450 I Clin Pathol 1996;49:450-452 Papers J Clin Pathol: first published as 10.1136/jcp.49.6.450 on 1 June 1996. Downloaded from

Value of colony forming unit- macrophage assay in predicting relapse in acute myeloid leukaemia

M C del Cafiizo, A Mota, A Orfao, J Galende, M D Caballero, M A Garcia Marcos, J F San Miguel

Abstract over the past 20 years, the majority of patients Aim-To evaluate the validity ofthe colony who have achieved complete remission will forming unit-granulocyte macrophage eventually relapse as a result of the persistence (CFU-GM) assay for predicting relapse in of residual leukaemic cells, below the detection patients with acute myeloid leukaemia limit of light microscopy.' 2 Several methods (AML). for detecting minimal residual disease have Methods-The study population com- been developed and evaluated-for example, prised 32 patients with AML in remission, cytogenetics, immunological marker analysis followed for a median of 18 months. A and molecular biological techniques," and in mean of four studies was carried out per vitro clonogenic assays.7 However, distin- patient. Three patterns of in vitro growth guishing between clonogenic leukaemic cells based on the number of CFU-GM in nor- (colony forming unit-leukaemic (CFU-L)) and mal were defined: 1 = normal myeloid progenitor cells (colony form- normal (normal number of CFU-GM and ing unit-granulocyte macrophage (CFU-GM)) a cluster:colony ratio <2); 2 = hypoplastic is problematic, because both types of cells (low number of CFU-GM and a cluster- respond to the same growth factors, despite :colony ratio <2); 3 = anomalous (low or displaying different growth characteristics in normal number of CFU-GM and a clus- vitro.""1' In order to identify precisely the http://jcp.bmj.com/ ter:colony ratio >2). existence of CFU-L, more sophisticated ap- Results-Eleven patients relapsed, all of proaches have to be applied-for example, whom had previously displayed an abnor- analysis of clonal gene rearrangements8 or in mal CFU-GM pattern: anomalous in nine situ analysis of surface markers on the and hypoplastic in two. The remaining 25 leukaemic colonies.9 In patients with preleu- patients were in complete remission at the kaemic syndromes, such as the myelodysplastic time of writing, 16 of whom had a normal syndrome, alterations in the growth patterns of on September 29, 2021 by guest. Protected copyright. growth pattern. The other nine had CFU-GM appear before the transformation anomalous (eight patients) or hypoplastic into acute leukaemia."2 (one patient) growth. The latter may be Servicio de vitro Methods false positive results. The in growth The population comprised 37 patients Hematologia y pattern was not constant during follow up study Hemoterapia, the with AML.'3 '4 Patients were uniformly treated Departamento de analysis. All 15 patients in whom with daunorubicin and Ara-C (3/7 days) plus Medicina, growth pattern switched from abnormal Hospital Universitario in remission. intensive consolidation treatment with high de to normal remain complete bone marrow samples Universidad the five cases in whom the dose Ara-C. Sequential Salamanca, By contrast, of were obtained once the patients achieved com- Salamanca, pattern changed from normal to abnor- remission. Five patients died and were Spain three have relapsed and the other two plete M C del Cafiizo mal, excluded from the study. Four ofthe remaining A Mota had other indicators of relapse. The in 32 patients achieved remission for a second J Galende growth pattern remained unchanged marrow samples from both remis- M D Caballero the remaining 16 patients. time; bone M A Garcia Marcos sion episodes in these patients were included in J F San Miguel Conclusion-The present data show that of the the study, giving a total of 36. A mean of four the sequential investigation studies was carried out per patient. Median Servicio Central de CFU-GM growth pattern may be of value Citometria, in with follow up was 18 months. Universidad de in predicting relapse patients Control bone marrow samples were ob- Salamanca AML. tained from 15 haematologically normal pa- A Orfao (_ Clin Pathol 1996;49:450-452) tients undergoing orthopaedic surgery who Correspondence to: gave informed consent to bone marrow aspira- Dr M C del Caiiizo, Keywords: colony forming unit-granulocyte macro- Department of Haematology, phage, acute myeloid leukaemia, relapse. tion for scientific purposes. University Hospital, 37007 Salamanca, COLONY ASSAY Spain Bone marrow samples were collected in sterile, in the treat- preservative free heparin tubes and separated Accepted for publication Despite significant improvements 16 January 1996 ment of acute myeloblastic leukaemia (AML) by Ficoll-Hypaque (d=1070) density gradient Predictive value ofCFU-GM in AML 451

centrifugation." The CFU-GM assay was car- analysis of growth of CFU-L in bone marrow ried as described by Iscove et al.'6 Briefly, 2 x cultures.! Moore et al'5 studied the predictive 10' mononuclear cells/ml in Iscove's modified value of analysing the in vitro growth pattern of J Clin Pathol: first published as 10.1136/jcp.49.6.450 on 1 June 1996. Downloaded from Dulbecco's medium (IMDM) were plated on CFU-L at diagnosis. Analysis of the growth 35 mm Petri dishes in 0.9% methylcellulose pattern of CFU-GM has generally been used containing 10% phytohaemagglutinin stimu- to assess residual haemopoietic damage as a lated leucocyte conditioned medium,"7 10% result of treatment'9 but not for predicting bovine serum albumin, and 10% human AB relapse. Our results show that sequential inves- serum. Cultures were incubated at 37°C in a tigation of the CFU-GM growth pattern may fully humidified atmosphere with 5% CO2 and be helpful in predicting relapse in patients with scored on day 14 under an inverted micro- AML. In our experience the proliferation scope. Aggregates containing more than 40 pattern does not remain constant during the cells were scored as colonies; aggregates evolution of the disease and frequent changes containing four to 40 cells were scored as clus- may occur. All patients who achieved a normal ters. growth pattern remain in complete remission, Based on the CFU-GM pattern observed in but a median of six months elapsed before a normal bone marrow, we defined three pat- stable, normal growth pattern was observed. terns of in vitro growth: 1 = normal (normal By contrast, three of the five patients whose number of CFU-GM and a cluster:colony ratio initial normal growth pattern became abnor- <2); 2 = hypoplastic (low number ofCFU-GM mal relapsed. The other two patients exhibited and a cluster:colony ratio <2); 3 = anomalous other indicators of imminent relapse.' (low or normal number of CFU-GM and a Sequential studies of the CFU-GM growth cluster:colony ratio >2). Normal values were pattern are mandatory if this technique is to be (mean (SD)) 142 (60) CFU-GM and a used to predict relapse. Thus, patients whose cluster:colony ratio of 1.22 (0.18). CFU-GM growth pattern changes from nor- mal to abnormal must be monitored frequently Results as they are at a high risk of relapse. It is highly Eleven patients relapsed, all of whom previ- probable that those patients with a persistant, ously displayed an abnormal CFU-GM pat- normal CFU-CM growth pattern of one years' tern: anomalous in nine and hypoplastic in duration will remain in remission and generally two. No false negative results were observed. will not need further intensive treatment. A The median time between detection of an larger patient cohort and a longer follow up are abnormal growth pattern and relapse was four needed to confirm these observations. + two months. The situation is not so clear for those The remaining 25 patients were in complete patients with a persistently abnormal growth remission at the time of writing: 16 had a nor- pattern, as only half have relapsed to date. http://jcp.bmj.com/ mal CFU-GM growth pattern, whereas the Fluctuations in CFU-GM numbers have been other nine showed anomalous (eight patients) observed previously in patients with AML and or hypoplastic (one patient) growth. These these have been interpreted differently. Chang may be false positive results. Of the nine et al'9 regarded these fluctuations as a result of patients with an abnormal CFU-GM growth haemopoietic damage following chemotherapy pattern, four also had other indicators ofimmi- with no particular pathological significance. nent relapse: in two patients the number of Sallefords and Olofson"o, however, suggested on September 29, 2021 by guest. Protected copyright. blast cells increased from undetectable levels that a fall in the number of these progenitor by morphological and immunophenotypical cells could precede a relapse episode. methods to 1.3% and 4%, respectively; throm- Chemotherapy results in a severe reduction bocytopenia was diagnosed in the other two in the number of clonogenic progenitor cells, patients. which may persist for a long period of time During the first three months of the study all after the patient has achieved complete remis- except one patient had an abnormal CFU-GM sion.'92" By contrast, Sallefors and Olofson'0 growth pattern. Stable, normal CFU-GM showed that most patients with AML in remis- growth patterns were observed a median of six sion have a normal number of CFU-CM cells, months after diagnosis. All 15 patients whose which is in keeping with the data presented growth pattern normalised remain in complete here. remission. By contrast, of the five patients In summary, sequential investigation of the whose pattern changed from normal to abnor- CFU-GM growth pattern may be of value in mal, three have relapsed and the other two predicting relapse in patients with AML. showed either an increase in the number of Patients with a persistently abnormal growth morphological blast cells present or a decrease pattern are at high risk of relapse. in their platelet count. The growth pattern 1 Champlin R, Gale RP. Acute myelogenous . Recent remained unchanged in the remaining 16 advances in therapy. Blood 1987;69:1551-62. patients: it was normal throughout the study in 2 Cheson BD, Casileth PA, Head DR, Schiffer CA, Bennett one and was always abnormal in the JM, Bloomfield CD, et al. Report of the National Cancer other 15. Institute-sponsored workshop on definitions of diagnosis Eight of the latter 15 patients subsequently and response in . J Clin Oncol 1990;8:813-19. relapsed. 3 Freirich EJ, Cork A, Stass AS, McCredie KB, Keating MJ, Elihu HE, et al. Cytogenetics for detection of minimal residual disease in acute myeloid leukemia. Leukemia Discussion 1 992;6:500-6. Investigations of minimal residual disease may 4 Van Dongen JJM, Breit TM, Adriansen HJ, Beshuizen A, Hoo- be of in ijkaas H. Detection of minimal residual disease in acute leu- great help managing patients with kemia by immunological marker analysis and polymerase AML. Most investigations have focused on chain reaction. Leukemia 1992;6(Suppl 1):47-59. 452 del Canlizo, Mota, Orfao, Galende, Caballero, Garcia Marcos, San Miguel

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