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HLA antigens and haplotypes in acute leukemia
Leukemia Research,Vol. 1. No. 4, pp. 261-269PergamonPress. 1977.Printedin Great Britain.
HLA A N T I G E N S AND HAPLOTYPES IN A C U T E L E U K E M I A * EKKEHARD D. ALBERT,t BRENDA N]SPEROS and E. DONNALL THOMAS~ Department of Medicine, University of Washington School of Medicine, and the Fred Hutchinson Cancer Research Center, Seattle, WA 98104, U.S.A. (Received 17 January 1977. Accepted 23 February 1977) Abstraet--HLA-antigen and hapiotype frequencies were determined in 198 patients with acute iymphoblastic leukemia (ALL) and 179 patients with acute myvlogenous leukemia (AML). In AML there were no significant deviations. A slight increase of HLA-A2 and A9 was found in patients with ALL which is in agreement with a number of reports in the literature. The deviation' for HLA-A2 was confirmed by segregation analysis in families, where healthy siblings served as a control. There was no increase in the number of homozygotes at the HLA-A and B loci in neither AML nor ALL. The number of families in which the parents share one HLA-A,B haplotype was not higher than expected in both diseases. Thus there is no evidence for a recessive mode of action for the assumed HLA-A2 associated gene. Key words: HLA-antigens, acute leukemia INTRODUCTION SUSCEt~rIB]LrrYto certain forms of leukemia has been found linked to the major histocompatibility complex (MHC) in the mouse [I 3]'. The high degree of analogy between the M H C in mice (H-2) and in man (HLA) has provided a strong argument for the existence of ~a similar relationship between the susceptibility to leukemia and the HLA-system in man. A linkage with susceptibility genes would be expected to manifest itself in the form of an association of certain HLA antigens with the disease, as it has indeed been found for a large number of nonmalignant diseases [18]. In spite of numerous investigations of leukemia patients (reviewed in [3]) it is still uncertain whether there is any significant association between leukemia and HLA. Clearly, if
* This investigation was supported by Grant Numbers CA 18029 and CA 15704, awarded by the National Cancer Institute, DHEW, and Contract AI 52515 from the National Institute of Allergy and Infectious Diseases. ~fDr. Albert is supported in part by grant AL 92/9/10 Forsehungsgemeinschaft and is a member of Sonderforschungsbereich 37, Mfinchen. ~: Dr. Thomas is a recipient of Research Career Award AI 02425 from the National Institute of Allergy and Infectious Diseases. Correspondence to: Ekkehard D. Albert, M.D., Tissue Typing Laboratory, Kinderpoliklinik der Universit~t, 8 Mfinchen 2, Pettenkoferstral3e 8a, Mtinchen, Germany. Abbreviations: ALL, Acute lymphoblastic leukemia; AML, Acute myeioid leukemia; MHC Major histocompatibility complex. 261
there is an association at all, it cannot be very strong. Therefore it is necessary to investigate a large sample of each of the different forms of leukemia. In connection with the search for a donor for bone marrow transplantation we have had the opportunity to carry out HLA typing on a large number of patients with leukemia and their families. This paper reports the phenotype and genotype data on 377 leukemic patients. MATERIALS AND METHODS Patients were referred to us by a large number of physicians from throughout the United States and Canada. There is a possibility that the patient population represents a selected sample particularly with respect to the time elapsed between diagnosis and HLA-typing. This problem is discussed in the appropriate section of the results. Diagnosis was based on the evaluation of bone marrow aspirates and/or biopsy as well as histochemical characterization of malignant cells. Tissue typing for 25 HLA antigens of the HLA-A and B loci was performed using routine procedures. Each sample was tested with at least 180 highly selected antisera including 60 sera from the NIH-tray provided by the Transplantation Immunology Branch of the NIAID, National Institutes of Health, Bethesda, Maryland. RESULTS Phenotypes HLA phenotype frequencies of 198 patients with acute lymphoblastic leukemia I(ALL) and 179 patients with acute myeiogenous leukemia (AML) are compared with those of a control population
EKKEHARDD. ALBERTet al.
262
consisting of 1070 American Caucasians [1]. The results are listed in Table 1, It can be seen that only HLA-A2 and A9 in patients with ALL show an increase (X2=5.74 and 4.77 respectively). If the corresponding P values are multiplied with the number of variables (i.e. antigens) tested, as is customarily done in HLA disease association studies, the increase of A2 and A9 does not reach the level of significance. Since the present study can be considered as a confirmation of earlier reports (reviewed in [18]) in which an increase of A2 and A9 was found, it is not necessary to apply the statistical safeguard of P value correction. Therefore it can be concluded that the uncorrected P values of P ~- 0.018 (for A2) and P = 0.032 (for A9) indicate a significant association of ALL with HLA-A2 and A9.
Genotypes The testing of families or family members of 198 patients with ALL and of 179 patients with AML allowed the deduction of the genotypes, In 17 ALL patients and in 38 A M L patients the family segregation did not allow an unequivocal deduction of haplotypes, so that in these cases the most probable genotypes had to be asserted by maximum likelihood procedures based on the haplotype frequencies obtained for the respective disease group. Thus it was possible to determine the HAL-A,B haplotype frequencies by haplotype counting. The matrix of haplotype frequencies derived from 198 ALL patients and from 179 A M L patients is given in Tables 2 and 3 respectively. The marginal totals in these tables represent the gene frequencies. Corresponding to the increase in phenotype frequencies in ALL there is also an increase in the gene frequencies of HLA-A2 and A9. All other antigens show frequencies within the normal range for American Caucasians. Looking at single haplotypes one can see that the A2 increase
TABLE 1. HLA
PHENOTYPE FREQUENCIES IN PER CENT OF 198 PATIENTS W I T H ACUTE LYMPHOCYTIC LEUKEMIA AND OF 1 7 9 PATIENTS W I T H ACUTE MYELOGENOUS LEUKEMIA IN COMPARISON W I T H A CONTROL POPULATION
ALL (N= 198)
AML (N= 179)
Controls (N= 1070)
A1 A2 A3 A9 AI0 All A28 A29 AW30,31 AW32
21.7 60.1 * 22.2 26.3$ 10.1 15.1 5.0 9.1 10.1 2.0
27.9 43.0 30.7 19.6 11.1 10.1 11.7 7.3 13.8 1.1
27.2 50.9 23.4 19.4 12.6 11.6 7.8 6.1 9.6 11.1
B5 B7 B8 BI2 BI3 BI4 BW15 BWI6 BWI7 BI8 BW21 BW22 B27 BW35 BW40
13.6 20.2 17.2 30.3 4.0 5.6 16.7 3.0 6.1 3.0 0.5 4.5 7.6 19.2 17.7
10.6 26.8 18.4 26.8 2.8 8.4 16.7 2.2 12.3 7.3 1.7 7.8 9.5 18.4 13.9
11.3 24.5 18.5 26.4 4.0 8.6 11.6 1.0 9.4 9.0 3.8 4.0 9.4 19.0 14.8
HLA
* x~=5.74 P=0.018 not corrected t x~=4.77 P=0.032 not corrected Control population: [4]. in ALL involves mostly the A2,B5; A2,BI2 and A2,blank hapiotypes (Table 2). These differences in hapiotype frequencies are however not large enough to reach statistical significance. The pattern of gene and haplotype frequencies in A M L (Table 3) is essentially normal.
TABLE2. HLA HAPLOTYPEF~QUENCI~ OF 198 PATENTSWITHALL DETERMINEDBY HAPLOTYPECOUNTING (Frequencies are given as numbers per 10,000) HLA
B5
A1 A2 A3 A9 AI0 All A28 A29 AW30/31 AW32
63 291 66 25 0 102 66 0 50 0
50 553 303 153 377 25 152 56 25 25 73 20 0 25 50 0 0 25 0 0
54 767 126 266 76 50 25 252 I0 0
0 25 26 25 0 25 0 25 51 0
0 25 25 0 50 0 77 0 25 0
76 353 126 202 50 25 0 0 50 0
26 25 0 25 51 0 0 0 25 0
50 76 68 0 25 50 0 0 0 0
0 0 25 51 25 0 0 25 26 0
0 25 0 0 0 0 0 0 0 0
25 67 38 0 0 25 0 0 25 25
0 163 0 I0 25 51 25 25 41 25
25 202 74 227 50 278 25 25 25 26
61 368 101 92 25 50 25 50 118 25
103 504 62 251 76 60 9 2 34 0
1086 3347 1139 1382 503 809 277 454 505 I01
25
0 25
57
25
76
2
0
34
0
0
22
25
78
25
3
397
688 1030 907 1683 202 278
884
152
303
152 25
227
390
1035
940
Blanks Total
B7
B8 BI2 B13 Bi4 BW15 BW16 BWI7BI8 BW21 BW22 B27 BW35 BW40 Blanks Total
1 1 0 4 10000
HLA antigens and haplotypes in acute leukemia
263
TABLE 3. H L A HAPLOTYPE FREQUENCIES OF 179 PATIENTS WITH A M L DETERMINED BY HAPLOTYPE COUNTING
(Frequencies are given as numbers per 10,000) B5
B7
B8
BI2 B13 B I 4 BWI5 BWI6 B W 1 7 B I 8 BW21 BW22 B27 BW35 BW40 Blanks Total
Al A2 A3 A9 AI0 All A28 A29 AW30/31 AW32 Blanks
55 248 28 55 0 28 28 0 90 0 4
39 693 307 61 726 108 228 0 0 28 28 53 28 28 55 0 28 6 0 0 30 5
57 542 84 196 66 0 155 140 74 28 40
0 28 0 0 0 0 0 0 112 0 0
28 44 0 47 28 0 72 0 65 0 134
112 199 211 168 84 55 0 0 34 3 1
28 0 28 28 0 0 28 0 0 0 0
164 291 10 15 55 0 0 28 47 0 32
0 84 48 11 112 0 28 0 17 0 64
16 28 0 0 0 0 0 0 28 0 12
28 28 31 45 0 112 28 28 59 25 8
25 196 84 0 28 55 28 0 55 0 3
146 112 248 55 11 167 155 0 0 0 46
55 201 112 107 9 0 28 28 133 0 1
88 100 2 50 138 3 19 84 10 0 30
1534 2469 1720 1005 559 501 625 363 758 56 410
Total
536 1469 982 1382
140
418
867
112
642
364
84
392
474
940
674
524
10000
Observed and expected frequency of homozygotes It h a s b e e n r e p o r t e d t h a t in p a t i e n t s w i t h A L L t h e r e is a n i n c r e a s e d n u m b e r o f H L A - A h o m o zygotes [4]. T h e r e f o r e , t h e n u m b e r o f h o m o z y g o t e s f o u n d a m o n g t h e 198 A L L a n d 179 A M L p a t i e n t s was c o m p a r e d to e x p e c t a t i o n s b a s e d o n t h e gene frequencies o b t a i n e d in T a b l e s 2 a n d 3. A c c o r d i n g to t h e H a r d y - W e i n b e r g law, t h e n u m b e r o f h o m o zygotes (h) is e x p e c t e d to b e h = L g ( a ) ] ~ . N, w h e r e g(a) is t h e gene f r e q u e n c y o f t h e allele a a n d N is t h e n u m b e r o f i n d i v i d u a l s u n d e r study. T h e results, w h i c h are s u m m a r i z e d in T a b l e 4, i n d i c a t e t h a t - - i f a n y t h i n g - - t h e r e is a slightly l o w e r n u m b e r o f H L A - A h o m o z y g o t e s in A L L t h a n expected. T h e f r e q u e n c y o f H L A - A locus h o m o z y g o t e s in A M L ,
as well as o f t h e H L A - B locus h o m o z y g o t e s in A L L a n d A M L are n o t significantly different f r o m expectation.
Families in which parents share one ttLA-A,B haplotype I n t h e family analysis it was o b s e r v e d t h a t in several families t h e r e was s h a r i n g o f a n H L A - A , B h a p l o t y p e , raising t h e q u e s t i o n o f w h e t h e r t h e r e c o u l d b e a n excess o f such families in A L L a n d / o r A M L . T h e e x p e c t e d n u m b e r o f families in w h i c h t h e p a r e n t s s h a r e o n e h a p l o t y p e was c a l c u l a t e d o n t h e basis o f t h e h a p l o t y p e f r e q u e n c i e s given in T a b l e s 2 a n d 3 u s i n g a f o r m u l a w h i c h was p r e v i o u s l y r e p o r t e d [1]. T h e c a l c u l a t i o n s ( T a b l e 5) r e v e a l e d t h a t n e i t h e r
TABLE 4. OBSERVED AND EXPECTED NUMBERSOF HOMOZYGOTES
HLA AI A2 A3 A9 AI0 A11 A28 A29 AW30/31 AW32
Total
ALL Obs.
LOCUSA Exp.*
AML Obs.
Exp.*
0 14 2 3 0 2 1 0 0 0
2.3 22.2 2.6 3.8 0.5 1.3 0.2 0.4 0.5 0.2
5 10 6 1 0 0 1 0 1 0
4.2 10.9 5.3 1.8 0.6 0.4 0.7 0.2 1.0 0.1
22 X==4.23
34.0 NS
24 X==0.06
25.2 NS
HLA
ALL Obs.
LOCUSB Exp.*
AML Obs.
Exp.*
B5 B7 B8 B12 BI3 BI4 BWI5 BWI 6 BW17 B 18 BW21 BW22 B27 BW35 BW40
0 1 1 8 0 0 2 0 0 0 0 0 1 3
0.9 2.1 1.6 5.6 0.1 0.2 1.5 0.0 0.2 0.0 0.0 0.10 0.30 2.10
0 4 2 2 0 0 1 0 0 0 0 0 0 1
4
1.7
0
0.5 3.9 1.7 3.4 0.0 0.3 1.3 0.0 0.7 0.0 0.0 0.3 0.4 1.6 0.8
Total
20 X==0.8
16.4 NS
10 X== 1.72
15.1 NS
* Expectations are calculated on the basis of the gene frequencies contained in Tables 2 and 3 respectively.
EKKEHARDD. ALBERTet al.
264
TABLE 5. FAMILIES SHARING ONE HLA-A,B HAPLOTYPE
ALL (150 families analyzed) Observed Expected* HLA-AI,B8 A2,BW40 A2,B 12 A2,B27 A2,B5 A9,BI2 All others
3 3 2 1 1 1 1
1.73 0.78 3.26 0.16 0.49 0.41 5.01
Total
12
11.84
X2----0.002
NS
AML (94 families analyzed) Observed Expected* HLA-AI,B8 A2,BI 2 A2,B5 A3,B7 A3,B5 A9,B7 A29,B 12 A2,BW 17 A9,B5 Other
2 1 1 3 1 1 1 1 I 0
1.68 1.05 0.23 1.84 0.003 0.19 0.07 0.31 0.10 2.58
Total
12
8.053
X2= 1.93
NS
* Expected values were calculated using the haplotype frequencies given in Tables 2 and 3. The expected numbers of families sharing a given HLA-A,B haplotype is Exp (A,B)=[2-hf(A,B). (l-hf(A,B))+ (hf(A,B))~] 2- N, where hf(A,B) is the haplotype frequency of A,B and N is the number of families investigated. in A L L nor in A M L was there a significant excess of families sharing an H L A - A , B haplotype.
Segregation analysis of HLA antigens and haplotypes In order to validate the findings of an H L A association in the phenotype data, a segregation analysis of H L A antigens Was performed in the families o f patients with A L L and A M L . Only families representing a simple backcross situation (+/-x-/-), where one parent is heterozygous and the other parent is negative for the antigen under study, were included in the analysis. In such a backcross situation one expects 50% of the children to be positive and 50 % to be negative for this antigen. The segregation analysis compares the segregation in patients with that in the healthy siblings. Thus, the segregation analysis is a method of detecting a disease association using the healthy siblings as a control. In A L L the segregation analysis (Table 6) reveals that H L A - A 2 has a significant predominance in the patients while the segregation in the healthy siblings is very close to the expected partition rate o f 50: 50. Thus, the association between A L L and H L A - A 2 observed in the phenotype data is also reflected in the segregation analysis. The increase in frequency for H L A - A 9 is not paralleled by a significant deviation in the segregation analysis and should therefore be considered with caution. All other antigens revealed a segregation which was not significantly different from expectation. The same type of segregation analysis was carried out considering hapiotypes which were frequent enough to yield sufficient numbers of informative
families. In A L L this haplotype segregation (Table 7) showed deviations for the hapiotypes A2, B5; A2,B12 and A2,BX. Although these deviations are only on the borderline of statistical significance-probably due to small n u m b e r s - - i t is interesting to note that exactly these three haplotypes accounted for the increase of H L A - A 2 frequency in A L L as seen in the analysis of haplotype frequencies in Table 2. As can be seen in Tables 8 and 9 there is no significant deviation in the families of patients with A M L , which is in agreement with the normal distribution of H L A antigens and haplotypes in this disease.
Frequency of HLA antigens in relation to the time elapsed between diagnosis and tissue typing It has been suggested by several authors [7, 9, 15] that deviations in H L A antigen frequencies could possibly reflect a selective influence which would favor the survival of such individuals, e.g. positive for HLA-A2. If that were true, we would expect to find different degrees of associations for groups of patients tested at different times in the course of the disease. A group of 119 patients with A L L , for which there was accurate information about the time span between diagnosis and tissue typing, was analyzed for the frequency of H L A - A 2 and A9 (Table 10). Three groups were formed: The first group consisted of 31 patients who had been typed up to 6 months following the diagnosis. This group might be considered as prospectively studied. The second group consisting of 37 patients must be considered a product of selection by the disease
H L A antigens and haplotypes m acute leukemia
265
TABLE 6. SEGREGATION ANALYSIS OF H L A ANTIGENS IN FAMILIES OF PATIENTS WITH A L L
HLA-
N u m b e r of informative families
Patients Positive Negative
X~
Healthy siblings Positive Negative
X2
A1 A2 A3 A9 AI0 All A28 A29 AW30/31 AW32
58 76 49 50 28 15 15 15 27 5
27 55 23 26 11 13 6 12 8 3
31 21 26 23 16 12 9 3 19 3
0.28 15.20" 0.18 0.18 0.92 0.04 0.60 5.40 4.48 0.00
76 84 59 61 34 29 22 23 25 9
61 93 . 60 60 42 27 17 17 28 7
1.64 0.44 0.008 0.008 0.84 0.06 0.64 0.94 0.16 0.25
5 7 8 12 13 14 15 16 17 18 21 22 27 35 40
23 45 47 59 11 18 33 8 15 9 3 15 24 38 32
17 22 18 31 6 8 19 6 6 4 1 6 8 22 18
6 23 29 28 5 10 14 2 9 4 2 9 16 16 14
5.26 0.02 2.56 0.14 0.08 0.22 0.76 2.00 0.60 0.00
30 63 50 67 21 22 41 8 12 10 2 18 25 46 35
33 51 55 70 9 23 36 8 14 16 3 18 27 58 31
0.14 1.26 0.24 0.06 4.80 0.02 0.32 0.00 0.14 1.40 0.00 0.06 1.38 0.24
0.60 2.66 0.94 0.50
* P < 0.001 ; corrected P < 0.025.
TABLE 7. SEGREGATION ANALYSIS OF H L A - A , B
HLAA2,B5 A2,B12 A2,BW40 A2,BX A2,BWI5 AI,B8 A3,B7
N u m b e r of informative families 11 29 12 21 17 40 17
HAPLOTYPES IN FAMILIES OF PATIENTS WITH A L L
Patients Positive Negative 10 21 6 16 10 16 8
1 8 6 5 7 24 9
since in t h e t i m e s p a n o f 7 - 2 4 m o n t h s a f t e r diagnosis a fair n u m b e r o f p a t i e n t s are e x p e c t e d to h a v e died f r o m t h e i r l e u k e m i a . T h i s selection m u s t b e e v e n m o r e p r o n o u n c e d in t h e t h i r d g r o u p o f 51 p a t i e n t s t y p e d 2 a n d m o r e years a f t e r t h e initial d i a g n o s i s o f l e u k e m i a . It c a n be seen f r o m T a b l e 10 t h a t t h e f r e q u e n c y o f b o t h H L A - A 2 a n d A 9 are h i g h e s t in t h e g r o u p s w i t h t h e longest t i m e s p a n b e t w e e n d i a g n o s i s a n d tissue typing. It m u s t b e stressed, h o w e v e r , t h a t t h e differences b e t w e e n these g r o u p s a r e statistically n o t significant.
X2 7.36 5.82 0.00 4.26 0.53 1.60 0.59
Healthy siblings Positive Negative 15 26 18 31 21 45 21
14 32 10 17 20 41 18
X~ 0.03 0.62 2.28 4.08 0.02 0.18 0.23
DISCUSSION O u r analysis o f t h e H L A p h e n o t y p c a n d g e n o t y p e frequencies in p a t i e n t s w i t h A M L h a s n o t r e v e a l e d a n y significant d e v i a t i o n s . This is in k e e p i n g w i t h results o b t a i n e d b y o t h e r g r o u p s [2, 16, 21]. T h e increase in H L A - A 2 r e p o r t e d b y J e a n n v t a n d M a g n i n [5], h a s n o t b e e n c o n f i r m e d in o u r m a t e r i a l . I t was n o t e d h o w e v e r , t h a t in T e r a s a k i ' s , v a n R o o d ' s , a n d in o u r m a t e r i a l t h e r e was a slight increase for H L A - A 3 , w h i c h was n o t statistically significant in
EKKEHARD D. ALBERT et al.
266
TABLE 8. SEGREGATION ANALYSIS OF H L A ANTIGENS IN FAMILIES OF PATIENTS WITH A M L Number of informative families
HLA-
Patients Positive Negative
X~
H e a l t h y siblings Positive Negative
Xz
A1 A2 A3 A9 AI0 All A28 A29 AW30/31 AW32
37 38 40 24 10 23 15 12 16 2
15 16 18 12 6 12 3 5 7 1
23 22 22 12 4 I1 12 7 9 1
1.68 0.94 0.40 0.00 0.40 0.04 5.40 0.33 0.25 -
49 39 50 32 18 26 19 6 16 1
42 40 46 16 12 17 16 16 22 I
0.54 0.01 0.16 5.33 1.20 1.88 0.25 4.54 0.94 -
B5 B7 B8 B12 BI3 BI4 BWI5 BWl6 BWI7 BI8 BW21
10 34 25 35 9 12 22 5 12 12 2 9 9 21 23
2 14 13 19 6 5 9 3 4 7 1 3 3 7 11
8 20 12 16 3 7 13 2 8 5 1 6 6 14 12
3.60 1.05 0.04 0.25 1.00 0.33 0.72 1.33 0.33 1.00 1.00 2.33 0.04
5 42 30 36 11 13 26 11 15 I1
11 38 32 41 8 15 23 6 12 12
2.25 0.20 0.06 0.32 0.47 0.14 0.18 1.47 0.33 0.04
BW22
B27 BW35 BW40
1
1
9 9 14 28
11 12 24 32
-
0.20 0.42 2.63 0.26
TABLE 9. SEGREGATION OF H L A - A B HAPLOTYPES IN FAMILIES OF PATIENTS WITH A M L
HLA-
Number of informative families
AI,B8 A3,B7 A2,BI2 A2,B7
25 17 19 10
Patients Positive Negative 12 5 10 4
13 12 9 6
Xz
0.004 2.88 0.05 0.40
H e a l t h y siblings Positive Negative 28 18 20 16
31 24 24 11
TABLE 10. FREQUENCY OF H L A - A 2 AND A 9 n~ RELATXON TO TIME ELAPSED BETWEEN DIAGNOSIS OF A L L AND TISSUE T~PINO T i m e elapsed f r o m d i a g n o s i s to t y p i n g : 0-6 months 7-24 m o n t h s
> 25 m o n t h s
H L A - A 2 positive
16/31 (A) 51.6
19/37 (B) 51.4
32/51 (C) 62.7
H L A - A 9 positive ~o
7/31 (D) 22.6
3/37 (E) 8.1
14/50 (F) 28.0
X~ test:
A vs B A vs C B vs C A + B vs C A vs B + C D vs E D vs F E vs F D + E vs F D vs E + F
not significant n o t significant n o t significant n o t significant n o t significant n o t significant n o t significant X~=4.16, p = O . 0 4 n o t significant n o t significant
X2 0.15 0.90 0.36 0.46
HLA antigens and haplotypes in acute leukemia either of the three studies. The segregation analysis in families did not confirm the deviation observed in the phenotype and genotype data. Thus we can conclude that, in patients with A M L on the basis of our data and of those in the literature, there is no evidence for an association with H L A - A or HLA-B. In contrast, the analysis of 198 patients with A L L indicates a relationship between H L A and A L L which is expressed by a very weak association with the antigen H L A - A 2 and perhaps A9. Our data are then in agreement with a survey conducted by Svejgaard et al. [18] and with the data of Rogentine et al. [15], Thorsby et aL [22], Sanderson et al. [17], Terasaki and Mickey [21], and Gluckman et al. [4], who all found a more or less significant increase of H L A - A 2 in ALL. The fact that so many independent studies find this one antigen increased makes it highly unlikely that this finding is due to chance alone, in spite of the rather low degree of significance in most of the single studies. Since this association with H L A - A 2 is so weak, it is not particularly astonishing that some authors do not find significant deviations in HLA-A,B antigen frequencies [6, 8, 14]. The possibility that serological problems and the increased reactivity of leukemia cells with B-cell specific antibodies in typing sera could influence the typing results is excluded by the genotyping of practically all patients included in this study. The analysis of the haplotype frequencies of A L L patients shows that at least in this material the increase of H L A - A 2 is largely due to an increase in the A2,BS; A2,B12 and A2,blank haplotypes. It is however not possible to establish a significant association of the disease with particular H L A - A , B haplotypes as this would imply a three factor interaction, the significance of which could only be assessed if one would have a good estimate of the frequency of the H L A linked A L L gene and a measure of the linkage disequilibrium of this gene with H L A - A and HLA-B. Such data are not available. Since the observed association of A L L with H L A - A 2 is so weak and since family data were available for practically all patients, we attempted to validate the result obtained in the phenotype analysis by segregation analysis, a procedure that uses the healthy siblings as a control, which eliminates the problematic choice of an unrelated control population. Quite in agreement with the phenotype data, we found in this analysis a significant preponderance of H L A - A 2 among the patients but not among the healthy siblings. The weak increase in H L A - A 9 phenotype frequency was not borne out by the segregation analysis. In the segregation analysis of HLA-A,B haplotypes the above men-
267
tioned haplotypes A2,B5; A2,BI2 and A2,blank showed a deviation in the expected direction, although the numbers are too small to reach statistical significance. We can therefore conclude that there is indeed a significant--although quite w e a k - association of H L A - A 2 with ALL. It is now generally accepted that an established H L A disease association implies the existence of an H L A linked gene which is in some way involved with the disease. In order to elucidate the mode of action of such a gene it is of considerable interest to establish whether the inheritance pattern of this gene is recessive or dominant.'Dominant inheritance usually suggests a positive action of the disease gene such as, for example, the production of a detrimental enzyme or the initiation of an unusually strong or wrongly directed immune response. In contrast the recessive mode rather implies a lack of a particular enzyme or the lack of immune response. It was therefore in keeping with the immune surveillance theory that the H-2 linked susceptibility to virus leukemia in mice [10-13, 19, 20] was recessive and resistance was dominant. F r o m the data in the mouse, one would expect that also in man susceptibility to leukemia would be recessive. If this were so, one would expect to find an increased number of homozygotes among the patients. Another indicator of a recessive action would be the finding of an increased number of families in which the parents share a rare genotype (one H L A haplotype) as an indication of an increased inbreeding coefficient in the parents of leukemic patients. However, the analysis revealed that there is no excess of homozygotes for H L A - A or B among the A L L patients. Likewise, the numbers of families in which the parents share one haplotype was in very good agreement with expectations calculated on the basis of the haplotype frequencies (Table 2) assuming random mating. These data are in contrast with a series of patients with A L L in which Gluckman et al. [4] found an excess of H L A - A homozygotes. This discrepancy might be due to the relatively small numbers of patients in their series. We therefore conclude that there is in our material no evidence for an increased frequency of homozygotes, which suggests that the H L A - A 2 associated A L L gene does not seem to have a recessive mode of action. In considering an explanation for the observed association with HLA-A2, there are two principally different possibilities: (l) The association reflects the presence of a disease susceptibility gene in linkage disequilibrium with the H L A - A locus, a mechanism which is widely accepted for the association between ankylosing spondylitis or coeliac disease and the HLA-B locus. (2) Alternatively it is conceivable that
268
EKKEHARD D. ALBERTet al.
this association could reflect the existence o f an A2 associated gene which conveys protection against an early death f r o m leukemia as was p r o p o s e d by Rogentine et al. [15]. A l t h o u g h the slight (but not significant) increase o f H L A - A 2 and A9 in the group o f patients who were typed m o r e than 2 years after the diagnosis o f leukemia (Table 10) would tend to f a v o r this second possibility, it must be stressed that a large e n o u g h prospective longitudinal study has not been d o n e which alone could resolve this question. W h a t e v e r the m e c h a n i s m may be, it is clear that the H L A linked gene has only a m i n o r influence on
the development o f leukemia or the course of the disease, which is in keeping with the data in tlae mouse, where besides the H-2 linked genes a n u m b e r o f i m p o r t a n t H-2 independent genes have been identified which influence leukemogenesis. If this is so in inbred mouse strains it is no surprise that in the outbred h u m a n species the situation is so m u c h m o r e difficult to resolve.
Acknowledgements--The authors would like to thank Ms. Luz Bennett and Mr. Richard Goodell for their excellent technical assistance in the typing laboratory.
REFERENCES 1. 2.
3. 4.
5. 6. 7. 8. 9. 10. 1I. 12. 13. 14.
15. 16. 17. 18. 19. 20.
ALBERTE. D., MICK~Y M. R., "riNG A. & TERASAK; P. I. (1973) Deduction of 2140 HL-A haplotypes and segregation analysis in 535 families. Transplant Prec. 5, 215-221. BE~m~XN. M., SAOLm~ M., O w ~ s A., HUSSON N., BUS~L A., I~BOUL M., DASTOT H. & CT_.AZ~ E. (1976) Acute my©locytic leukemia (AML), chronic myeloid leukemia and H L A antigens. 1st Int. Congr. H L A and Disease, Paris (Book of Abstracts, p. 217). DAUSSETJ. & HOPS J. 0975) Some contributions of the HL-A complex to the genetics of human diseases. Transplant Rev. 22, 44-74. GLUCKMANE., LEMARCHANDF., NUNEZ-ROLDANA., HOPS J. & DAU~ET J. (1976) Possible excess of H L A - A homozygous among aplastic anemia and acute lymphoblastic leukemia (ALL). 1st Int. Congr. H L A and Disease, Paris (Book of Abstracts, p. 226). JEANNETM. & MAOmN C. (1971) HL-A antigens in malignant diseases. Transplant Prec. 3, 1301-1303. JOHNSONA. H., WARD F. E., AMtm D. B., L~rdN S. & ROGENTINE N. (1976) HLA and acute lymphocytic leukemia. 1st Int. Congr. HLA and Disease, Paris (Book of Abstracts, p. 227). KLOUDAP. T., LAWLERS. D., TILL M. M. & HARDIS'rYR. M. (1974) Acute lymphoblastic leukemia and HL-A: a prospective study. Tissue Antigens 4, 262-265. KOURILSKYF. M., DAussmr J., F~C_.,OLD N., DUPYU J. M. & BERNAnD J. (1967) Etude de ta repartition des antigenes leucoytaires chez malades atteints de leucemie aigue en remission. Adv. Transplantation, 515-522. LAWLERS. D., KLOt,'DA P. T., SMITHP. G., MORWENNAM. T. & HARDIS'rYR. M. (1974) Survival and the HL-A system in acute lymphoblastic leukaemia. Br. med. J. 1, 547-548. LILLYF. (1966) The histocompatibility-2 locus and susceptibility to tumor induction. Natn. Cancer Inst. Monogr. 22, 631-642. LILLYF. (1966) The inheritance of susceptibility to the Gross leukemia virus in mice. Genetics 53, 529-539. LILLY F. (1967) Genetic determination of susceptibility to friend virus in mice. Prec. Am. Ass. Cancer Res. 8,41 LILL~'F., BOYSE E. A. & OLD L..L (1964) Genetic basis of susceptibility to viral leukaemogenesis. Lancet 2, 1207-1209. REEND. J., MURTAGH T. J., GI~EALLYJ. & DEVLIN J. G. (1976) HL-A antigen distribution in acute childhood lymphatic leukaemia and in Hodgldn's disease. 1st Int. Congr. HLA and Disease, Paris (Book of Abstracts, p. 233). ROGENTtNEG. N., TRAI'ANI R. J., YANKIm R. A. & HENDERSONE. S. (1973) HL-A antigens and acute lymphocytic leuekmia: the nature of the HL-A2 association. Tissue Antigens 3, 470--476. VAN ROOD J. J., VAN HOOFF J. P. & K~UNING J. J. (1975) Disease predisposition, immune responsiveness and the fine structure of the HL-A supergene. A need for a reappraisal. Transplant Rev. 22, 75-104. SANDERSONA. R., MAHOURG. H., JAFFEN. & DAS L. (1973) Incidence of HL-A antigens in acute lymphocytic leukemia. Transplantation 16, 672-674. SVEJGAARDA., PLATZ P., RYDER L. P., NIELSENL. S. & THOMSEN M. (1975) HL-A and disease associations--a survey. Transplant Rev. 22, 3-43. TENNANT J. R. (1965) Susceptibility and resistance to viral leukemogenesis in the mouse. II. Response to the virus relative to histocompatibility factors carried by the prospective host. J. natn. Cancer Inst. 34, 633-641. TENNAI'¢r J. R. (1967) Additional evidence for implication of histocompatibility factors in resistance to viral leukemogenesis in the mouse. In Advance in Transplantation J. DAUSSET,J. HAMBURGER,& G. MATH/.:,(Eds.), pp. 507-514. Williams & Wiikins, Baltimore, 1968.
HLA antigens and haplotypes in acute leukemia 21. 22.
269
TeRAS^K! P. I. & M1OCI~Y M. R. (1975) H L - A haplotypes of 32 diseases. Transplant Rev. 22, 105-119. THOP,SBY E., ENor.scr A. & LXE S. O. (1971) HL-A antigens and susceptibility to diseases. A study of patients with acute lymphoblastic ieukaemia, Hodgkin's diasese, and childhood asthma. Tissue Antigens 1, 147-152.
Note from the Editors: One important aim of Leukemia Research is to put papers in proper perspective. This can often be done by a concise but broad introduction by the authors. In some cases, referees come up with a very positive statement that may be of interest to the readers. Such remarks will be printed after obtaining permission from the referee and the author. Objections from a referee goes the usual way directly to the author and are of course not printed.
Dissidents are welcome to criticize in later issues of the journal as "Letters to the Editors", which must be signed. Concerning the present paper, the journal received the following: Comment from a referee: "This is an important study of large numbers of patients which (almost) brings the final proof that ALL is associated with HLA. Moreover, the extensive family studies are the largest reported so far and bring important information on haplotype frequencies."
HLA A N T I G E N S AND HAPLOTYPES IN A C U T E L E U K E M I A * EKKEHARD D. ALBERT,t BRENDA N]SPEROS and E. DONNALL THOMAS~ Department of Medicine, University of Washington School of Medicine, and the Fred Hutchinson Cancer Research Center, Seattle, WA 98104, U.S.A. (Received 17 January 1977. Accepted 23 February 1977) Abstraet--HLA-antigen and hapiotype frequencies were determined in 198 patients with acute iymphoblastic leukemia (ALL) and 179 patients with acute myvlogenous leukemia (AML). In AML there were no significant deviations. A slight increase of HLA-A2 and A9 was found in patients with ALL which is in agreement with a number of reports in the literature. The deviation' for HLA-A2 was confirmed by segregation analysis in families, where healthy siblings served as a control. There was no increase in the number of homozygotes at the HLA-A and B loci in neither AML nor ALL. The number of families in which the parents share one HLA-A,B haplotype was not higher than expected in both diseases. Thus there is no evidence for a recessive mode of action for the assumed HLA-A2 associated gene. Key words: HLA-antigens, acute leukemia INTRODUCTION SUSCEt~rIB]LrrYto certain forms of leukemia has been found linked to the major histocompatibility complex (MHC) in the mouse [I 3]'. The high degree of analogy between the M H C in mice (H-2) and in man (HLA) has provided a strong argument for the existence of ~a similar relationship between the susceptibility to leukemia and the HLA-system in man. A linkage with susceptibility genes would be expected to manifest itself in the form of an association of certain HLA antigens with the disease, as it has indeed been found for a large number of nonmalignant diseases [18]. In spite of numerous investigations of leukemia patients (reviewed in [3]) it is still uncertain whether there is any significant association between leukemia and HLA. Clearly, if
* This investigation was supported by Grant Numbers CA 18029 and CA 15704, awarded by the National Cancer Institute, DHEW, and Contract AI 52515 from the National Institute of Allergy and Infectious Diseases. ~fDr. Albert is supported in part by grant AL 92/9/10 Forsehungsgemeinschaft and is a member of Sonderforschungsbereich 37, Mfinchen. ~: Dr. Thomas is a recipient of Research Career Award AI 02425 from the National Institute of Allergy and Infectious Diseases. Correspondence to: Ekkehard D. Albert, M.D., Tissue Typing Laboratory, Kinderpoliklinik der Universit~t, 8 Mfinchen 2, Pettenkoferstral3e 8a, Mtinchen, Germany. Abbreviations: ALL, Acute lymphoblastic leukemia; AML, Acute myeioid leukemia; MHC Major histocompatibility complex. 261
there is an association at all, it cannot be very strong. Therefore it is necessary to investigate a large sample of each of the different forms of leukemia. In connection with the search for a donor for bone marrow transplantation we have had the opportunity to carry out HLA typing on a large number of patients with leukemia and their families. This paper reports the phenotype and genotype data on 377 leukemic patients. MATERIALS AND METHODS Patients were referred to us by a large number of physicians from throughout the United States and Canada. There is a possibility that the patient population represents a selected sample particularly with respect to the time elapsed between diagnosis and HLA-typing. This problem is discussed in the appropriate section of the results. Diagnosis was based on the evaluation of bone marrow aspirates and/or biopsy as well as histochemical characterization of malignant cells. Tissue typing for 25 HLA antigens of the HLA-A and B loci was performed using routine procedures. Each sample was tested with at least 180 highly selected antisera including 60 sera from the NIH-tray provided by the Transplantation Immunology Branch of the NIAID, National Institutes of Health, Bethesda, Maryland. RESULTS Phenotypes HLA phenotype frequencies of 198 patients with acute lymphoblastic leukemia I(ALL) and 179 patients with acute myeiogenous leukemia (AML) are compared with those of a control population
EKKEHARDD. ALBERTet al.
262
consisting of 1070 American Caucasians [1]. The results are listed in Table 1, It can be seen that only HLA-A2 and A9 in patients with ALL show an increase (X2=5.74 and 4.77 respectively). If the corresponding P values are multiplied with the number of variables (i.e. antigens) tested, as is customarily done in HLA disease association studies, the increase of A2 and A9 does not reach the level of significance. Since the present study can be considered as a confirmation of earlier reports (reviewed in [18]) in which an increase of A2 and A9 was found, it is not necessary to apply the statistical safeguard of P value correction. Therefore it can be concluded that the uncorrected P values of P ~- 0.018 (for A2) and P = 0.032 (for A9) indicate a significant association of ALL with HLA-A2 and A9.
Genotypes The testing of families or family members of 198 patients with ALL and of 179 patients with AML allowed the deduction of the genotypes, In 17 ALL patients and in 38 A M L patients the family segregation did not allow an unequivocal deduction of haplotypes, so that in these cases the most probable genotypes had to be asserted by maximum likelihood procedures based on the haplotype frequencies obtained for the respective disease group. Thus it was possible to determine the HAL-A,B haplotype frequencies by haplotype counting. The matrix of haplotype frequencies derived from 198 ALL patients and from 179 A M L patients is given in Tables 2 and 3 respectively. The marginal totals in these tables represent the gene frequencies. Corresponding to the increase in phenotype frequencies in ALL there is also an increase in the gene frequencies of HLA-A2 and A9. All other antigens show frequencies within the normal range for American Caucasians. Looking at single haplotypes one can see that the A2 increase
TABLE 1. HLA
PHENOTYPE FREQUENCIES IN PER CENT OF 198 PATIENTS W I T H ACUTE LYMPHOCYTIC LEUKEMIA AND OF 1 7 9 PATIENTS W I T H ACUTE MYELOGENOUS LEUKEMIA IN COMPARISON W I T H A CONTROL POPULATION
ALL (N= 198)
AML (N= 179)
Controls (N= 1070)
A1 A2 A3 A9 AI0 All A28 A29 AW30,31 AW32
21.7 60.1 * 22.2 26.3$ 10.1 15.1 5.0 9.1 10.1 2.0
27.9 43.0 30.7 19.6 11.1 10.1 11.7 7.3 13.8 1.1
27.2 50.9 23.4 19.4 12.6 11.6 7.8 6.1 9.6 11.1
B5 B7 B8 BI2 BI3 BI4 BW15 BWI6 BWI7 BI8 BW21 BW22 B27 BW35 BW40
13.6 20.2 17.2 30.3 4.0 5.6 16.7 3.0 6.1 3.0 0.5 4.5 7.6 19.2 17.7
10.6 26.8 18.4 26.8 2.8 8.4 16.7 2.2 12.3 7.3 1.7 7.8 9.5 18.4 13.9
11.3 24.5 18.5 26.4 4.0 8.6 11.6 1.0 9.4 9.0 3.8 4.0 9.4 19.0 14.8
HLA
* x~=5.74 P=0.018 not corrected t x~=4.77 P=0.032 not corrected Control population: [4]. in ALL involves mostly the A2,B5; A2,BI2 and A2,blank hapiotypes (Table 2). These differences in hapiotype frequencies are however not large enough to reach statistical significance. The pattern of gene and haplotype frequencies in A M L (Table 3) is essentially normal.
TABLE2. HLA HAPLOTYPEF~QUENCI~ OF 198 PATENTSWITHALL DETERMINEDBY HAPLOTYPECOUNTING (Frequencies are given as numbers per 10,000) HLA
B5
A1 A2 A3 A9 AI0 All A28 A29 AW30/31 AW32
63 291 66 25 0 102 66 0 50 0
50 553 303 153 377 25 152 56 25 25 73 20 0 25 50 0 0 25 0 0
54 767 126 266 76 50 25 252 I0 0
0 25 26 25 0 25 0 25 51 0
0 25 25 0 50 0 77 0 25 0
76 353 126 202 50 25 0 0 50 0
26 25 0 25 51 0 0 0 25 0
50 76 68 0 25 50 0 0 0 0
0 0 25 51 25 0 0 25 26 0
0 25 0 0 0 0 0 0 0 0
25 67 38 0 0 25 0 0 25 25
0 163 0 I0 25 51 25 25 41 25
25 202 74 227 50 278 25 25 25 26
61 368 101 92 25 50 25 50 118 25
103 504 62 251 76 60 9 2 34 0
1086 3347 1139 1382 503 809 277 454 505 I01
25
0 25
57
25
76
2
0
34
0
0
22
25
78
25
3
397
688 1030 907 1683 202 278
884
152
303
152 25
227
390
1035
940
Blanks Total
B7
B8 BI2 B13 Bi4 BW15 BW16 BWI7BI8 BW21 BW22 B27 BW35 BW40 Blanks Total
1 1 0 4 10000
HLA antigens and haplotypes in acute leukemia
263
TABLE 3. H L A HAPLOTYPE FREQUENCIES OF 179 PATIENTS WITH A M L DETERMINED BY HAPLOTYPE COUNTING
(Frequencies are given as numbers per 10,000) B5
B7
B8
BI2 B13 B I 4 BWI5 BWI6 B W 1 7 B I 8 BW21 BW22 B27 BW35 BW40 Blanks Total
Al A2 A3 A9 AI0 All A28 A29 AW30/31 AW32 Blanks
55 248 28 55 0 28 28 0 90 0 4
39 693 307 61 726 108 228 0 0 28 28 53 28 28 55 0 28 6 0 0 30 5
57 542 84 196 66 0 155 140 74 28 40
0 28 0 0 0 0 0 0 112 0 0
28 44 0 47 28 0 72 0 65 0 134
112 199 211 168 84 55 0 0 34 3 1
28 0 28 28 0 0 28 0 0 0 0
164 291 10 15 55 0 0 28 47 0 32
0 84 48 11 112 0 28 0 17 0 64
16 28 0 0 0 0 0 0 28 0 12
28 28 31 45 0 112 28 28 59 25 8
25 196 84 0 28 55 28 0 55 0 3
146 112 248 55 11 167 155 0 0 0 46
55 201 112 107 9 0 28 28 133 0 1
88 100 2 50 138 3 19 84 10 0 30
1534 2469 1720 1005 559 501 625 363 758 56 410
Total
536 1469 982 1382
140
418
867
112
642
364
84
392
474
940
674
524
10000
Observed and expected frequency of homozygotes It h a s b e e n r e p o r t e d t h a t in p a t i e n t s w i t h A L L t h e r e is a n i n c r e a s e d n u m b e r o f H L A - A h o m o zygotes [4]. T h e r e f o r e , t h e n u m b e r o f h o m o z y g o t e s f o u n d a m o n g t h e 198 A L L a n d 179 A M L p a t i e n t s was c o m p a r e d to e x p e c t a t i o n s b a s e d o n t h e gene frequencies o b t a i n e d in T a b l e s 2 a n d 3. A c c o r d i n g to t h e H a r d y - W e i n b e r g law, t h e n u m b e r o f h o m o zygotes (h) is e x p e c t e d to b e h = L g ( a ) ] ~ . N, w h e r e g(a) is t h e gene f r e q u e n c y o f t h e allele a a n d N is t h e n u m b e r o f i n d i v i d u a l s u n d e r study. T h e results, w h i c h are s u m m a r i z e d in T a b l e 4, i n d i c a t e t h a t - - i f a n y t h i n g - - t h e r e is a slightly l o w e r n u m b e r o f H L A - A h o m o z y g o t e s in A L L t h a n expected. T h e f r e q u e n c y o f H L A - A locus h o m o z y g o t e s in A M L ,
as well as o f t h e H L A - B locus h o m o z y g o t e s in A L L a n d A M L are n o t significantly different f r o m expectation.
Families in which parents share one ttLA-A,B haplotype I n t h e family analysis it was o b s e r v e d t h a t in several families t h e r e was s h a r i n g o f a n H L A - A , B h a p l o t y p e , raising t h e q u e s t i o n o f w h e t h e r t h e r e c o u l d b e a n excess o f such families in A L L a n d / o r A M L . T h e e x p e c t e d n u m b e r o f families in w h i c h t h e p a r e n t s s h a r e o n e h a p l o t y p e was c a l c u l a t e d o n t h e basis o f t h e h a p l o t y p e f r e q u e n c i e s given in T a b l e s 2 a n d 3 u s i n g a f o r m u l a w h i c h was p r e v i o u s l y r e p o r t e d [1]. T h e c a l c u l a t i o n s ( T a b l e 5) r e v e a l e d t h a t n e i t h e r
TABLE 4. OBSERVED AND EXPECTED NUMBERSOF HOMOZYGOTES
HLA AI A2 A3 A9 AI0 A11 A28 A29 AW30/31 AW32
Total
ALL Obs.
LOCUSA Exp.*
AML Obs.
Exp.*
0 14 2 3 0 2 1 0 0 0
2.3 22.2 2.6 3.8 0.5 1.3 0.2 0.4 0.5 0.2
5 10 6 1 0 0 1 0 1 0
4.2 10.9 5.3 1.8 0.6 0.4 0.7 0.2 1.0 0.1
22 X==4.23
34.0 NS
24 X==0.06
25.2 NS
HLA
ALL Obs.
LOCUSB Exp.*
AML Obs.
Exp.*
B5 B7 B8 B12 BI3 BI4 BWI5 BWI 6 BW17 B 18 BW21 BW22 B27 BW35 BW40
0 1 1 8 0 0 2 0 0 0 0 0 1 3
0.9 2.1 1.6 5.6 0.1 0.2 1.5 0.0 0.2 0.0 0.0 0.10 0.30 2.10
0 4 2 2 0 0 1 0 0 0 0 0 0 1
4
1.7
0
0.5 3.9 1.7 3.4 0.0 0.3 1.3 0.0 0.7 0.0 0.0 0.3 0.4 1.6 0.8
Total
20 X==0.8
16.4 NS
10 X== 1.72
15.1 NS
* Expectations are calculated on the basis of the gene frequencies contained in Tables 2 and 3 respectively.
EKKEHARDD. ALBERTet al.
264
TABLE 5. FAMILIES SHARING ONE HLA-A,B HAPLOTYPE
ALL (150 families analyzed) Observed Expected* HLA-AI,B8 A2,BW40 A2,B 12 A2,B27 A2,B5 A9,BI2 All others
3 3 2 1 1 1 1
1.73 0.78 3.26 0.16 0.49 0.41 5.01
Total
12
11.84
X2----0.002
NS
AML (94 families analyzed) Observed Expected* HLA-AI,B8 A2,BI 2 A2,B5 A3,B7 A3,B5 A9,B7 A29,B 12 A2,BW 17 A9,B5 Other
2 1 1 3 1 1 1 1 I 0
1.68 1.05 0.23 1.84 0.003 0.19 0.07 0.31 0.10 2.58
Total
12
8.053
X2= 1.93
NS
* Expected values were calculated using the haplotype frequencies given in Tables 2 and 3. The expected numbers of families sharing a given HLA-A,B haplotype is Exp (A,B)=[2-hf(A,B). (l-hf(A,B))+ (hf(A,B))~] 2- N, where hf(A,B) is the haplotype frequency of A,B and N is the number of families investigated. in A L L nor in A M L was there a significant excess of families sharing an H L A - A , B haplotype.
Segregation analysis of HLA antigens and haplotypes In order to validate the findings of an H L A association in the phenotype data, a segregation analysis of H L A antigens Was performed in the families o f patients with A L L and A M L . Only families representing a simple backcross situation (+/-x-/-), where one parent is heterozygous and the other parent is negative for the antigen under study, were included in the analysis. In such a backcross situation one expects 50% of the children to be positive and 50 % to be negative for this antigen. The segregation analysis compares the segregation in patients with that in the healthy siblings. Thus, the segregation analysis is a method of detecting a disease association using the healthy siblings as a control. In A L L the segregation analysis (Table 6) reveals that H L A - A 2 has a significant predominance in the patients while the segregation in the healthy siblings is very close to the expected partition rate o f 50: 50. Thus, the association between A L L and H L A - A 2 observed in the phenotype data is also reflected in the segregation analysis. The increase in frequency for H L A - A 9 is not paralleled by a significant deviation in the segregation analysis and should therefore be considered with caution. All other antigens revealed a segregation which was not significantly different from expectation. The same type of segregation analysis was carried out considering hapiotypes which were frequent enough to yield sufficient numbers of informative
families. In A L L this haplotype segregation (Table 7) showed deviations for the hapiotypes A2, B5; A2,B12 and A2,BX. Although these deviations are only on the borderline of statistical significance-probably due to small n u m b e r s - - i t is interesting to note that exactly these three haplotypes accounted for the increase of H L A - A 2 frequency in A L L as seen in the analysis of haplotype frequencies in Table 2. As can be seen in Tables 8 and 9 there is no significant deviation in the families of patients with A M L , which is in agreement with the normal distribution of H L A antigens and haplotypes in this disease.
Frequency of HLA antigens in relation to the time elapsed between diagnosis and tissue typing It has been suggested by several authors [7, 9, 15] that deviations in H L A antigen frequencies could possibly reflect a selective influence which would favor the survival of such individuals, e.g. positive for HLA-A2. If that were true, we would expect to find different degrees of associations for groups of patients tested at different times in the course of the disease. A group of 119 patients with A L L , for which there was accurate information about the time span between diagnosis and tissue typing, was analyzed for the frequency of H L A - A 2 and A9 (Table 10). Three groups were formed: The first group consisted of 31 patients who had been typed up to 6 months following the diagnosis. This group might be considered as prospectively studied. The second group consisting of 37 patients must be considered a product of selection by the disease
H L A antigens and haplotypes m acute leukemia
265
TABLE 6. SEGREGATION ANALYSIS OF H L A ANTIGENS IN FAMILIES OF PATIENTS WITH A L L
HLA-
N u m b e r of informative families
Patients Positive Negative
X~
Healthy siblings Positive Negative
X2
A1 A2 A3 A9 AI0 All A28 A29 AW30/31 AW32
58 76 49 50 28 15 15 15 27 5
27 55 23 26 11 13 6 12 8 3
31 21 26 23 16 12 9 3 19 3
0.28 15.20" 0.18 0.18 0.92 0.04 0.60 5.40 4.48 0.00
76 84 59 61 34 29 22 23 25 9
61 93 . 60 60 42 27 17 17 28 7
1.64 0.44 0.008 0.008 0.84 0.06 0.64 0.94 0.16 0.25
5 7 8 12 13 14 15 16 17 18 21 22 27 35 40
23 45 47 59 11 18 33 8 15 9 3 15 24 38 32
17 22 18 31 6 8 19 6 6 4 1 6 8 22 18
6 23 29 28 5 10 14 2 9 4 2 9 16 16 14
5.26 0.02 2.56 0.14 0.08 0.22 0.76 2.00 0.60 0.00
30 63 50 67 21 22 41 8 12 10 2 18 25 46 35
33 51 55 70 9 23 36 8 14 16 3 18 27 58 31
0.14 1.26 0.24 0.06 4.80 0.02 0.32 0.00 0.14 1.40 0.00 0.06 1.38 0.24
0.60 2.66 0.94 0.50
* P < 0.001 ; corrected P < 0.025.
TABLE 7. SEGREGATION ANALYSIS OF H L A - A , B
HLAA2,B5 A2,B12 A2,BW40 A2,BX A2,BWI5 AI,B8 A3,B7
N u m b e r of informative families 11 29 12 21 17 40 17
HAPLOTYPES IN FAMILIES OF PATIENTS WITH A L L
Patients Positive Negative 10 21 6 16 10 16 8
1 8 6 5 7 24 9
since in t h e t i m e s p a n o f 7 - 2 4 m o n t h s a f t e r diagnosis a fair n u m b e r o f p a t i e n t s are e x p e c t e d to h a v e died f r o m t h e i r l e u k e m i a . T h i s selection m u s t b e e v e n m o r e p r o n o u n c e d in t h e t h i r d g r o u p o f 51 p a t i e n t s t y p e d 2 a n d m o r e years a f t e r t h e initial d i a g n o s i s o f l e u k e m i a . It c a n be seen f r o m T a b l e 10 t h a t t h e f r e q u e n c y o f b o t h H L A - A 2 a n d A 9 are h i g h e s t in t h e g r o u p s w i t h t h e longest t i m e s p a n b e t w e e n d i a g n o s i s a n d tissue typing. It m u s t b e stressed, h o w e v e r , t h a t t h e differences b e t w e e n these g r o u p s a r e statistically n o t significant.
X2 7.36 5.82 0.00 4.26 0.53 1.60 0.59
Healthy siblings Positive Negative 15 26 18 31 21 45 21
14 32 10 17 20 41 18
X~ 0.03 0.62 2.28 4.08 0.02 0.18 0.23
DISCUSSION O u r analysis o f t h e H L A p h e n o t y p c a n d g e n o t y p e frequencies in p a t i e n t s w i t h A M L h a s n o t r e v e a l e d a n y significant d e v i a t i o n s . This is in k e e p i n g w i t h results o b t a i n e d b y o t h e r g r o u p s [2, 16, 21]. T h e increase in H L A - A 2 r e p o r t e d b y J e a n n v t a n d M a g n i n [5], h a s n o t b e e n c o n f i r m e d in o u r m a t e r i a l . I t was n o t e d h o w e v e r , t h a t in T e r a s a k i ' s , v a n R o o d ' s , a n d in o u r m a t e r i a l t h e r e was a slight increase for H L A - A 3 , w h i c h was n o t statistically significant in
EKKEHARD D. ALBERT et al.
266
TABLE 8. SEGREGATION ANALYSIS OF H L A ANTIGENS IN FAMILIES OF PATIENTS WITH A M L Number of informative families
HLA-
Patients Positive Negative
X~
H e a l t h y siblings Positive Negative
Xz
A1 A2 A3 A9 AI0 All A28 A29 AW30/31 AW32
37 38 40 24 10 23 15 12 16 2
15 16 18 12 6 12 3 5 7 1
23 22 22 12 4 I1 12 7 9 1
1.68 0.94 0.40 0.00 0.40 0.04 5.40 0.33 0.25 -
49 39 50 32 18 26 19 6 16 1
42 40 46 16 12 17 16 16 22 I
0.54 0.01 0.16 5.33 1.20 1.88 0.25 4.54 0.94 -
B5 B7 B8 B12 BI3 BI4 BWI5 BWl6 BWI7 BI8 BW21
10 34 25 35 9 12 22 5 12 12 2 9 9 21 23
2 14 13 19 6 5 9 3 4 7 1 3 3 7 11
8 20 12 16 3 7 13 2 8 5 1 6 6 14 12
3.60 1.05 0.04 0.25 1.00 0.33 0.72 1.33 0.33 1.00 1.00 2.33 0.04
5 42 30 36 11 13 26 11 15 I1
11 38 32 41 8 15 23 6 12 12
2.25 0.20 0.06 0.32 0.47 0.14 0.18 1.47 0.33 0.04
BW22
B27 BW35 BW40
1
1
9 9 14 28
11 12 24 32
-
0.20 0.42 2.63 0.26
TABLE 9. SEGREGATION OF H L A - A B HAPLOTYPES IN FAMILIES OF PATIENTS WITH A M L
HLA-
Number of informative families
AI,B8 A3,B7 A2,BI2 A2,B7
25 17 19 10
Patients Positive Negative 12 5 10 4
13 12 9 6
Xz
0.004 2.88 0.05 0.40
H e a l t h y siblings Positive Negative 28 18 20 16
31 24 24 11
TABLE 10. FREQUENCY OF H L A - A 2 AND A 9 n~ RELATXON TO TIME ELAPSED BETWEEN DIAGNOSIS OF A L L AND TISSUE T~PINO T i m e elapsed f r o m d i a g n o s i s to t y p i n g : 0-6 months 7-24 m o n t h s
> 25 m o n t h s
H L A - A 2 positive
16/31 (A) 51.6
19/37 (B) 51.4
32/51 (C) 62.7
H L A - A 9 positive ~o
7/31 (D) 22.6
3/37 (E) 8.1
14/50 (F) 28.0
X~ test:
A vs B A vs C B vs C A + B vs C A vs B + C D vs E D vs F E vs F D + E vs F D vs E + F
not significant n o t significant n o t significant n o t significant n o t significant n o t significant n o t significant X~=4.16, p = O . 0 4 n o t significant n o t significant
X2 0.15 0.90 0.36 0.46
HLA antigens and haplotypes in acute leukemia either of the three studies. The segregation analysis in families did not confirm the deviation observed in the phenotype and genotype data. Thus we can conclude that, in patients with A M L on the basis of our data and of those in the literature, there is no evidence for an association with H L A - A or HLA-B. In contrast, the analysis of 198 patients with A L L indicates a relationship between H L A and A L L which is expressed by a very weak association with the antigen H L A - A 2 and perhaps A9. Our data are then in agreement with a survey conducted by Svejgaard et al. [18] and with the data of Rogentine et al. [15], Thorsby et aL [22], Sanderson et al. [17], Terasaki and Mickey [21], and Gluckman et al. [4], who all found a more or less significant increase of H L A - A 2 in ALL. The fact that so many independent studies find this one antigen increased makes it highly unlikely that this finding is due to chance alone, in spite of the rather low degree of significance in most of the single studies. Since this association with H L A - A 2 is so weak, it is not particularly astonishing that some authors do not find significant deviations in HLA-A,B antigen frequencies [6, 8, 14]. The possibility that serological problems and the increased reactivity of leukemia cells with B-cell specific antibodies in typing sera could influence the typing results is excluded by the genotyping of practically all patients included in this study. The analysis of the haplotype frequencies of A L L patients shows that at least in this material the increase of H L A - A 2 is largely due to an increase in the A2,BS; A2,B12 and A2,blank haplotypes. It is however not possible to establish a significant association of the disease with particular H L A - A , B haplotypes as this would imply a three factor interaction, the significance of which could only be assessed if one would have a good estimate of the frequency of the H L A linked A L L gene and a measure of the linkage disequilibrium of this gene with H L A - A and HLA-B. Such data are not available. Since the observed association of A L L with H L A - A 2 is so weak and since family data were available for practically all patients, we attempted to validate the result obtained in the phenotype analysis by segregation analysis, a procedure that uses the healthy siblings as a control, which eliminates the problematic choice of an unrelated control population. Quite in agreement with the phenotype data, we found in this analysis a significant preponderance of H L A - A 2 among the patients but not among the healthy siblings. The weak increase in H L A - A 9 phenotype frequency was not borne out by the segregation analysis. In the segregation analysis of HLA-A,B haplotypes the above men-
267
tioned haplotypes A2,B5; A2,BI2 and A2,blank showed a deviation in the expected direction, although the numbers are too small to reach statistical significance. We can therefore conclude that there is indeed a significant--although quite w e a k - association of H L A - A 2 with ALL. It is now generally accepted that an established H L A disease association implies the existence of an H L A linked gene which is in some way involved with the disease. In order to elucidate the mode of action of such a gene it is of considerable interest to establish whether the inheritance pattern of this gene is recessive or dominant.'Dominant inheritance usually suggests a positive action of the disease gene such as, for example, the production of a detrimental enzyme or the initiation of an unusually strong or wrongly directed immune response. In contrast the recessive mode rather implies a lack of a particular enzyme or the lack of immune response. It was therefore in keeping with the immune surveillance theory that the H-2 linked susceptibility to virus leukemia in mice [10-13, 19, 20] was recessive and resistance was dominant. F r o m the data in the mouse, one would expect that also in man susceptibility to leukemia would be recessive. If this were so, one would expect to find an increased number of homozygotes among the patients. Another indicator of a recessive action would be the finding of an increased number of families in which the parents share a rare genotype (one H L A haplotype) as an indication of an increased inbreeding coefficient in the parents of leukemic patients. However, the analysis revealed that there is no excess of homozygotes for H L A - A or B among the A L L patients. Likewise, the numbers of families in which the parents share one haplotype was in very good agreement with expectations calculated on the basis of the haplotype frequencies (Table 2) assuming random mating. These data are in contrast with a series of patients with A L L in which Gluckman et al. [4] found an excess of H L A - A homozygotes. This discrepancy might be due to the relatively small numbers of patients in their series. We therefore conclude that there is in our material no evidence for an increased frequency of homozygotes, which suggests that the H L A - A 2 associated A L L gene does not seem to have a recessive mode of action. In considering an explanation for the observed association with HLA-A2, there are two principally different possibilities: (l) The association reflects the presence of a disease susceptibility gene in linkage disequilibrium with the H L A - A locus, a mechanism which is widely accepted for the association between ankylosing spondylitis or coeliac disease and the HLA-B locus. (2) Alternatively it is conceivable that
268
EKKEHARD D. ALBERTet al.
this association could reflect the existence o f an A2 associated gene which conveys protection against an early death f r o m leukemia as was p r o p o s e d by Rogentine et al. [15]. A l t h o u g h the slight (but not significant) increase o f H L A - A 2 and A9 in the group o f patients who were typed m o r e than 2 years after the diagnosis o f leukemia (Table 10) would tend to f a v o r this second possibility, it must be stressed that a large e n o u g h prospective longitudinal study has not been d o n e which alone could resolve this question. W h a t e v e r the m e c h a n i s m may be, it is clear that the H L A linked gene has only a m i n o r influence on
the development o f leukemia or the course of the disease, which is in keeping with the data in tlae mouse, where besides the H-2 linked genes a n u m b e r o f i m p o r t a n t H-2 independent genes have been identified which influence leukemogenesis. If this is so in inbred mouse strains it is no surprise that in the outbred h u m a n species the situation is so m u c h m o r e difficult to resolve.
Acknowledgements--The authors would like to thank Ms. Luz Bennett and Mr. Richard Goodell for their excellent technical assistance in the typing laboratory.
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TeRAS^K! P. I. & M1OCI~Y M. R. (1975) H L - A haplotypes of 32 diseases. Transplant Rev. 22, 105-119. THOP,SBY E., ENor.scr A. & LXE S. O. (1971) HL-A antigens and susceptibility to diseases. A study of patients with acute lymphoblastic ieukaemia, Hodgkin's diasese, and childhood asthma. Tissue Antigens 1, 147-152.
Note from the Editors: One important aim of Leukemia Research is to put papers in proper perspective. This can often be done by a concise but broad introduction by the authors. In some cases, referees come up with a very positive statement that may be of interest to the readers. Such remarks will be printed after obtaining permission from the referee and the author. Objections from a referee goes the usual way directly to the author and are of course not printed.
Dissidents are welcome to criticize in later issues of the journal as "Letters to the Editors", which must be signed. Concerning the present paper, the journal received the following: Comment from a referee: "This is an important study of large numbers of patients which (almost) brings the final proof that ALL is associated with HLA. Moreover, the extensive family studies are the largest reported so far and bring important information on haplotype frequencies."