during therapy and therapeutic selection is described below.
Chronic myeloid leukemia (CML), typical in older adults, is a monoclonal expansion of bone marrow stem cells. The disease is due to an acquired genetic alteration, termed Philadelphia chromosome (described in 1961 in this city), which causes translocation of gene material between the long arm of chromosome 9 and the short arm of chromosome 22. The translocation causes the formation of a fusion gene (BCR-ABL) that encodes an oncogene with tyrosine kinase functions that promotes uncontrolled stem proliferation. The disease is characterized by a chronic course that can evolve toward a blastic phase. Until a few years ago, the diagnosis of CML was morphological (peripheral blood smear and marrow analysis), made more specific by the analysis (first cytological and then cytofluorimetric) of some specific sur face markers of CML cells. Cytogenetics (in vitro culture of cells from peripheral blood or marrow and analysis of the karyogram), through the identification of the Philadelphia chromosome, has made the diagnosis more specific. Subsequently, clinical molecular biology technologies allowing BCR-ABL transcript analysis (i.e., messenger RNA analysis by RT-PCR) favored the strong increase of the analytical sensitivity, allowing recognition of a cell expressing the fusion gene among more than 100,000 normal cells. In the meantime, a class of molecular drugs capable of selectively inhibiting the BCR-ABL protein has been developed, drastically changing the disease’s prognosis. At present, BCR-ABL transcript analysis has three main applications:
• Diagnosis of disease by searching for the transcript in circulating blood or bone marrow cells.
• Monitoring during therapy, which allows the identification of minimal residual disease. Currently, we speak of complete cytogenetic remission to indicate patients in whom the cytogenetic analysis for the Philadelphia chromosome is negative; significant molecular remission, when the molecular analysis (more sensitive than cytogenetics) shows a residue of positive cells below a thresh old; complete molecular remission, when the molecular analysis is negative.
• Identification of therapy-resistant patients. During therapy with a tyrosine kinase inhibitor, some patients with CML develop resistance to therapy due to the development of somatic mutations in the BCR-ABL gene that make the protein resistant to the inhibitor. In these cases, there is a recurrence (also molecular) of the disease. In these patients, it is possible to analyze the gene in the leukemic cells (by PCR and sequencing) and identify the mutation and the percentage of mutated cells, planning to use a different inhibitor.
In addition to chronic myeloid leukemia, aberrant transcripts from fusion genes or other genetic abnormalities have been identified in several other hemolymphoproliferative diseases. In analogy to CML, analysis of these transcripts may be helpful in monitoring the patient during therapy.
