M.C. Cox Froncillo 7 L. Maffei 7 M. Cantonetti 7 G. Del Poeta R. Lentini 7 A. Bruno 7 M. Masi 7 M. Tribalto 7 S. Amadori Division of Hematology, “Tor Vergata” University, Rome, Italy M.C. Cox Froncillo (Y) Cattedra di Ematologia, Ospedale S. Eugenio, P. le dell’Umanesimo 10, I-00144, Rome, Italy Ann Hematol (1996) 73 : 113–119 Q Springer-Verlag 1996 ORIGINAL ARTICLE M.C. Cox Froncillo 7 L. Maffei 7 M. Cantonetti G. Del Poeta 7 R. Lentini 7 A. Bruno 7 M. Masi M. Tribalto 7 S. Amadori FISH analysis for CML monitoring ? Received: 29 February 1996 / Accepted: 7 May 1996 Abstract Conventional cytogenetics is considered the gold standard for evaluating CML during interferon (IFN) treatment. Drawbacks to this approach are the small number of metaphases available during IFN ther- apy and the impossibility of scoring interphase cells. We applied, besides cytogenetics, double-color FISH (dc-FISH) detection of BCR-ABL gene fusion to mon- itor 20 CML patients on IFN. dc-FISH easily detected 200 cells per specimen, while with cytogenetic examina- tion a mean of 16.1 mitoses per sample were scored. Though the correlation of dc-FISH and cytogenetic data was good (rp0.77, p~0.001), the discrepancy be- tween the two methods as regards the proportion of leukemic cells in the marrow was often important. dc- FISH detected a relevant proportion of BCR-ABLc cells in three patients classified as complete cytogenetic responders and showed that, after 9–12 months of IFN treatment, a significant reduction of BCR-ABLc cells was present in all the 20 patients tested. This might sug- gest that all CML patients are potentially responsive to IFN. Though more data are required, we think that dc- FISH is more informative than cytogenetic analysis for CML monitoring. Notably because of the simplicity of the procedure, this method could be easily standar- dized among different laboratories, thus permitting cross-comparison in therapeutic trials. Key words IFN therapy 7 CML monitoring 7 BCR-ABL 7 Philadelphia chromosome Introduction Detection and quantification of residual leukemia dur- ing therapy is a main concern of hematologists. Con- ventional cytogenetics is considered the gold standard for evaluating response to IFN therapy in CML [1–3]. By general agreement, 20–30 analyzed metaphases are considered adequate for assessing the remission rate. The main limitations to this approach are: (a) the small number of evaluable metaphases generally found in IFN-treated subjects and (b) the inability of metaphase cytogenetics to score interphase cells, possibly leading to erroneous conclusions regarding the actual propor- tion of Phc cells [4, 5]. Recently, fluorescent in situ hybridization (FISH) has been introduced in hematolo- gy: this method is widely used for gene mapping, for identifying complex chromosome translocations (paint- ing), for detecting gene deletions, and for verifying chromosome number aberrations and gene rearrange- ment [6, 7]. The monitoring of leukemic disease is an important application of FISH and might soon become very widespread owing to the ease of the procedure and the few technical devices required [8]. We applied, in conjunction with conventional cytogenetics, double- color FISH (dc-FISH) detection of BCR-ABL genes fusion [9–17] to monitor 20 CML patients during the course of IFN therapy. To our knowledge, except for a few preliminary reports [17–19], this is the first paper comparing serial cytogenetic and interphase dc-FISH analyses in CML during IFN treatment. Patients, materials and methods Study design Marrow samples from 20 CML patients were collected at diagno- sis and serially during IFN therapy. Cytogenetic and dc-FISH analysis were performed on each sample. For cytogenetics we aimed at analyzing the greatest number of mitoses, while for FISH detection we fixed the number at 200 cells to be scored. Besides CML samples, we tested 50 AML specimens as negative