Document 37Ga16daB8DYJV0Z58QO1Ekan

ras Oncogene Activation and Occupational Exposures in Acute Myeloid Leukemia I EXHlBlT [sa Jack A. Taylor,* Dale P. Sandler, Clara D. Bloomfield, David L. Shore, Edward D. Ball, Andreas Neubauer, 0. Ross Mclntyre, Edison Liu Pri only cupal I risks been ? tiona' Background: Epidemiologic studies of acute myeloid leu- kemias (AMLs)show small increases in risk of disease as- sociated with certain occupations and chemical exposures. Purpose: This study was designed to determine whether the presence of mutationally activated ras oncogenes in A M L are associated with occupational and chemical ex- posures. Methods: We interviewed 62 patients with newly diagnosed A M L (or their next-of-kin), all of whom were enrolled in a national multicenter clinical trial, and 630 healthy control subjects. DNA extracted from patients' pretreatment bone marrow samples was amplified by using the polymerase chain reaction and probed with allele-specific oligonucleotides for activating point muta- tions at the 12th, 13th, and 61st codons of three protooncogenes: H-ras (also known as HRAS), K-ras (also known as KRAS2), a n d N-ras (also known as NRAS). Re- sults: Patients with ras mutation-positive A M L had a higher frequency (six of 10 patients) of working 5 o r more years in an a priori high-risk occupation than did patients with ras mutation-negative A M L (eight of 52; odds ratio [OR]= 6.8; 95% confidence interval [CI]= 1.3-36). Patients with ras mutation-positive A M L were more likely than patients with ras mutation-negative A M L to have breathed chemical vapor on the j o b (OR = 9.1; 95% CI = r.3-64) or to have had skin contact with chemicals (OR = 6.9; 95% CI = 1.3-37). When ras-positive patients were compared with healthy control subjects, the ORs for occupation and occupational exposures remained elevated, while patients with ras mutation-negative A M L showed no Canellos, CA-32291); Dartmouth College, Hanover, N.H. (Gibbons G. Cornwell 111, CA-04326); Duke University Medical Center, Durham, N.C. I (Donald L. Trump, CA-47577); University of Iowa, Iowa City (Gerald H. 'POnt Received April 27, 1992; revised July 13, 1992; accepted July 31, 1992. Supported by Public Health Service grants CA-37027 (C. D. Bloomfield), CA-49240 (A. Neubauer), and CA-31946 and CA-33601 (0. R. McIntyre) from the National Cancer Institute, National Institutes of Health, Department of Health and Human Services (CA grant Nos.); by Deutsche Forschungsgemeinschaft N.E. 310/4-1 and the Deutsche Krebs-gesellschaft Berlin (A. 'Journal of the National Cancer Instilule "O1. . -. __ __ increased risk when compared with control subjects. Con- patients were jointly enrolled in both studies and thus had chision: Activation of ras prolo-oncogenes may identify an both detailed exposure information and oncogene analysis. t etiologic subgroup of AML caused by occupation and Patients were enrolled between January 30, 1986, and January chemical exposure. implication: Disease etiology may be 31, 1989. Written informed consent was obtained from the better understood if epidemiologic measures of exposure patients at the time of enrollment. i ure integrated with molecular assays of the genetic defects Interviewers contacted patients initially in the hospital responsible for cancer initiation and promotion. [J Natl within a day or two of diagnosis and either completed the Cancer Inst 84:16261632, 19921 telephone interview at that time or arranged an appointment ' , Prior case-control studies of leukemia have demonstrated only a slight increase in risk of disease for persons with oc- for a later interview. For the few patients who were too ill to participate or who died shortly after diagnosis, telephone interviews were carried out with next-of-kin. With the structured questionnaire used in the interview, we cupational or chemical exposures. Except for special groups obtained data on demographics, medical history, medication ' exposed to high levels of benzene or radiation, the reported use, family medical history, smoking, occupational history, ' ribks associated with occupation and chemicals have generally hobbies, residential history, and environmental exposures. We been less than twofold (I), making these exposures of ques- requested`a lifetime occupational history and asked, for each I tionable etiologic significance. Since acute myeloid leukemia job, whether the patient had been exposed to six broad classes I (AML)appears to be a heterogeneous disease at the molecular of chemicals, radiation, or other agents associated with cancer and cytogenetic level, it is possible that certain environmental risk. In addition, patients were asked specifically whether agents might be linked to molecular subtypes. If so, the asso- they had been employed in one or more of 32 occupations ciation between an exposure and a specific subtype of AML (Table 1) previously reported to be associated with increased Founda- could be strong, while the association between that same ex- leukemia risk (1,9). Within this set of 32 a priori "at-risk" Group B idy: Uni- Bowman Cooper, . Green, Ellison. posure and all types of AML might be weak. Animal studies (2) suggest that mutation of ras family proto-oncogenes, which creates activated ras oncogenes, is frequently an initiating, or at least an early, event in some chemically induced tumors. The frequency of such muta- occupations, we also identified a subset of 22 "high-risk" occupations in which, based on previous industrial hygiene review, either the frequency or intensity of chemical exposure was considered to be greater. We also obtained information :orge P. lionally activated ras genes is much higher in some chemi- # b u n sG . mi, N.C ierald It. cally induced rodent tumors compared with that in spontaneous tumors (3),and the pattern of mutational activa- Table 1. A priori at-risk occupations ,de Park, tion can show chemical specificity (3,4). In human tumors, a ilt imorc, relationship between oncogene activation and environmental Bruce A. exposures has not been reported except for limited studies of I2. Perry, lames F. , er, New lung cancer and smoking (5,6) and the skin and sunlight exposure (7). another of melanoma of rolina at Mutationally activated ras family oncogenes occur in widencc 15%-30% of patients with AML and commonly occur in mye- Occupation Artistic painting Auto mechanics or repair Automobile or heavy equipment manufacturing Beautician, barber, or cosmetologist Biological or medical laboratory technician Chemical manufacturing Dry cleaning hinBtonv Iodysplastic syndrome, a preneoplastic precursor of AML (8). Dye manufacturing miology Myelodysplastic syndrome may be more likely to progress to Electrician Electronic industry or manufacturing I AML in patients who have activated ras oncogenes (8). A Farming variety of chemical agents have been identified as risk factors Funeral director or embalmer nslitute, ' for ieukemia, although, with the exception of benzene, the Furniture manufacturing, repair, or refinishing Gasoline station attendant associations have not been strong (Z,9). This study was de- Landscaping or gardening Iligh-risk occupation * + + + ,crsity of signed to test whether AML patients with mutationally acti- vated ras oncogenes are more likely to have a history of f North occupational exposure to chemicals. Jniver"Y. t chcock illy Dr. y Chair of the lALGB :tt, and Health nvironch l r i - Methods Study `Population The study took place in collaboration with Cancer and Leukemia Group B (CALGB), a multi-institutional cooperative cancer treatment group. The study population consisted of patients who were jointly enrolled in two independent CALGB investigations: a case-control study of risk factors for acute leukemias in adults with 625 case patients (CALGB protocol 8661) and a study of the prognostic significance of`oncogene activation in 99 patients (CALGB protocol 8765). Sixty-two Leather/shoe industry or shoe repair Munitions/explosives manufacturing Nuclear power industry Nursing Other professional arts Other health professions with patient care Paint manufacturing or application Paper, pulp, saw mill, or lumbering Pest extermination Petroleum industry or manufacturing Pharmaceutical worker Plastics manufacturing Printing Rubber industry Textile manufacturing Truck, bus, or taxi driver X-ray technician * + = a priori high-risk occupations with increased frequency or intensity of chemical or radiation exposurc. islitutc Vol. 84, No. 21, November 4. 1992 ARTICLE 1627 o n exposure to a series of specific chemicals, but the number of reported exposures to individual agents was too small to permit analysis. Patients were registered to treatment protocols on the basis of clinical and laboratory data available at the time of presentation; their tumors were later classified histopathologically using the French, American, and British (FAB) classification (10) following central review of their slides. Bone marrow samples obtained at the time of hospital admission were frozen for later oncogene analysis. As par1 of the larger case-control study (CALGB protocol 8661), disease-free control subjects were selected from the general population by a two-stage random telephone sampling procedure. Control subjects were frequency matched to case patients by age (10-year age intervals), race (WhitehonWhite), sex, and region of residence (six regions in the United States plus Canada). Control subjects and case patients completed the same questionnaire; the questionnaire was administered over the telephone by trained study interviewers. A total of 630 control subjects completed an interview. We used all of the available control subjects and adjusted for appropriate demographic factors in the analysis. Statistical Analysis Associations between disease and exposure were assessed by calculating the odds ratio (OR) and 95% confidence intervals (CIS). The OR is the odds of exposure in one group divided by the odds of exposure in a referent group. When there were other variables that needed to be controlled for in the analysis, an adjusted OR was calculated on the basis of Mantel-Haenszel's formula ( 2 1 ) . This adjusted OR is a weighted average of the stratum-specific ORs. Chi-squared statistics can be used to test whether the ORs and adjusted ORs differ from their null value of unity. Comparisons were made between ras mutation-positive (ras-positive) patients and ras mutation-negative (ras-negative) patients and between these two groups and healthy control subjects. When healthy control subjects are the referent group and it is thus assumed that disease is rare, the OR closely approximates relative risk (RR). The RR is the ratio of the probability of disease given exposure to the probability of disease given no exposure. This ratio cannot be estimated directly because of our study design (11). We used Student's t test to assess differences in coptinuous variables between groups and the Wilcoxon-Mann-Whitney test to compare medians. The latter test is simply a Student's t test performed on the ranks of the continuous variables and is especially useful for small samples (Z2). Polymerase Chain Reaction DNA from bone marrow cells was extracted using standard procedures (13). The polymerase chain reaction (PCR) was performed as described previously (14). Briefly, 200-500 ng of high-molecular-weight DNA was used to amplify the areas surrounding codons 12, 13, and 61 of three proto-oncogenes: N-ras (also known as NRAS), K-ras (also known as KRAS2), and H-ras (also known as HRAS), respectively. The primer$ allele for the PCR (final concentration, 0.5 pM) were from Clontech (Palo Alto, Calif.). The PCR was performed for 35 cycles i n , ( 1 4 ~ an automatic thermocycler (Perkin-Elmer Corp., Norwalk. Conn.) in a 100-pL solution containing the following: 10 mM R,,,, Tris-HC1 (pH 8.3), 50 mM KCI, 1.5 mM MgCI,, 0.001%(wJ vol) gelatin (Sigma Chemical Co.. St. Louis, Mo.), 200 p M of' Chai each of the four deoxynucleoside triphosphates (pH 7.0: Oncc Boehringer Mannheim Biochemicals, Indianapolis, Ind.), and 2 U AmpliTaq polymerase (Cetus Corp.. Emeryville, Calif.)I Of activ. In the first cycle, denaturing was performed at 94 "C for 5 minutes, annealing at 55 "C for 1 minute, and extension at Threi None 72 "C for 1 minute. In all other cycles, denaturing time W ~ Fhave 1 minute. Otherwise, cycle times and temperatures were simi- five ( lar to those used in the first cycle. After the pleted, 5-10 pL of the reaction mixture was PCR was comelectrophoresed activ, Blacl on a 3% NuSieve (FMC Corp., Rockland, Me.) and 18 Amei agarose gel to amplification. ascertain the effectiveness of the PCR ras-p years Oligonucleotide Hybridization nosis signi The remaining 90-95 pL of the PCR solution was de. spec1 natured with 150 p L of 0.4 N NaOH, heated to 95 "C for 2 were minutes, neutralized with 200 pL of 2 M Tris (pH 7.5). and in tht slotted on nylon filter paper (Hybond N; Amersham Corp.. male: Arlington Heights, Ill.) according to the manufacturer's in- M4, structions. DNA was cross-linked using UV light. The M4. oligonucleotide probes covering all possible mutations and 3.2; the wild-type sequence were from Clontech. For initial activ; screening, a mixture of 3-6 specific oligonucleotides (each probe, 7 pmol) was end-labeled with T4 polynucleotide' ASSO# kinase and [X-32P]adenosine triphosphate (DuPont NEN Re- Acth search Products, Boston, Mass.) (14). The filters were then hybridized overnight in 5 X (Le., five times the standard concentration) sodium chloride-sodium Af founc phosphate ethylenediamine tetraacetic acid, 5X Denhardt's -solution, 100 mM sodium pyrophosphate, and 0.5% sodium dodecyl sulfate and washed with 3 M tetramethylammonium chloride. The filters were exposed for one to several hours to an XOMAT film (Kodak. Rochester, N.Y.) at -70 "C using -Age, Y intensifying screens. To confirm the specific mutation, we re- 24 amplified the DNA samples from ras-positive case patients and analyzed them by oligonucleotide hybridization with indi- 31 vidual oligonucleotide probes after Southern blot transfer. For 35 the Southern blot transfer, 7 p L of the PCR solution was 46 electrophoresed in a 3% NuSieve and 1% agarose gel. and the 55 transfer was performed in a vacuumblotter (Hoefer, San Fran- 58 cisco, Calif.) onto Hybond-N membranes. After the transfer 59 was completed, DNA was cross-linked by using UV light and hybridized with individual mutation-specific oligonucleotide 65 probes (Z4.Z-5). 7s Two ras-positive samples were selected for further confir- mation using direct sequencing of the PCR products as de- 16 scribed previously (14). In both cases, the allele-specific ' oligonucleotide hybridization analysis concurred with the di- , rect sequencing results (data not shown). The sensitivity of these two procedures for detecting a ras mutation in the presence of normal cells is one mutant to 10 normal cells when +w t Bet SA Bun, Journal of the National Cancer Institrite Vol. 8 -* .* - men allele-specific oligonucleotide hybridization is used and one negative patients to have been employed in an a priori at-risk [tech mutant to four normal cells when direct sequencing is used occupation (Tables 1, 3). Crude and adjusted ORs are similar :s in-(i4,16), ialk, in all analyses; only the adjusted ORs are presented. When we classified only those patients who had worked 5 or more years as exposed, the OR increased from 2.2 to 4.1 (95% CI = M of Characteristics of AML Patients With Activated ras 7 .O; Oncogenes and lif.). for I Of the activated 62 K- patients in this or N-ras genes study, 10 at codon (16%) were positive 12, 13, or 61 (Table for 2). n at1 Three patients had activation of both K- and N-ras genes. was None had activated H-ras. Non-Whites were more likely to imi- have ras activation than Whites (OR = 9.4; 95% C1 = 2.0-44); om- five (50%) of 10 non-Whites and five (10%) of 52 Whites had :sed activated ras genes. Among the non-White group, two of five 1% Blacks, two of four American Indians, and the only Asian 'CR American in the study had activated ras genes. Patients with ras-positive tumors were older (mean age at diagnosis, 52.4 yrars) than those with ras-negative tumors (mean age at diag- nosis, 44.5 years), although this difference is not statistically significant (P = .17). Median ages (56.5 versus 42 years, re- de- spectively) also did not differ significantly (P = .17). There )r 2 were slight, but not statistically significant, gender differences and in the frequency of ras activation (13% for females; 19% for w , males; P = .73). Of the ras-positive tumors, 40% were FAB in- M4, whereas only 17% of those that were ras negative were The M4. This difference was not statistically significant (OR = and 3.2; 95% CI = 0.7-14). There was no association between ras tial activation and smoking (data not shown). ach ide Association Between Occupational Exposure and ras Re- Activation 0.7-23). The OR increased further to 6.8 (95% CI = 1.3-36) for 5 or more years of employment in a priori high-risk occupations (Table 3). Six of 10 ras-positive patients worked in an a priori high-risk occupation for 5 or more years, compared with only eight of 52 ras-negative patients. After adjusting for age and race, we found that, compared with healthy control subjects, ras-positive patients were four times as likely (95% CI = 1.1-15) to report 5 or more years in an at-risk occupation and 5.9 times as likely (95% CI = 1.7-20) to report 5 or more years in a high-risk occupation. The employment histories of ras-negative patients and control subjects did not differ from each other: For 5 or more years' employment in an at-risk occupation, the adjusted OR was 0.9 (95% CI = 0.5-lS), and in the high-risk occupations, it was 0.6 (95% CI = 0.3-1.4). Patients with ras-positive leukemia were particularly more likely than ras-negative patients to report skin contact with chemicals (OR = 6.9; 95% CI = 1.3-37), breathing chemical vapors (OR = 9.1; 95% CI = 1.3-64), and exposure to dusty conditions (OR = 6.3; 95% CI = 1.1-35) while working (Table 4). The adjusted OR for exposure to solvents and degreasers (OR = 3.2; 95% CI = 0.5-20) was also elevated in ras-positive patients compared with that in ras-negative patients. The two remaining categories of occupational exposure, non-ionizing radiation and ionizing radiation, had too few exposed raspositive patients to permit meaningful analysis (none and one, respectively). When ras-positive patients were compared with healthy i ve um After adjusting for age and race (Whitelnon-White), we found that ras-positive patients were more likely than ras- control subjects, the ORs for all exposures remained greater than one, although only in the case of exposure to chemicals It's urn JI11 Table 2. Characteristics of AML patients with activated ras genes 10 ng Age, y re- 24 11s di- II 'or 35 as 46 he 55 n- $8 er $9 nd Sex Male Male Female Male Male Female Male Race* W AI W A B W W FAB M4 MA M5A M2 M2 M6 M4 Gene K-ras N-ras K-ras N-ras N-ras K-ras N-ras K-ras N-ras Codon 61 61 12 61 13 12 12 12 61 Mutation? CAA-CAT CAA-AAA GGT+GTT CAA-rAAA GGT-rGAT GGT-rGAT GGT-+CAT GGT-rGAT CAA-CGA At-risk occupation Auto mechanic$ Paper mill* and nuclear power industry$ Other health profession Electronics industry$ 65 Female W M4 N-ras 12 GGT+AGT Textile industry$ and nursing 75 re- 16 ic Female AI M2 K-ras N-ras Male B Unclassified$ K-ras 12 GGT-+GAT Textile industry,$ rubber industry,$ 12 GGT- GAT and auto manufacturing$ 13 GGC-GAC Furniture manufacturing.$ paint manufacturing,$ and truck driver I- )f 'W = White; AI = American Indian; B = Black: and A = Asian. ,- t Because three individuals had two ras mutations concurrently, the number of mutations exceeds the number of case patients $ A priori high-risk occupations. I1 5 Unclassifiable acute leukemia. Risk category Ever exposcdt versus never a5 y exposed$ versus <5 y 3 5 y high risks versus <5 y Tnhle 3. Association between occupational risk category and ras activation in AMI, patients ras+ (N = 10) 70 (7) 70 (7) 60 (6) % exposed (No.) ras- (N = 52) 56 (29) 37 (19) 15 (8) Control subjects (N = 630) 59 (373) 39 (247) 22 (137) Adjusted OR (95% CI)* ras+ versus ras- ras+ versus control subjects 2.2 (0.4.-12) 4.I (0.7-23) 6.8 (1.3-36) I .9 (0.5-7.6) 4.0 (1 .I -15) 5.9 (1.7-20) ras- versus control subjects 0.9 (0.5-1.5) 0.9 (0.5-1.5) 0.6 (0.3-1.4) ,I c: ; tl. 1 E1 tc pi ! SI , C( *Adjusted for age and race. tEver worked in an at-risk occupation. $Worked a5 years in an at-risk occupation. Worked 3 5 years in a high-risk occupation. Table 4. Association between general occupational exposures and ras activation in AML patients ai cl e: , ni C( in e, 0 Exposure Chemicals on skin Chemical vapor Dusty conditions Solvents and degreasers % exposed (No.) ras+ ras(N = 10) (N = 52) 60 (6) 29 (15) 60 (6) 35 (18) 7 0 (7) 44 (23) Control subjects (N = 630) 33 (207) 40 (249) 50 (316) 50 (5) 38 (20) 40 (255) ras+ versus ras- 6.9 (1.3-37) 9.1 (1.3-64) 6.3 (1.1-35) 3.2 (0.5-20) Adjusted OR (95% CI)* ras+ versus control subjects ras+ versus control subjects, protective clothing reclassified 4.5 (1.3-16) 3.0 (0.8-11) 2.9 (0.7-12) 7.2 (2.2-24) 5.4 (1.5-19) 4.2 ( I . 1- 17) 1.6 (0.5-5.9) 2.5 (0.7-9.0) O( ras- versus control sub- jects 0.8 (0.4-1.4) 0.8 (0.4-15) 0.8 (0.5-1.5) 0.9 (0.5-1.5) W I st I SL , O' W , (t . cc ac ea fit *Adjusted for age and race. on the skin (OR = 4.5; 95% CI = 1.3-16) did the 95% CI exclude one (Table 4). The use of protective masks and clothing by some workers could lead to misclassification of exposure status; workers who were exposed to an agent but who wore protective clothing would be biologically unexposed. Because such uncorrected misclassification could artificially lower risk estimates, we reclassified as unexposed those subjects who reported wearing masks or other protective garments. In every instance, the risk estimates for ras-positive leukemia associated with individual exposures increased (Table 4). In contrast to ras-positive patients, ras-negative patients did not differ from control subjects with regard to occupational exposures (Table 4). Taking into account protective clothing did not change this result (data not shown). Specificity of ras Mutations The most common activating mutation was a G to A transition, which accounted for eight of 13 mutations (Table 2). Because three individuals had two ras mutations concurrently, the number of mutations exceeds the number of ras-positive case patients. The first patient with two mutations had two G to A mutations, the second had a G to A and an A to G mutation, and the third had an A to T and a C to A mutation. After we excluded the case patient with both a G to A and an L gc PC re A to G mutation, we found that six of six case patients with G , to A mutations were older than 45 years of age, while three of j ar three case patients with other mutations (A to T, C to A, or G I th 'to T) were 35 years old or younger. The mean age of patients cL with G to A mutations (62.6 years; SD, 12) was significanlly ar different (P = .003) from that of patients with other mutations ' ei (30.0 years; SD, 5.6). Median ages (61.5 versus 31 years) also di differed significantly (Pc.05).There were no associations be- lii tween occupational exposure and the specific ras prolo- I in oncogene activated, codon mutated, or base pair change. Discussion i; rn, We found that patients with ras mutation-positive AML had,l ( 1 Ihigher rates of occupational exposure to chemicals than pa- tients with ras mutation-negative disease. Of those exhibiting , (thI activated ras genes, 60% worked 5 or more years in a priori A high-risk occupations, whereas only 15% of patients with ras th mutation-negative AML had this same exposure. The link be- , th 1tween chemical exposure and ras-positive AML is reinforced + by evidence of increasing risk with duration of employmenl lo, and likelihood of solvent exposure and is supported by Ihe 1 fr( results for specific categories of occupational exposures. The 1 co apparent association with occupational exposure is unlikely to tic be the result of recall bias, since both ras-positive and ras- I negative case patients have disease. 1 va I 630 Journal of the National Cancer Inslitulc v o -. -! j Although the ras-positive versus ras-negative comparisons !can identify exposure differences, i t is the comparison of 1 ihese two groups with healthy control subjects that allows us IO estimate the RR of exposure. In our data, the ORs for com- --its , parisons involving ras-positive patients versus healthy control subjects were elevated and similar to the ORs found for comparisons involving ras-positive patients versus ras-negative patients. In contrast, ras-negative patients had the same frequency of exposure as healthy control subjects. This result is consistent among the many different exposures we examined e and offers additional evidence that occupational exposure to chemicals is strongly linked to ras-positive AML. It is interesting to note that, had we combined ras-positive and rasnegative patients and compared the entire group with healthy control subjects, there would have been no apparent increase in risk of disease associated with chemical or occupational exposures. For our two strongest ras-positive associations, the ' OR was reduced to 1.02 for 5 or more years in a high-risk I occupation and to 1.05 for exposure to chemicals on the skin - when we compared all combined case patients with control , subjects. 1- ! Although recall bias is an unlikely explanation for our re- - sults, it is possible that there is some random misclassification : uf exposure status. Data were obtained by questionnaire and were not verified. Random misclassification errors usually i (but not always) tend to bias the ORs toward the null. Our conclusions are based primarily on associations between ras , activation and employment in specified jobs. Specific jobs are easily recalled, whereas occupational exposures are more dif- = ficult to recall. In addition, the associations between the categories of occupational exposures are not independent, so that ' people who reported exposure in one category also tended to report exposure in other categories. G Because the frequency of ras activation differed with age Of , imd race, we stratified by age and race to evaluate whether G these factors explained the observed associations with oc- Its cupational exposures. The associations between ras activation lY and employment in high-risk occupations were not affected in ns ' either case. Conversely, stratifying on occupational exposures $0 did not change the age or race associations. Thus, within the e- limits of the small sample, it appears that these factors are J- , independently associated with ras activation. Mutationally activated ras family oncogenes (N-ras, K-ras, I and H-ras) have been reported in 15%-30%of patients with !I primary AML, making aberrant ras genes the most common i molecular genetic abnormality in this form of acute leukemia id,* (17). Since ras mutations are found in preleukemic disorders a- (18) and in affected pluripotent stem cells (19), it is likely '6 I that mutant ras genes have a role in the initiation of some ri AMLs. Considerable experimental evidence exists showing 1s that specific carcinogens can cause a variety of cancers :- through the induction of point mutations in ras (20). d 1 Our overall frequency of ras mutations (16%) is somewhat It lower than in previously published series (21-23),where the le frequency is usually between 20% and 30%. This difference ,e could be due in part to the young age of our patient popula0 tion; among patients under age 55, four (10%) of 41 had actii- vated ras, whereas in those aged 55 or older, six (29%) of 21 had activated ras. Alternatively, earlier studies may have elevated frequencies because they included AML patients with histories of antecedent hematologic disorders or used detection systems that can identify mutations which exist in only a tiny fraction of blast cells (17). In the larger group of 99 patients enrolled in the oncogene prognosis study (but for whom we do not have exposure data on all subjects), a similar frequency of ras mutations has been detected (la%), indicating that sampling bias did not occur. We found significant associations with other common demographic parameters not previously noted. First, our analysis revealed a higher frequency of ras activation in nonWhites than in Whites, although the number of non-Whites in our study is small. Other published studies have not specified the race of tas-positive and ras-negative case patients. Second, we ,found an increased frequency of ras mutation in older patients, although this finding was not statistically significant, and one study which reported age data for both ras-positive and ras-negative patients did not show any difference (21). Finally, including double ras mutations, eight of 13 activating mutations in N- and K-ras genes were G to A transitions. The predominance of this specific point mutation has been reported by other investigators (21,24-28);however. our data also revealed that patients with G to A mutations were statistically significantly older than those with other mutations. While this finding has not been previously noted, our review of other published studies with age data for ras-positive case patients shows similar trends, with G to A mutations more common in older patients (21,24,26-29).although childhood AML includes both G to A and other mutations (29,30).The reason for the predominance of the G to A mutation in older age groups remains unclear, but it may reflect subtle differences in age-related exposure, accumulation of DNA damage, or reduction in DNA repair. There was no evidence of an association between occupation or occupational exposures and the specific ras gene activated, codon mutated, or base pair substitution. This finding is not surprising, given the small number of ras-positive patients in our study, the variety of occupations these patients held, and the large number of potential exposures within each occupation. A larger follow-on study may have sufficient raspositive case patients and highly specific exposure information, which would enable us to explore associations between particular agents and specific patterns of DNA damage. AML with activated ras oncogenes may represent a pathogenetically distinct subset of AML that is strongly associated with chemical exposure. The risk estimates that we describe for occupation and chemical exposure are larger than those found in other epidemiologic studies that do not separate AML by ras oncogene status. While the estimates of these risks may be uncertain because of small sample size, the fact that such consistent associations can be detected with SO few case patients illustrates the value of using oncogene activation to define subclasses of disease. Such studies linking epidemiologic measures of exposure with molecular assays of the genetic defects responsible for cancer initiation and promotion may provide a more integrated understanding of disease etiology. r References ( I ) LINETMS: Thc Leukemias: Epidcmiolngic Aspects. Ncw York: Oxford Univ Press, 1985 (2) KUMAR R, SUKUMAR S, BARRACIMD: Activation of ras oncogenes preceding the onset of neoplasia. Science 248:llOl-1104, 1990 (3) REYNOLDS SH, PATTERSON RM, MENNEAJRH, ET AL: ras Gene activation in rat tumors induced by benzidine congeners and derived dyes. Cancer Res 50:26&272, 1990 (4) You M, CANDRIAN U, MARONPORTR, ET AL: Activation of the Ki-ras protooncogene in spontaneously occurring and chemically induced lung tumors of the strain A mouse. Proc Natl Acad Sci U S A 86:307&3074, 1989 (5) RODENHUISS, SLEBOSRJ, B o o r AJ, ET AL: Incidence and possible clinical significance of K-ras oncogene activation in adenocarcinoma of the human lung. Cancer Res 485738-5741, 1988 (6) SLEBOSRJ, HRUBAN RH, DALESIO0, ET AL: Relationship between K-ras oncogene activation and smoking in adenocarcinoma of the human lung. J Natl Cancer Inst 83:1024-1027. 1991 (7) VAN'T VEER W, BIJRGERING BM, VERSTEEG R, ET AL: N-ras mutations in human cutaneous melanoma from sun-exoosed bodv sites. Mol Cell Biol 93114-3116, 1989 Liu E, HJELLE B, MORGAN R, ET AL: Mutations of the Kirsten-ras proto-oncogene in human preleukaemia. Nature 330:186-188, 1987 SANDLER DP, COLLMAN GW: Cytogenetic and environmental factors in the etiology of the acute leukemias in adults. Am J Epidemiol 126~1017-1032, 1987 BENNETT JM, CATOVSKYD, DANIEL MT, ET AL: Proposed revised criteria for the classification of acute myeloid leukemia. A report of the French-American-British Group. Ann Intern Med 103:626-629, 1985 KLEINBAUDMG, KUPPERLL, MORGENSTERN H: Epidemiologic Re- search Principles and Quantitative Methods. New York: Van Nostrand, 1982 HOLLANDER M, WOLFEAD: Nonparametric Statistical Methods. New York: Wiley, 1973 MANiATIS T, FRISCHEF, SAMBROOJ: KMolecular Cloning: A Laboratory Manual. Cold Spring Harbor, NY: Cold Spring Harbor Laboratory, 1982 NEUBAUER A, SHANNON K, Liu E: Mutations of the ras protooncogenes in childhood monosomy 7. Blood 77594-598, 1991 LUBBERMT , MIRROR J JR, MILLER CW, ET AL: N-ras gene point mutations in childhood acute lymphocytic leukemia correlate with poor prognosis. Blood 75:1163-1169, 1990 COCSWELLPC, MORGAN R, DUNNM, ET AL: Mutations or the r u proto-oncogenes in chronic myelogenous leukemia: A high frequency of ras mutations i n bcrlabl rearrangement-nega:ive chronic my. Ielogenous leukemia. Blood 74:2629-2633, 1989 ILIUET: The role of ras gene mutations in myeloproliferative disorders 1Clin Lab Med 10:797-807, 1990 I YUNISJJ, BOOT AJ, MAYER MG, ET AL: Mechanisms of ras mutation in myelodysplastic syndrome. Oncogene 4:609-614, 1989 JANSSENJW, BUSCHLE M, LAYTONM, ET AL: Clonal analysis or myc. ' -lodysplastic syndromes: Evidence of multipotent stem cell origin. iBlood 73: 24S254, 1989 BARBACIMD: ras Oncogenes: Their role in neoplasia. Eur J Clin lnvcsi 20~225-235, 1990 * RADICHJP, KOPECKYKJ, WILLMANCL. ET AL: N-ras mutations in adult de novo acute myelogenous leukemia: Prevalence and clinical sig- ; Ro nificance. Blood 76:801-807, 1990 La A nBos JL, TOKSOZD, MARSHALL CJ, ET AL: Amino-acid substitutionsat codon 13 of the N-ras oncogene in human acute myeloid leukaemia. I GrNature 315:726-730, 1985 Bos JL, VERLAAN-DE VRIESM, VAN DER ER AJ, ET AL: Mutations in I! M;N-ras predominate in acute myeloid leukemia. Blood 69:1237-1241. 1987 TOKSOZD, FARR CJ, MARSHALL CJ:ras Gene activation in minor pro- portion of the blast cell population i n acute myeloid leukemia. On. cogene 1:409-413, 1987 SENN HP, TRAN-THANG CH, WODNAR-FILIPOWACZ A, ET AL: Mutalion analysis of the N-ras proto-oncogene in active and remission phase of human acute leukemias. Int J Cancer 4159-64, 1988 M(. Gr - FARR CT, S A K IRK, ERLICH HA, ET AL: Analysis of RAS gene mulations i n acute myeloid leukemia by polymerase chain reaction and Bat oligonucleotide probes. Proc Natl Acad Sci U S A 85:1629-1633, 1988 BROW- PJ, NORTON JD: Analysis of ras gene mutations and methyla. tion state in human leukemias. Oncogene 4:1029-1036, 1989 AHUJA HG, F o n A, BAR-ELI M, ET AL: The pattern of mutational in. izat gro con volvement of RAS genes in human hematologic malignancies detcrmined by DNA amplification and direct sequencing. Blood 75:1686 1690, 1990 seq haj BARTRAMCR, LUDWIG WD, HIDDEMANWN, ET AL: Acute myeloid ane leukemia: Analysis of ras gene mutations and clonality defined by polymorphic-linked loci. Leukemia 3:247-256, 1989 FARR C, GILL R, KATZ F, ET AL: Analysis of ras gene mutations in of i (ha childhood myeloid leukaemia. Br J Haematol 773323-327. 1991 ma. ant P"' mo act, fro1 con ReporterResearchResources Only $9 (U.S.) or $11.25 (Foreign Address, Via Airmail) for 12 issues filled with current biomedical research findings. Fill out the order form below and mail to: The Superintendentof Documenls. U.S. Government Printing Office, Washington, D.C. 20402 Send 12 issues of the Research Resources Reporter to: Don't Miss an Issue of the Research Resources Reporter! 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