[CANCER RESEARCH 42, 4276-4279,
0008-5472/82/0042-OOOOS02.00
October 1982]
Increase of HLA-DR4 in Melanoma Patients from Alabama1
Bruce 0. Barger,2 Ronald T. Acton, Seng-Jaw Soong, Jeffrey Roseman, and Charles Balch
Departments of Microbiology ¡B.O. B.. R, T. A., C. B.I, Epidemiology [R. J. A.. J. R.. B. O. B.], Biostatistics
Birmingham, Birmingham, Alabama 35294
ABSTRACT
This report describes the association of HLA-DR phenotypes
in a population of 91 Caucasian melanoma patients compared
with 106 Caucasian controls from the Sunbelt region of the
United States. Over 75% of both patients and controls were
born in Alabama or a surrounding state. There was a significant
increase in the frequency of HLA-DR4 (x2 = 12.8; p = 0.0003).
This was present in 38.5% of the patients compared to a 16.0%
frequency in the controls, producing a relative risk of 3.3. The
difference in DR4 distribution remained significant after cor
recting for the number of antigens ( pc = 0.0018). The patients
were then grouped into two categories, "low risk" and "high
risk," based on their clinically assessed risk at presentation
for metastatic involvement. The decrease of DR3 in the highrisk group (x2 = 5.2; p = 0.02) suggested that it may represent
a marker for long-term survival. Thus, it appears that suscep
tibility to developing melanoma may be associated with DR4
while survival may be associated with DR3.
INTRODUCTION
A number of studies have examined the relationship between
HLA phenotypes and the occurrence of melanoma (1,5,8,10,
14, 16, 19, 22, 24, 25, 28, 29). To date, the only studies
describing a significant HLA association with melanoma have
been in the ethnically homogeneous populations of Italy and
France. Pellegris ef a/. (20) described an Italian population of
melanoma patients where the B40 antigen was increased and
Bw35 was decreased significantly. This report also described
a significant increase in A and B locus blanks over that seen in
their control population. In a French population described by
Cavalier (7), an HLA-A9 association was found with the recur
rent forms of melanoma. Reports of D and DR typing in mela
noma patients have been limited to studies in Norway (5) and
California (18). Neither study identified any significant associ
ations after correction, although there was an increase in the
D-locus marker, LD108, in the Norwegian population. A recent
report providing additional evidence implicating the products
of the region of the major histocompatibility
complex as a
contributing factor in the susceptibility of malignant melanoma
was that of Hawkins ef a/. (14). In a study of a large kindred
with a history of melanoma, data were presented suggesting
an autosomal dominant pattern of inheritance and segregation
with the HLA complex.
The evidence to date is conflicting about the role of HLA in
the susceptibility for developing melanoma or the clinical
course of the patients. Indeed, there is sufficient reason to
'This
investigation
was supported
in part by USPHS Grants
CA15338,
CA09128, CA18609, CA27197, CA30467, and CA13148 awarded by the Na
tional Cancer Institute, Department ot Health and Human Services, and by a
grant from the Cancer Research Institute, Inc.
2 To whom requests for reprints should be addressed.
Received June 18, 1981 ; accepted July 12. 1982.
4276
¡S-J.SJ, and Surgery [C. B], University of Alabama in
suspect that susceptibility to melanoma might be influenced or
controlled by genes in the region of the major histocompatibility
complex since there is a well-documented tumor-specific im
mune response in many melanoma patients. Both humoral
immune responses with specific anti-melanoma antibodies and
cellular immune response in the form of cytotoxic and prolif
erating lymphocytes have been demonstrated (6, 11, 13, 15,
17, 23).
While the Alabama melanoma population does not exhibit
the ethnic homogeneity of patient populations studied in Italy,
France, and Norway (5, 7, 20), it is a relatively nonmigratory
group that exhibits the highest melanoma mortality rate in the
United States. The inability to detect an HLA association in
previous reports from the United States might be due to the
extreme genetic heterogeneity of the population being studied.
Since the population in the state of Alabama is relatively stable,
they facilitated the opportunity to determine whether a DR
association with melanoma might be discernible in a more
genetically stable patient population. This report contains the
results of HLA-DR typing on 91 melanoma patients and 106
normal individuals, mainly from the southeastern United States.
MATERIALS
ANO METHODS
Patients. The 91 melanoma patients described herein were all
treated at the University of Alabama Melanoma Clinic, and diagnosis
was confirmed by histological examination. All patients were Cauca
sian. The criteria for assessing risk status at presentation have been
published (2-4). Briefly, low-risk patients are those with a decreased
risk for métastases. These patients are characterized by a clinically
localized melanoma (Stage I) with a tumor thickness :S4.0 mm and no
ulcération. Patients with a high risk for métastasesare those with Stage
I melanoma with a tumor thickness a4.0 mm or ulcération or any
patient with regional or distant métastases (Stages II or III). Over 93%
of the patients were born in the southeastern United States (Alabama,
Georgia, Arkansas, Florida, Louisiana, North Carolina, Mississippi,
Tennessee, Texas). All are long-term residents of the state of Alabama.
Controls. The controls were selected from a pool of individuals
typed during the time span of the study. They were selected from
hospital personnel, paternities, and acquaintances and represent a
major sampling from the Birmingham metropolitan area with no restric
tions on place of residence, either for controls or patients. The 106
normal controls were all Caucasians, with no personal history of cancer
and with no history of cancer in a first-degree family member. Over
76% were born in Alabama or in the southeastern United States.
Typing Methods. HLA typing was performed with the microdroplet
lymphocytotoxicity test using B-lymphocytes isolated by the nylon wool
column procedure (26). The antisera were obtained from Dr. Paul
Terasaki. The specificities tested were DR1, DR2, DR3, DR4, DR5,
and DR7, using at least 4 sera for each. The criteria for typing the Bcells was 85% or greater purity after the nylon wool separation pro
cedure.
Statistical Analysis. A maximum likelihood estimation of antigen
frequencies was used in analysis of our data. Estimation of the degree
of association of HLA antigens and melanoma was made by the method
of Woolf (30) for relative risk (odds ratio). The likelihood ratio asymp-
CANCER
RESEARCH
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VOL. 42
DR4 Association
totic x2 was used to test the significance
of association
of the various
HLA antigens. The attributable risk percentage was that described by
Cole and MacMahon (9). The tests of homogeneity were based upon
X2 analysis (1 2).
RESULTS
The frequency distribution for the HLA-DR locus determi
nants for melanoma patients and controls is shown in Table 1.
The only HLA-DR determinant that deviated significantly from
the normal frequency was the DR4 antigen. This was present
in 38.5% of the patient population compared to 16.0% in the
control population (p = 0.0003) with a relative risk of 3.3 and
an attributable risk of 69.4%. This difference is still significant
after correction for the number of DR antigens (pc = 0.0018).
DR2 was decreased compared to controls (19.8 versus
30.2%), but the difference was not significant.
The patients were then categorized into low-risk and highrisk groups based on their clinically defined risk status at
presentation. The DR4 antigen was increased in the low-risk
patients (x2 - 15.37; p = 0.0009) when compared to controls,
for a relative risk of 4.0 (Table 2). This was still significant after
correction (pc = 0.005). There was no significant DR4 differ
ence between the low- and high-risk melanoma patients. How
ever, the frequency of DR3 was significantly decreased in the
high-risk patients with respect to normal controls (x? = 6.62;
p = 0.01). There was a similar decrease in DR3 when com
paring low-risk patients with high-risk patients (x? = 5.21; p
= 0.02). The latter comparisons were not significant after
correction.
It was possible that the patient sample in this study might be
skewed because of selective deaths of potential participants.
To address this question, the patient pool was divided into 4
groups based upon the time interval from their initial presen-
tation with melanoma to the date they were HLA-typed (Table
3). Group 1 patients were typed within 1 year of diagnosis;
Group 2, within 2 years; Group 3, within 3 years; and Group 4,
within 4 years or greater. DR4 was significantly increased in
each of the patient groups, and there were no statistically
significant differences among the groups (p > 0.3). It seems
likely, therefore, that the pooled data do not skew the data
presentation and that the combined data could be treated as
incident data to yield the values shown in Table 1.
DISCUSSION
In this study of 91 melanoma patients, there was a highly
significant increase in DR4. This is significant after correcting
for the total number of antigens examined (6). No other signifi
cant HLA-DR associations were identified. As a means of
assessing the influence of any variation in the HLA-DR fre
quencies in patients based on the selective loss of patients that
might occur due to death in the time from onset to the time of
typing, the patients were divided into those typed within 1, 2,
3, and 4 years of diagnosis, respectively, and analyzed for
significant differences by the homogeneity index (12). There
are no significant differences (p > 0.3) among the 4 groups.
This can be seen in Table 3 wherein it is noted that, although
the frequencies for DR4 do exhibit some variation, they are
consistently increased over that seen in the control group.
Considering the lack of significant differences among the var
ious groups, it would appear that the relative risk of 3.3 is a
suitable estimate of risk based on the entire set of data rather
than using the incident cases represented by Group 1 alone.
Table 3
Analysis of homogeneity among subgroups of melanoma patients
(%)HLA-DR
Table 1
Comparison of HLA-DR phenotype frequencies in controls and melanoma
patients
2
19.8
30.2
2.837
1.151
3
32.1
24.2
4
16.0
38.5
12.795
3.8
5.5
0.331
5
7Controls 25.5Patients 19.8x20.001 0.906p
An
tigens1
Frequencies
(W=106)18.9
2b
1°
3C
4rf
(W42)19.019.0
(N -27)7.1 (W=
10)30.050.0
(N12)25.016.7
-
2
3
4e
(uncorrected)0.9733
(%)
<%)
HLA-DR1 (W 106)18.9
=
(W91)18.7
=
with Melanoma
risk1.0
0.0921
0.2188
0.0003
0.5468
0.3412Relative
0.6
0.73.3
1.5
0.7
30.2
7.1
32.1
30.9
18.5
30.0
8.3
16.0
35.7
48.1
20.0
41.7
3.8
4.8
5
3.7
0.0
16.7
7Controls
25.5Group
19.0Group 22.2Group 10.0Group 25.0
* Typed within 1 year of onset.
b Typed within 2 years of onset.
c Typed within 3 years of onset.
Typed within 4 years of onset
e Test of homogeneity among groups: p > 0.3.
Table 2
Comparison of HLA-DR frequencies between controls and risk groups of melanoma patients
(%)HLA-DR
comparedLow
Frequencies
risk vs.
controlx20.01
antigens1
risk
(N 67)19.4
(N 106)18.9
-
30.2
17.9
32.1
29.8
43.3
16.0
6.0
57Controls 3.8
16.4High
25.5Low
" RR, relative risk,
"p = 0.01 uncorrected.
c p - 0.02 uncorrected.
" p = 0.0009 uncorrected.
234
OCTOBER 1982
risk vs
risk vs.
controlX*0.060.26riskx'0.08
low
risk
IN 24)16.7
=
0.54
25.0
3.37
0.5
0.8
1.5
6.62b
5.21C
8.3
0.09
0.9
0.2
0.2
15.37"
1.01
2.60
25.0
4.0
1.7
0.4
0.44
0.01
012
4.2
1.0
1.1
0.7
29.2Groups 2.02RRa1.0
0.6High0.14RR0.9
1.2High 1.71RR0.8
2.1
4277
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S. O. Barger et al.
The attributable risk for DR4 is 69.4%, which suggests that
while the genetic component is a strong factor there are other
factors that may exert a very important influence as well.
A point of particular concern that should be addressed is the
composition of the control population and the frequencies of
the DR antigens. No attempt was made to match for age and
sex, since no specific association with HLA and age has been
shown to occur, nor was any effort made to match for ethnic
background.
With regard to DR antigen frequencies in the controls, those
of DR1, DR2, DR3, and DR7 are not appreciably different from
reports of North American Caucasians in the Eighth Interna
tional Histocompatibility Workshop (27). The DR4 frequency is
lower than that of the North American Caucasians (27.3%) but
does fall well within the range of frequencies found in the
European population, where it is 12.3% in the Italians and
27.6% in the English. DR4 is recognized as a marker that
exhibits considerable variation between populations. Even so,
we did analyze the patient data against the Eighth International
Histocompatibility Workshop Data and found that DR4 remains
significantly increased (x? = 5.16; p = 0.02) with a relative
risk of 1.7. Since the criteria for B-cell typing were those of
Terasaki et al. (26) and were uniformly applied throughout the
term of this study, the difference between these populations
and that of the Workshop is unlikely to be due to technical
error or, if it is due to technical bias, the error is uniformly
represented throughout. If the latter is the case, the results
would still remain valid.
Another antigen frequency that is decreased is DR5, which
may well be a unique feature of the local Caucasian population.
It does not appear to reflect a lack of ability to detect DR5,
since the DR5 frequencies in our local black population were
not appreciably different from those of the North American
blacks reported in the recent Workshop (21). These results
were obtained using the same sera over the same interval as
when the melanoma study was conducted. Consequently, it
appears that the low frequency for DR5 may be characteristic
of the local population. As a further comment, it should be
noted that DR5 is not significantly associated with melanoma;
thus, should it be a problem with the reagents, it is uniformly
distributed throughout the study.
The subgrouping of the patients into a low-risk or high-risk
group at presentation was based on the thickness of the
melanoma, the occurrence of ulcérationat the lesion site, and
the pathological stage of the disease (2-4). From Table 2, it
can be seen that low-risk patients have DR4 at very significantly
increased frequency over that of the controls for a relative risk
of 4.0. DR4 has been consistently increased in the high-risk
patients throughout the duration of this study. However, it is
not a significant association, nor is it significantly different
when comparing low-risk and high-risk patients. There is a
decrease in the frequency of DR3 in the high-risk patients
when compared to controls and low-risk patients (p = 0.01).
This association with DR3 has persisted, although not signifi
cantly after correction, since the first report on 69 patients (1 ).
The decrease of DR3 in the high-risk group is consistent with
an increase of DR3 in a group with the most rapidly progressing
form of melanoma. However, of 91 patients typed, 10 have
died (a mortality rate consistent with previous reports); only
one of these patients was DR3. Consequently, we are led to an
alternative possibility that DR3 patients, rather than being
4278
prone to a more rapidly progressing form of the disease, are
less likely to progress to the more severe phases of melanoma
and, in fact, may possess a determinant for long-term survival.
This possibility will be examined in a group of patients followed
for 5 or more years.
In summary, this report describes the increased association
of HLA-DR4 with melanoma in a Caucasian population residing
in a subtropical region based on a comparison with locally
derived controls and with North American Caucasians reported
in the Eighth International Histocompatibility Workshop. In ad
dition, evidence is presented which indicates that DR3 may be
a genetic factor associated with the long-term survival of mel
anoma patients.
ACKNOWLEDGMENTS
The authors thank Jayne Roberson, Virginia Vittor, Linda Baker, and James
Shoaf for their excellent technical assistance. Mary Estock for her secretarial
help, Anna Lee Ingalls for her assistance in identifying and interviewing patients,
and Kathy Hetch for her assistance in processing the data.
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Increase of HLA-DR4 in Melanoma Patients from Alabama
Bruce O. Barger, Ronald T. Acton, Seng-Jaw Soong, et al.
Cancer Res 1982;42:4276-4279.
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