The aim of this study was to demonstrate the value of immunocytochemical staining of Ki67 antigen expression in blast cells of children with acute myeloid leukemia (AML) and to evaluate its correlation with treatment failure. The material included bone marrow specimens obtained during induction treatment from 46 children treated for AML between 1998-2003. Immunocytochemical staining for Ki67 was based on the ABC technique. Expression of Ki67 antigen on day 0 of induction treatment was confirmed in all patients. The percentage of immunopositive blasts ranged from 88.4% to 99.8% (mean 91.8%). On day 15, according to chemotherapy response, patients were divided into two groups: G1-36 children who responded to induction treatment and reached remission (blast level 5%, low risk group) and G2-10 patients who did not meet remission criterion (blast level > 5%) and were assigned to the high risk (HR) group. Out of 10 children assigned to this group, Ki67 expression in blast cells was confirmed in 4 cases. The fraction of immunopositive blasts ranged from 78.4% to 88.6%. In the other 6 cases, blasts were Ki67-negative. In 12-month period after beginning the treatment, 18 cases of treatment failure (including 7 deceases) were observed in both groups. Five deaths, observed in the HR group, concerned the patients characterized by Ki67-negative blasts. The results indicate a possible correlation between the Ki67-immunonegative blast pattern on day 15 of treatment induction and early decease of AML children assigned to HR group

Miśkowiak, Bogdan

Nowicki, Michał

Ostalska-Nowicka, Danuta

English

Via Medica Journals

FOLIA HISTOCHEMICAET CYTOBIOLOGICAVol. 44, No. 1, 2006pp. 49-52Prognostic significance of Ki67-negative blast cell clone in the high risk group of children treated for acute myeloid leukaemiaMichał Nowicki1, Danuta Ostalska-Nowicka2 and Bogdan Mis´kowiak1,3Departments of 1Histology and Embryology, 2Pediatric Cardiology and Nephrology and 3Optometry and Biology of the Visual System, University of Medical Sciences, Poznan´, PolandAbstract: The aim of this study was to demonstrate the value of immunocytochemical staining of Ki67 antigen expression inblast cells of children with acute myeloid leukemia (AML) and to evaluate its correlation with treatment failure. The materialincluded bone marrow specimens obtained during induction treatment from 46 children treated for AML between 1998-2003.Immunocytochemical staining for Ki67 was based on the ABC technique. Expression of Ki67 antigen on day 0 of inductiontreatment was confirmed in all patients. The percentage of immunopositive blasts ranged from 88.4% to 99.8% (mean 91.8%).On day 15, according to chemotherapy response, patients were divided into two groups: G1 - 36 children who responded toinduction treatment and reached remission (blast level  5%, low risk group) and G2 - 10 patients who did not meet remissioncriterion (blast level > 5%) and were assigned to the high risk (HR) group. Out of 10 children assigned to this group, Ki67 expressionin blast cells was confirmed in 4 cases. The fraction of immunopositive blasts ranged from 78.4% to 88.6%. In the other 6 cases,blasts were Ki67-negative. In 12-month period after beginning the treatment, 18 cases of treatment failure (including 7 deceases)were observed in both groups. Five deaths, observed in the HR group, concerned the patients characterized by Ki67-negative blasts.The results indicate a possible correlation between the Ki67-immunonegative blast pattern on day 15 of treatment induction andearly decease of AML children assigned to HR group. (www.cm-uj.krakow.pl/FHC)Key words: Ki67  - Acute myeloid leukemia - PrognosisIntroductionAcute myeloid leukemia (AML) constitutes up to 15%of all leukemias in children [16]. Definition of AMLencompasses a number of proliferative diseases develo-ping secondary to neoplastic transformation of non-lym-phocytic and poorly differentiated bone marrow cells.The current classification was established in 1976 by aFrench-American-British (FAB) team [2]. Modified in1985 by supplementation with erythroleukemia andmegakaryocytic leukemia [3, 4], it is based first of all onmorphological criteria related to neoplastically trans-formed hematopoietic cells. In children with AML, among other factors, theoriginal leukocytosis in peripheral blood  50 G/l can beregarded as unfavourable [20]. Patient age < 2 yr, formsM4 and M5 [19] or genetic abnormalities are also usedto be listed [21, 24]. In the Polish Pediatric Leuke-mia/Lymphoma Study Group, patients are included tothe high risk group if the M5 subtype is preliminarilydiagnosed or when leukemic blasts constitute > 5% nu-cleated cells of bone marrow on day 15 after commenc-ing the treatment (AML-98 regimen) [5, 6]. In ourobservations, high risk group criteria are met by around20% of patients [7]. Although the hematological re-mission is obtained in 70%-85% of those patients on day29, follow-up survival without relapses and decease canbe obtained in no more than 50% of patients [9]. Thismay indicate that the high histological and clinical ma-lignancy in this group has not yet been counteracted withan appropriate therapeutic protocol. In earlier study performed in a group of children withacute lymphoblastic leukaemia, we have found that thepresence of Ki67-positive blast cells before the start ofthe treatment represents a potential index of favourableprognosis and can assist qualification of the patients torisk groups [14]. Ki67 antigen expression appears to occur in themiddle of the G1 phase of the cell cycle. It increasesCorrespondence: M. Nowicki, Dept. Histology and Embryology,University of Medical Sciences, S´wie˛cickiego 6, 60-781 Poznan´,Poland; e-mail: mnowicki@amp.edu.pl during the S and G2 phases to reach a peak during mitosis[23]. The Ki67 molecule becomes rapidly hydrolysedsoon after the end of the M phase. Together with prolif-erating cell nuclear antigen (PCNA), the Ki67 antigen isone of the most sensitive markers of undifferentiated cellproliferation [1, 10, 23]. It is absent during the G0 phase- the stage at which neoplastic cells are totally unrespon-sive to chemotherapy [23]. In line with the abovementioned findings, the aim ofthe present report was to demonstrate the value of Ki67antigen expression in blast cells of patients with AMLduring chemotherapy regimen and to evaluate its corre-lation with early treatment failure.Materials and methodsMaterial used in this study consisted of bone marrow samples ob-tained from children treated at the Department of Pediatric Oncology,Hematology and Transplantology, Poznan´ University of MedicalSciences, between 1998 and 2003. The research protocol was ap-proved by the local Ethical Commission of the University. Sixty-fivechildren referred to our Department were included in the investiga-tion. Bone marrow biopsies were performed in all of them andevidence for neoplasia was found in 46 cases. The remaining 19children, all of whom presented only one enlarged lymph node,served as the control group. In this group, histological examinationof the enlarged node indicated an inflammatory response only.Subsequent observation of the children for around 12 months in theOutpatient Clinic for Hyperplastic Diseases, detected no clinicaltraits of a neoplastic disease. Four children were diagnosed as AML type M0, 8 as AML M1,26 as AML M2, 4 as AML M3 and 4 as AML M4. There were nopatients diagnosed with AML M5, M6 and M7 types. The relevantdata are presented in Table 1. Bone marrow, according to ANLL-98 protocol, was sampled ondays 0, 15 and 29 after starting the treatment. Its aspiration wascarried out under intravenous anesthesia (pethidine 1mg/kg b.w.,midazolam 0.1mg/kg b.w.). The samples were collected from the pos-terior superior iliac spine. The bone marrow smears were fixed in 96%ethanol (30 min, room temperature) within 24 hours of sampling andkept at -80˚C until immunophenotyping was performed.Ki67 antigen detection involved an immunocytochemical proce-dure with the use of mouse monoclonal antibodies against humanKi67 (Dako, M 7187) and StreptABComplex/HRP technique ampli-fied by the use of biotinylated tyramine (Dako Catalysed SignalAmplification System, Peroxidase, K 1500). Heat-induced antigendemasking pre-treatment was carried out (Target Retrieval Solution,Dako S 1699) [15, 17, 18]. The endogenous peroxidase activity wasblocked by 10 min preincubation in 10% hydrogen peroxide. Thesmears were then treated with anti-Ki67 antibodies, diluted 1:500,for 12 h at 4˚C. Incubation with the secondary antibody (biotinylatedgoat anti-mouse, Dako E 0433, diluted 1:300) was performed at roomtemperature for 60 minutes. This was followed by the incubation withdiaminobenzidine (DAB, Dako S 3000). The number of immunoposi-tive blasts, as compared to the total number of blast cells, was establishedunder a light microscope (magnification × 100). Quantitative analysiswas carried out using MultiScan® computer software.Immunocytochemical staining of the following antigens was usedto verify leukemic and normal cells: HLA-DR, CD13, CD14, CD15,CD33, CD34, CD41 and CD61 [9]. The diagnosis of AML remissionwas confirmed by blood morphology (clinical remission), micro-scopic analysis of subsequent bone marrow samples (May-Grüun-wald-Giemsa staining, hematologic remission) and the secondimmunophenotyping of bone marrow nucleated cells (immunologi-cal remission).In every case of Ki67 immunocytochemical detection, each la-boratory test was performed at least twice. In addition, each exam-ination of bone marrow was accompanied by positive and negativecontrols [8, 13]. All results which were doubtful, nonspecific, ordifficult to interpret were excluded from the study.Since the studied group was relatively small, the statistical ana-lysis was based on the Fisher exact test.ResultsExpression of Ki67 antigen on day 0 of initial treatmentwas confirmed in all patients with AML. The percentageFig. 1. Diagrammatic representation of the sample outcome.50 M. Nowicki et al.of immunopositive blasts ranged from 88.4% to 99.8%(mean of 91.8% blast cells) and did not significantlydiffer between AML subtypes. Ki67 was absent (< 5%nucleated cells) in the normal hematopoietic cells of 19children in the control group. The AML remission was diagnosed in 36 children(78.3%, low risk group) on day 15 (mean 3.2% leukemiccells). In 10 children (21.7%, high risk group) diseaseprogression was diagnosed (4 cases of AML type M1, 5of M2 and 1 of M3, respectively) with a poor responseto cytoreducing treatment on days 15 and 29 of treatmentwith cytosine arabinoside, idarubicin and etoposide (areduction of the bone marrow blast level by less than5%). In 4 out of 10 children assigned to the high risk group,Ki67 expression in blast cells was confirmed on day 15(40.0%). The fraction of immunopositive blasts rangedfrom 78.4% to 88.6%. In the remaining 6 cases (60.0%),blasts were Ki67-negative.During the observation period (12 months), 18 casesof early leukemia treatment failure were noted. In thelow risk group one child died due to Pseudomonasaeruginosa infection. In 7 cases, the subsequent bonemarrow examination was performed due to relapse ofthe disease. It revealed Ki67-positive blasts (mean88.5%) in 3 children and Ki67-immunonegative blastpattern in the other 4 children. The treatment protocolwas changed to a more aggressive Ida-FLAG program[11] followed by bone marrow transplantation and, ex-cept of one Ki67 immunonegative patient, these childrenare still alive in their second remission. In the high riskgroup, 6 children died due to progression of the diseaseresulting from the pan-drug resistance. Apart from onechild who was Ki67-positive on day 15, all other patientsrevealed an immunonegative Ki67 blast pattern on thatday of treatment. The detailed information is presentedin Figure 1.The probability of the first remission was significant-ly higher in the low risk group patients (p=0.0001), butindependent from the initial Ki67 expression.The distribution of mortality in high risk group pa-tients was Ki67-dependent (regardless of AML type).Statistical analysis of these children revealed that theprobability of permanent leukemia remission is signifi-cantly higher in patients with Ki67-immunopositiveblast pattern on day 15 (p=0.0194). It has been alsoshown that the risk of death following leukemia progres-sion in patients with Ki67-immunonegative blast patterntreated according to only ANLL-98 programme is sig-nificantly higher (p= 0.0194) as compared to the patientswho were Ki67-negative but were treated according toIda-FLAG scheme.DiscussionKi67 antigen, which is regarded as a marker of prolif-erative cells, has been identified as a free peptide in thenucleus or complexed with DNA [12]. Despite the opti-mal fixation and the amplified antigen detection, someneoplastic cells in the G1 and G2 phases fail to revealimmunocytochemical expression of the marker. In theexperimental study on cell populations with surfacephenotyping of human T-cell leukemia (Molt-4 humanleukemia cells), the cell cycle was arrested at G1 and G2phases using 10 nM 12-0-tetra-decanoylphorbol-13-acetate. When applied to cell cultures, it reduced Ki67expression to nondetectable levels, if standard immuno-cytochemical methods were employed [24]. Thus, somecells with a relatively long G1 phase gradually lose theirability to express Ki67 during G1 and G2. There is,therefore, a population of G1 Ki67 negative blasts whichcan still proliferate. The above observations correspond to results of thepresent study. In the majority of the high risk groupchildren in whom Ki67-immunonegative blast patternhas been detected on day 15 of induction regimen, thetreatment has ended in a failure. It is interesting that the high percentage of Ki67-posi-tive blasts has been found in bone marrow before treat-ment in all the children, regardless of AML type or futurecomplications. It seems to indicate that originally allneoplastic cells in AML may exhibit high sensitivity tochemotherapy. However, in the course of the undertakeninduction therapy the originally homogenous populationof blasts becomes selected into Ki67-positive and Ki67-negative cells. At this stage of treatment, the most im-portant day is the day 15, on which the potential to reachstable remission may become determined. Table 1. Clinical characteristics and data on disease course in patients with acute myeloid leukaemia (AML)Studygroup AML type (markers)Number ofpatients Age (yr)Risk groupHR (%) LR (%)1 M0 (HLA-DR, CD33)  4 11.3  1.2 0 (0) 4 (100)2 M1 (HLA-DR, CD13, CD33)  8 10.8  5.2 4 (50) 4 (50)3 M2 (HLA-DR, CD13, CD33, CD15, CD34) 26 9.8  4.6 5 (19) 21 (81)4 M3 (CD13, CD33, CD15)  4 8.9  4.8 1 (25) 3 (75)5 M4 (HLA-DR, CD13, CD14, CD15, CD33)  4 9.4  6.7 0 (0) 4 (100)Ki67-negative blast cells clone in AML 51The present protocol of AML treatment (ANLL-98)does not introduce Ida-FLAG regimen to children whodid not respond to induction treatment on day 15. Itbecomes recommended when the AML relapse is rec-ognised. In our study, seven early relapses were notedin low risk patients. Although in 4 of them Ki67-negativeclone of blast cells was selected, 3 children have reachedthe second remission and after bone marrow transplan-tation they are still alive in their second remission. Itshould be emphasized that such a remission was reachedonly by the single child of high risk group who wasKi67-negative on day 15 and had undergone the AML-98, no-Ida-FLAG, protocol.Without doubt, oncological treatment may be effec-tive only when it is adjusted to aggressiveness of thedisease. Therefore, recognition of risk factors is of keyimportance for designing the effective therapy in anyoncological disease. In this aspect, the AML status is lessfavourable than that of ALL. This reflects less frequentmanifestation of AML and its higher pathogenetic vari-ability. The selection of Ki67-negative clone of blastscells seems to be a potent unfavourable factor of child-hood myeloid leukemia. However, introduction of Ida-FLAG protocol to all children who are Ki67-negative onday 15 needs a further multi-centre investigation.In conclusion, Ki67 immunonegative blast pattern onday 15 of the induction treatment in acute myeloidleukemia may represent a new index of unfavourableprognosis in the high risk group children. The obtainedresults may also justify routine determination of Ki67antigen before and during the treatment regimen of acutemyeloid leukemia.References[ 1] Ball LM, Lannon CL, Yhap M (1999) PCNA bearing structuresare retained in apoptotic phase of childhood ALL cell cycle. AdvExp Med Biol 457: 289-296[ 2] Bennett JM, Catovsky D, Daniel MT, Flandrin G, Galton DA,Gralnick HR, Sultan C (1976) Proposals for the classificationof the acute leukemias. French-American-British (FAB) co-operative group. Br J Hematol 33: 451-458[ 3] Bennett JM, Catovsky D, Daniel MT, Flandrin G, Galton DA,Gralnick HR, Sultan C (1985) Criteria for the diagnosis of acuteleukemia of megakaryocyte lineage (M7). A report of theFrench-American-British Cooperative Group. Ann Int Med103: 460-462[ 4] Bennett JM, Catovsky D, Daniel MT, Flandrin G, Galton DA,Gralnick HR, Sultan C (1985) Proposed revised criteria for theclassification of acute myeloid leukemia. A report of the French-American-British Cooperative Group. Ann Int Med 103: 626-629.[ 5] Creutzig K, Ritter J, Schellong G (1990) Acute myelogenousleukemia in childhood. Implications of therapy studies for futurerisk-adapted treatment strategies. Springer Verlag, Berlin[ 6] Cyklis R, Armata J, Dłuz˙niewska A (1988) Preliminary evalu-ation of treatment of acute myeloblastic leukemia in childrenaccording to the AML-BFM-83 protocol in the material ofPolish Group for Treatment of Leukemias and Lymphomas inChildren (in Polish). Pol Tyg Lek 43: 376-379[ 7] Cyklis R, Armata J, Dłuz˙niewska A (1996) Effectiveness ofAML-BFM-83 protocol in children with acute lymphoblasticleukemia. More than 10 year experience of the Polish PediatricGroup for treatment of Leukemias and Lymphomas (in Polish).Acta Hematol Pol 27: 131-137[ 8] Elias JM, Gown AM, Nakamura RM (1989) Special report:quality control in immunohistochemistry. Am J Clin Pathol 92:836-843[ 9] Hurwitz CA, Mounce KG, Grier HE (1995) Treatment of pa-tients with acute myelogenous leukemia: review of clinical trialsof the past decade. J Pediatr Hematol Oncol 17: 185-197[10] Kawahira K (1999) Immunohistochemical staining of prolif-erating cell nuclear antigen (PCNA) in malignant and nonma-lignant skin diseases. Arch Dermatol Res 291: 413-418[11] Keating M et al. (1998) In: Acute Leukemias VII - ExperimentalApproaches and Novel Therapies. Hiddemann et al. [Eds],Springer-Verlag, Heidelberg, pp 828-833[12] Lopez F, Belloc F, Lacombe F (1994) The labelling of prolif-erating cells by Ki67 and MIB-1 antibodies depends on thebinding of a nuclear protein to DNA. Exp Cell Res 210: 145-153[13] National Committee for Clinical Laboratory Standards (1991)Internal quality control testing: principles and definitions; ap-proved guideline Villanova, PA.[14] Nowicki M, Miskowiak B, Kaczmarek-Kanold M (2002) Corre-lation between early treatment failure and Ki67 antigen express-ion in blast cells of children with acute lymphoblastic leukemiabefore commencing treatment. A retrospective study. Oncology62: 55-59[15] Pileri SA, Roncador G, Ceccarelli C (1997) Antigen retrievaltechniques in immunohistochemistry: comparison of differentmethods. J Pathol 183: 116-123[16] Radwan´ska U (1992) Distinct therapeutic and clinical featuresof leukemia and malignant lymphomas in children (in Polish).In: Hematologia kliniczna. Janicki K [Ed], PZWL, Warszawa,pp 280-302[17] Shi SR, Cote RJ, Taylor CR (1997) Antigen retrieval immuno-histochemistry: past, present and future. J Histochem Cytochem45: 327-343[18] Shi SR, Cote RJ, Young LL et al. (1997) Antigen retrievalimmunohistochemistry: practice and development. J Histotech-nol 20: 145[19] Steuber CP, Civin C, Ruymann F (1991) Therapy of childhoodacute nonlymphoblastic leukemia (AML): a Pediatric OncologyGroup study (POG 8101). J Clin Oncol 9: 247-258[20] Steuber CP, Culbert SJ, Ravindranath Y et al. (1990) Therapyof childhood nonlymphoblastic leukemia: the Pediatric Onco-logy Group experience (1977-1988). Hematol Blood Transfus33: 198-209[21] Stevens RF (1996) Acute myeloid leukemia. Br Med Bull 62:764-777[22] Tsurusawa M, Fujimoto T (1995) Cell cycle progression andphenotypic modification of Ki67 antigen-negative G1- andG2-phase cells in phorbol ester-treated Molt-4 human leukemiacells. Cytometry 20: 146-153[23] Tsurusawa M, Ito M, Zha Z, Kawai S, Takasaki Y, Fujimoto T(1992) Cell-cycle-associated expression of proliferating cellnuclear antigen and Ki67 reactive antigen of bone marrow blastcells in childhood acute leukemia. Leukemia 6: 669-674[24] Woods WG, Nesbit ME, Buckley J et al. (1985) Correlation ofchromosome abnormalities with patient characteristics, histo-logic subtype and induction success in children with acutenonlymphoblastic leukemia. J Clin Oncol 3: 3-8Received: June 29, 2005Accepted after revision: July 18, 200552 M. Nowicki et al.

Prognostic significance of Ki67-negative blast cell clone in the high risk group of children treated for acute myeloid leukaemia.

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Prognostic significance of Ki67-negative blast cell clone in the high risk group of children treated for acute myeloid leukaemia.

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