Document 0q4qY0KO3XooxJOzN48MEk15d

00. tr `Y `g d. :e 3C- 0) !Is 1 `$ ic f 1.eukemin Rescurch Vol. 16. Nu. 8. pp. 789-7%. 1W. Printed in Great Dritain. MORPHOLOGIC, IMMUNOLOGIC AND CYTOGENETIC STUDIES IN ACUTE MYELOID LEUKEMIA FOLLOWING OCCUPATIONAL EXPOSURE TO PESTICIDES AND ORGANIC SOLVENTS ANTONIO CUNEO, FRANCA FAGIOLII,SABELLPAAZZI,ANTONELLA TALLARICO, RITA PREVIATI, NADIA PIVA, M.GRETEL CARLl, MASSIMOBALBONaInd GIANLUIGCAI STOLD1 Institute of Hematology, Via Savonarola 9, University of Ferrara, Italy (Receioed 10 February 1992. Revision uccepred 31 March 1992) Abstract-In order to analyze the correlation between environmental exposure and the clinicopathological picture in acute myeloid leukemia (AML), cytogenetic, cyto-immunologic and clinical studies were performed in 70 newly diagnosed AML patients, 30 of which were anamnestically exposed to pesticides (21 cases) or to organic solvents (9 cases). Clonal chromosome aberrations, with involvement of chromosome 5 and/or 7 were more frequently encountered among exposed patients. While the classical t(15;17), t(8;21) and t(9;ll) were detected more frequently among non-exposed patients, other recurring chromosome changes in the exposed group were: rearrangements leading to total or partial monosomy 17p (5 cases), structural aberrations involving the band 16q22 (4cases), trisomy l l q (2 cases), breaks involving bands 6p23,7p14,llq13 (2 cases each). Cytologically, trilineage myelodysplasia was observed in 21 exposed patients, whereas morphologic aberrations of the nonblast cell population were confined to a minority of cells in most patients non-exposed. Immunologic studies revealed positivity for the CD34 stem cell marker in 80% exposed patients vs 22% in the nonexposed group. Conventional chemotherapy achieved complete remission in 3/21 patients exposed and in 16/32 patients non-exposed. Median survival was 2 months in the former group and 9 months in the latter group. These findings show that AML following occupational exposure to pesticides and organic solvents may represent a distinct cytogenetic and clinicopathological entity. Key words: AML, cytogenetics, pesticides, organic solvents. INTRODUCTION A DISTINCT pattern of chromosome aberrations has been shown to be associated with acute myeloid leukemia (AML) following exposure to organic solvents, petroleum products and to pesticides [l-31. In these patients the frequent involvement of the long arms of chromosomes 5 and/or 7 recall the cytogenetic picture of so called `secondary leukemia' (SL) [4], suggesting that both toxic agents present in the environment and cytotoxic drugs or radiation therapy m y preferentially involve some chromosome regions [SI. However, description of cytogenetic findings in correlation with cytologic and clinical parameters in patients environmentally exposed has not been reported previously and it is not known whether AML following exposure to toxic agents represents Abbreoiations: A M L , acute myeloid leukemia; S L , secondary leukemia; BM, bone marrow; TMD, trilineage myelodysplasia;PB, peripheral blood; MA KA, major karyotype aberrations; M D S , myelodysplastic syndrome; WSC, white blood cells. a distinct cytogenetic and clinicopathological entity with respect to AML in patients non-exposed. Therefore, we retrospectively analyzed cytogenetic, cytoimmunologic and clinical findings in 70 consecutive AML patients, 30 of which were anamnestically exposed to pesticides or to organic solvents. The aim of the present report is two-fold: (1) to describe cytogenetic findings in patients exposed to pesticides and organic solvents and (2) to compare the cytogenetic and clinicopathologic findings in patients exposed to myelotoxic agents and in patients non-exposed. PATIENTS AND METHODS Patient selection Eighty-six consecutive patients with `de nouo' AML, admitted to our Institution during a 6-year period (19861991) were routinely submitted to immunologic and cytogenetic analysis. Seventy patients with BM smears available for cytologic review and with evaluable immunologic and cytogenetic data form the basis of the present report. Environmental exposure On admission each patient was interviewed about his 789 ! ' 790 A . CIJNEOet ai. occupational history and his hobbies. All patients were questioned on previous contacts with organic solvents, pesticides, chemicals, metals and petroleum products and were subsequently categorized as `exposed' or `nonexposed'. Exposed subjects were carefully questioned on the type and duration of exposure and were asked as to whether or not they employed protection measures (masks, gloves, pressurized cabins) in the workplace. An `exposure index' was calculated as follows: hours/day X days/year X years. Cytology Besides recommended procedures for the classification of each patient in the FAB system [6,7] bone marrow (BM) smears were reviewed to assess the presence of associated myelodysplastic features. According to previously proposed criteria 181, trilineage myelodysplasia (TMDS) was defined by the presence of more than 25% dysplasticerythroblasts and more than 50% abnormal granulocytes and megakaryocytes. lmmunophenotyping BM cytospin preparations and/or peripheral blood (PB) smears were stained by an inimunocytochemical method using alkaline phosphatase anti-alkaline phosphatase (APAAP) complexes [9]. In order to minimize Fc aspecific binding the slides were previously incubated with rabbit serum (Dakopatts). Reactivity to a panel of monoclonal antibodies purchased from various firms was tested: HPCA-l(CD34), My9(CD33), My7(CD13). My4(CD14), GpIIb/IIIa(CD41), Leul2(CD19), OKBCalla(CDlO), OKT16(CD7), OKTll(CD2). The cut-off point for positivity was 20%. Cytogenetics Chromosome analysis was performed at leukemia presentation in all patients. BM samples were cultured for 24 and 48 h without mitogens. Synchronization with methotrexate and thymidine was carried out. Metaphases were G banded with Wright stain [lo]. At least 10 karyotypes were studied in each patient and chromosome aberrations were described according to the ISCN [I I]. RESULTS Environmental exposure Thirty patients (21 farmers, 5 factory painters, 2 shoe workers, 2 hairdressers) were categorized as `exposed', based on interviews revealing unequivocal contact pcsticidcs (2 I cases) and with organicsolvents (9 cases). All patients with a history of exposure to pesticides were farmers who had been spraying carbamates and organophosphates [12, 131 for several years without effective protection measures. Pesticides were usually dissolved into water, and no significant exposure to organic solvents could be documented in these patients. The mean `exposure index' for patients exposed to pesticides and to organic solvents was 20 000 h and 38 000 h, respectively. Nineteen patients were actively working when AML was diagnosed, while in 11 patients direct exposure had ceased 3-9 years before referral to our center. In 40 patients (16 housewives, 14 white-collar workers, 2 students, 2 teachers, 3 operators and 3 farmers), here referred to as `non-exposed', no evidence of exposure to myelotoxic agents was anamnestically documented. Clinical features The salient clinical data in patients exposed to pesticides and to organic solvents and in patients nonexposed is summarized in Table 1. Overall, patients categorized as exposed had a median age (68 years) similar to that of non-exposed patients (64 years), while the male/female ratio was 3.5 in the former group and 0.9 in the latter group. Except for median age, no important difference emerged when comparing clinical features in patients exposed to pesticides and to organic solvents (see Table 1). As compared with the non-exposed group, exposed patients presented with lower leukocyte counts and with lower blast cell percentage in the bone marrow. Erythroleukemia and megakaryoblastic leukemia were encountered more frequently among exposed patients, while other FAB subtypes of AML were almost uniformly distributed in both categories of patients. Outcome of remission induction therapy and sur- vival in exposed and non-exposed subjects is reported in Table 1. Of 21 exposed patients treated with conventional myeloablative chemotherapy 3 achieved complete remission, with a median survival of 2 months. Complete remission was obtained in 50% non-exposed subjects with a median survival of 9 months. Cytology and irnmrtnophenotype (a) Morphologicfirzdirzgs. Among subjects exposed to pesticides and to organic solvents morphologic abnormalities affecting multiple cell lineages were apparent in 21 patients and were not evaluable in 9 patients because of overwhelming blast infiltrate. Abnormal cytologic features included the classical signs of dysmyelopoietic syndrome [14], Le. nuclear irrcgularitics iI1jd dcfcctive hemoglobinization in the red cell series, neutrophil hypogranulation, pseudoPelger forms, micromegakaryocytes, large mononuclear mcgakaryocytes. According to the stringent criteria by Bnto-Babapulle et al. [8], 15 patients in this group could be classified as A M L with trilineage myelodysplasia. In the `non-exposed' group morphologic abnormalities of the non-blast cell population were mostly confined to the granulocytic lineage. A minority of morphologically abnormal megakaryocytes was detected in 5 patients, while dyserythropoiesis involving more than 25% erythroblasts was present I a r f d o C I' d 7 P a a: P in P` C; ai e, c. re m4 Environmental exposure and acute myeloid leukemia 791 r TABLE 1. FAB CLASSES, CLINICAL FEATURES AT PRESENTATION, RESPONSE TO d CHEMOTHERAPY AND SURVIVAL IN AML PATIENTS 'EXPOSED' TO PESTICIDES. TO ORGANIC SOLVENTS AND IN PATIENTS ANAMNESTICALLY "ON-EXPOSED'* 3 Exposed Pesticides Solvents Non-exposed FAB subtypest Ml(1); M2(7); Ml(2); M2(2); Ml(4); M2(1L); M4(6); M5(4); M3(1); M4( 1); M3(2); M4(13); 3 M6(1); M7(2) M5(2); M6(1) M5(9); M6(1) 50-82 2674 14-79 S (69) (55) (64) ) , 5.0-10.5 5.4-9.5 5.6-13.7 r (8.8) (7.6) (9.6) n 2.1-83.9 1.3-12.3 I .0-249.0 (7.7) (4.8) (15.h) 20.0-488 .O (85) 18-243.0 (85) 26.0-240.0 (71) , 4-82 3-70 2-95 J., (39) (30) (64) I 30-90 34-85 35-90 (50) (58) (70) Y 3/15 016 16/32 S 1-78+ 1-8 1-36+ 1 (2) (2) (9) * Results are reported as variation range, median value in parentheses. J t Number of cases in each FAB category. $ Number of complete remissions/Number of patients treated with myeloablative chemotherapy. 1 2 7 in 6 patients. In this patient group, myelodysplasia exposed to pesticides, in 9/!! patients exposed to affecting more than one cell lineage was seen in 7 organic solvents and in 13/40 patients anamnestically patients only, 1of which fulfilled the cytologic criteria non-exposed. Ten 'exposed' patients ( 5 to pesticides for the diagnosis of AML with trilineage myelo- and 5 to organic solvents) had 3 or more events of dysplasia. translocation or non-disjunction in the same clone, i (b) Immunophenoiype. In general, immunophen- thus fulfilling the definition of 'major karyotype aberorype was in agreement with the cytologic classifi- rations' (MAKA) [171. Kecurring chromosome aber- cation according to the FAB criteria I151 (Table 2). rations in exposed patients were -5/5q- (8 1 Positivity for the CD34 stem cell marker [16] was patients), deletions or translocations of 17p (5 detected in 20/25 patients in the exposed group and in palients), deletion or translocations of 16q22 (4 I 7/32 patients in the non-exposed group. Unequivocal patients), -7/7q- and trisomy of 21q (3 patients), r positivity for the CD41 platelet antigen was found in trisomy of I l q (2 patients). Breaks at bands 6 ~ 2 3 , ii minority of cells (5-10%) in 7 patients categorized 7pl4, llp14 - 15, llq13 were found in 2 patients as exposed and in 1 patient non-exposed. Inap- each. propriate expression of lymphoid antigens was tested In the non-exposed group, two patients had the 1 in 25 exposed patients, 6 of which were found to be t( 15;17), 2 patients had aberrations of the long arms I positive for the CDlO (4 cases), and for the CD7 (2 of chromosome 3 and 6, and 3 patients had a 5q- cases). CD7 and CD19 positivity was also found in 2 chromosome, 1 of which had MAKA. and 1 non-exposed patients respectively (32 non- exposed patients tested). DISCUSSION C'yrogenetics Detailed results i n each category of patients are reported in Tables 3 and 4. Overall, clonal chromosome aberrations were detected in 17/21 patients This report extends previous observations on the correlation between environmental exposure and cytogenetic patterns in AML [ I , 2). contirniing that (a) patients exposed t o organic solvents and to pesti- TABLE 2. IMMUNOLOGIC FINDINGS IN AML PATIENTS CLASSIFIED ACCORDING TO THE FAB SYSTEM (BENNETT 1985): 'EXPOSED' SUBJECTS AND "ON-EXPOSED'SUBJECTS' ~____ Immunologic FAB subtype markers M1 M2 M3 M4 M5 M6 M7 CD34 Exposed Non-exposed CD33 Exposed Non-exposed CD 13 Exposed Non-exposed CD14 Exposed Non-exposed CD4lat Exposed Non-exposed Lymphoid Exposed Non-exposed 212 8/8 0/1 617 214 1/1 112 113 519 0/1 1/10 018 011 010 212 818 111 717 314 111 2/2 2/3 9/9 1/1 10/10 3/8 1/1 010 212 8/8 1/1 717 214 1/1 2/2 2/3 619 111 10/10 318 111 0/0 012 218 011 7/7 3/4 0/1 112 0/2 016 011 9/9 717 011 010 112 216 0/1 417 0/2 011 212 113 016 0/1 019 017 011 0/0 112 218 011 317 1/4 011 012 013 119 011 1/10 118 011 010 * Number of positive patients/Number of patients tested. t Patients with 5-10% unequivocally positive blasts (minor megakaryoblasticcomponent)are classified as positive cases in this table. Only AML-M7 had more than 20% positive cells. cides have a higher incidence of clonal aberrations than patients anamnestically non-exposed; (b) chromosomes 5 and 7 are non-randomly involved in A M L following occupational exposure to toxic agents; (c) complex karyotypes of the 'MAKA' type (defined by the presence of at least 3 aberrant events in the same clone) are found not only in SL, but in patients exposed to environmental hazards as well. In addition, our study shows that other recurring chromosome changes, whose presence was not emphasized before, may be frequently encountered in exposed patients. In 5 cases we could detect aberrations involving the short arm of chromosome 17 (either translocations of total/partial monosomy). Chromosome I7p aberration was the sole anomaly in 1 patient and was seen in all abnormal cells in patients with multiple related clones, thus outruling the possibility that this chromosome change may represent a late event in the cytogenetic evolution of highly abnormal cell lines. At present the molecular defect associated with 17p aberrations is unknown; it should be outlined, however, that a tumor suppressor gene encoding the p53 protein was mapped to this chromosome region [181. The detection of p53 gene point mutations in some patients with hematologic neoplasias [19] suggests the possibility that this molecular event, in association with the loss of the normal allele on the deleted chromosome 17, may play a role in leukemogenesis, as already demonstrated for the retinoblastoma gene [20]. Interestingly, the short arm of chromosome 17 has recently been added to the list of chromosomal sites non-randomly involved in SL (211. Four patients in this series showed structural aberrations involving chromosome 16, with breakpoint at band q22. Unlike the classical inv(l6) [22] aberrations of 16q22 were observed in the context of complex karyotypes and were associated with trilineage myelodysplasia, in the absence of abnormal eosinophils. The association of 16q22 breaks and professional or iatrogenic exposure was not documented previously, however the existence of clinicopathological differences between AML with the classical inv(l6) and with de1(16)(q22) were recently emphasized, the latter chromosome change having been found in AML preceded by myelodysplasia without bone marrow eosinophilia [23]. Other recurring cytogenetic aberrations in exposed subjects were structural changes of chromosome 6p and 7p. trisomy l l q , breaks at band llq13. These chromosome regions are commonly involved in a spectrum of myeloid stem cell disorders, including MDS, SL and myeloproliferative syndromes [24-271 and may thus be related to the transformation of an early progenitor cell rather than being specifically associated with a subset of AML [28]. Interestingly, both in this study and in the literature [1-4] the classical t(8;21), t( 15;17) and t(9;ll) were detected unfrequently in exposed patients. Globally, the suggestion can be drawn from these data that specific chromosome regions may be the target both of myelotoxic agents present in the workplace and of cytotoxic therapy [29]. According to recent findings, however, llq23 rearrangements may be associated with a clinicopathological subset of SL I301 whereas no patient with breaks involving band l l q 2 3 was detected in our exposed patients. At present it is unclear whether this finding reflects true heterogeneity in the cytogenetic pattern of SL and environmentally induced AML. or whether 11~123 breaks, the presence of which was not emphasized in the former studies of SL, will become obvious in environmentally induced AML as more cases will be studied. Among exposed patients with an abnormal karyotype, only abnormal metaphases were detected in 67% (6/9) patients exposed to organic solvents and in 29% (5/17) patients exposed to pesticides, thus suggesting that an entirely abnormal karyotype may be frequently associated with a history of exposure Environmental exposure and acute myeloid leukemia TABLE 3. CLONAL CHROMOSOME ABERRATIONS IN 30 AML PATIENTS CATEGORIZED AS 'EXPOSED' (4 PATIENTS EXPOSED TO PESTICIDES HAD NORMAL KARYOTYPE) Patient, age* FAB Karyotype Abnormal cells/ normal cells Pesticides (1) 64 (2) 73 (3) 61 (4) 61 (5) 51 (6) 73 (7) 74 (8) 69 (9) 67 (10) 74 (11) 82 (12) 50 (13) 69 (14) 56 (15) 72 (16) 68 (17) 77 M7 46,XY,de1(7)(q22435)/46,XY,de1(7)(q22q35),-9 M7 45,XY,- 17 M2 45,XY,-5,i( 17q) M4 47,XY ,+8 M4 45,XX,-5,t(22;?)(qll;?) M2 46,XY,del(ll)(q13q23) M4 46,XY,t(ll;?)( p15;?) M4 46,XX,de1(6)(q22) M4 46;XX,i(lq+)/46,XX,i(lq+),i(llq) M4 44,XY,del(5)(qllq34),-7,-1 4 416;?)(q22;?) M1 45,XY,t(1;7)( pl4;pll),del(3)(pllp15),-5, +der(5)t(5;17)(q31;qll), -17,t(17;?)(p?;?)/ same without the der(5)chromosome M4 4+4d,XerY( 1, 6-)6t(,1+1d;e1r6()6()qt1(63;;7q)2(2p),2-31;p91,+4)M,-17/ ,-16, same without M1 M2 47,XY,+M(E-size) M2 4 . 4 ,,-~3,~-5 ,t(15;?)(q21;?),i(21q)/45,XY, -3,t( is;? M2 4d3e,l(X1Y,6-)5(q,2de2l)(,tl(l1)7(;q?2)(3p)1,t2(;?1)5,;-121;81,3-)2(1pll;q13;q13), M5 46,XY,t@;?)(q23;?) M5 47.XY,+20 12/0 4/6 7/3 515 616 8/4 2/10 14/6 813 813 1010 10/0 1010 1210 10/4 3/11 3/15 Organic solvents (18) 70 M3 (19) 63 (20) 33 M5 M6 (21) 55 M2 (22) 43 (23) 40 (24) 74 (25) 26 (26) 71 M2 M6 M5 M2 M1 47,XY,ins(l4;2)(q23,ql3q37),t(5;?)(q31 ;?),t(l1;12) (p14;q12),+21 47,XY,+ 10 49,XY,+11,t(13;?)(pl1;?),+Ml ,+M2/50,XY,+ 11, t( 13;?)(p l l ;?),+MI, +M2,+M3 44,XX,del(5)(ql3q33),del(l6)(q22),t(17;?)(pl2;?),-18,t(20;?) ( p l l ;?), -21/45,XX,de1(5)(q13q33),t( 17;?)(p12;?),t(20;?) (p l 1;?) ,-18,-21,+M 46,XY,t(6;9)( p23;q34) 46,XY ,t( 13;?)(q14;?),de1(20)(qllq13),-8 47,XY, +21 47,XX,t(8;21)(q22;q22),del(l5)(qllq15),+20 45,XX,-21 1010 1210 1017 lO/O 1010 1215 10/0 10/0 3/10 * Years. 793 to organic solvents. Since in vitro culture conditions were the same in all patients studied, the difference of the karyotype status in patients exposed to organic solvents and to pesticides is unlikely to represent a technical artifact and it may in fact reflect heterogeneity of cytogenetic evolution of preleukemic/ leukemic clones in the two groups of patients. Comparative analysis of morphologic, immu- 194 A. CUNEOet al. TABLE 4. CLONAL CHROMOSOME CHANGES IN 40 AML PATIENTS CATEGORIZED AS "ON-EXPOSED' (27 PATIENTS HAD A NORMAL KARYOTYPE) Patient, age' FAB Karyotype Abnormal cells/ normal cells (4) 25 (5) 21 (6) 62 (7) 71 ( 8 ) 24 (9) 64 (10) 60 (11) 31 (12) 63 (13) 56 47,XX,+4 46,XY,deI(S)(q14q21) 46,XY ,t(15;17)(q22;912)/46, XY,t( 15;17)(q22;q12),+22 M1 45,XY,inv(3)(q21q26),-8/46, XY,inv(3)(q21q26) ,del(8q) M3 46,XX,t(15;17)(q22;q12) M2 46,XY,t(8;21)(q22;q12) M2 45,XX,-4,de1(5)(q21q32),+10,-14,de1(17p),+M M2 46,XY,del(3)(q21q26),d~p1(7)(ql2+q14) M2 46,XX,de1(6)(q22)/46,XX,de1(6)(q22),+22 M4 46,XX,t(6;?)(q15;?) M5 46,XX ,t(9;11)(p21 ;q23) M1 46,XX,de1(5)(q13q14)/sarne with +21 M2 46,XX,de1(4)( plS)/sarne with de1(6)(q12qIS) 614 5/8 10/0 912 10/0 4/10 8/3 1214 10/0 717 7/10 11/12 5/15 * Years. nologic and clinical data in the two patient groups documents that AML following environmental exposurc to pesticides and organic solvents may show distinct clinicopathological features as compared with AML in non-exposed subjects. Morphologically, patients exposed to pesticides and to organic solvents had unequivocal signs of disordered maturation of the non-blast cell population. Since identification of myelodysplasia in leukemic bone marrow may be somewhat arbitrary, we classified our patients according to previously published criteria for the diagnosis of AML with TMDS [8]. While over 60% of patients exposed fulfilled the stringent criteria for the diagnosis of AML with TMDS, morphologic aberrations were confined to a minority of erythroid, myeloid and megakaryocytic cells in the majority of patients nonexposed. These findings in AML following environmental exposure recall the cytologic picture of therapy-related leukemia, a disorder commonly regarded as a panmyelosis [31] and seem to indicate that AML in exposed patients may frequently be associated with a pre-clinical myelodysplastic phase, possibly due to the involvement of a multipotent progenitor cell retaining the capability to differentiate along multilinage pathways [32]. The frequent positivity for the CD34 stem cell marker in exposed patients (80% of cases tested), along with the presence of a minor megakaryoblastic component documented immunologically, seem to support this argument. The occurrence of multipotcnt stem cell involvement with multilineage differentiation of the leukemic clone has been documented previously in secondary leukemia and therapy related MDS [33]. According to our data, not only AML in exposed patients represents a cytologic and cytogenetic entity, but may show distinct clinical features as well. Globally, these patients may present with lower white blood cell count and lower percentage of bone marrow blasts than patients non-exposed. Interestingly, one of these patients, reported in detail elsewhere [34], showed a classical MDS with the features of refractory anemia with excess of blasts after remission induction with conventional chemotherapy. This pattern of relapse was previously described in some patients with AML with TMDS PI. 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