Flow cytometric DNA ploidy and S-phase fraction in canine leukaemias

Besides immunophenotypic characterization of hematologic neoplasms, flow cytometry plays an important role in human oncology with the analysis of cell cycle and DNA content in neoplastic cells. In veterinary medicine DNA ploidy and cell cycle (S-phase) determination have been characterized in few studies on some solid tumours and no researches on leukemias are present. The aim of the present study is to evaluate flow cytometric DNA ploidy and S-phase fraction in canine leukemias. Forty-six cases selected among the samples received in our laboratory were enrolled in the study. Inclusion criteria were a diagnosis (based on CBC, morphologic evaluation and flow cytometric analysis of peripheral blood, bone marrow and lymph node aspirates) of leukemia or lymphoma with blood involvement and the availability of fresh samples (within 4h since collection). Each case was allocated to one of the following groups: acute myeloid leukaemia (AML, n=11), chronic myeloid leukemia (CML, n=2), acute lymphoblastic leukemia (ALL, n=16), chronic lymphocytic leukemia (CLL, n=12), lymphoma with blood involvement (LSA, n=5). Samples were processed as fresh or stored at -80°C until analysis. Cell suspensions were stained with propidium iodide using a commercial kit (DNACon3, ConsulTS), acquired at a Epics XL fow cytometer (Coulter) and analyzed with a dedicated software (Multicycle for Windows, Coulter). Blood from healthy dogs was used as control to set the G0/G1 diploid peak and to calculate DNA index (DI) of the tumors. DI and S-phase fraction were recorded and used for statistical comparison between groups. According to the guidelines from the 1993 DNA Cytometry Consensus Conference, 3 AML cases reporting BAD>20% and/or CV>8% were excluded from the statistics. Hyperdiploidy (DI>1) was detected in 2 out of 8 AML (25%), 2/16 ALL (12.5%, 1 B-ALL and 1 T-ALL) and 1/5 LSA, while CML and CLL were euploid. No hypodyploid samples (DI<1) were identified. S-phase fraction was significantly higher in AML and ALL (particularly in aneuploid samples) compared to CLL and LSA. No statistical significant differences were detected between AML and ALL and between B- and T-ALL. CLL and LSA reported S-phase fraction similar to control samples. Present data suggest that flow cytometric DNA ploidy and cell cycle analysis with S-phase determination could be useful in discriminating acute versus chronic leukemias and acute leukemias versus V-stage LSA. ROC curves were calculated to identify a useful cut off value and showed encouraging results particularly in the second case. To confirm these results, a higher number of cases is needed; upon confirmation, the effects on clinical decisions will have to be carefully evaluated to decide whether to get a higher specificity or sensibility from the test.