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1355
Tumor-Associated Antigen TA-90 Immune Complex
Assay Predicts Subclinical Metastasis and Survival
for Patients with Early Stage Melanoma
Mark C. Kelley, M.D.
Ralph C. Jones, M.D., LCDR
Rishab K. Gupta, Ph.D.
Reynold Yee, B.S.
Stacey Stern, M.S.
Leslie Wanek, Ph.D.
Donald L. Morton, M.D.
MC USN
Roy E. Coats Research Laboratories, John Wayne
Cancer Institute, Saint John’s Health Center, Santa
Monica, California.
Presented as a poster at the 49th Annual Cancer
Symposium of the Society of Surgical Oncology,
Atlanta, Georgia, March 21–24, 1996.
Supported by Grants CA-12582, CA-29605, and
CA-0989 from the National Cancer Institute, and
funding from the Wrather Family Foundation, Los
Angeles, California.
Mark Kelley’s current address: Division of Surgical
Oncology and Section of Surgical Sciences,
Vanderbilt University Medical Center, 21st Ave.
South, Nashville, TN 37232-2729.
Ralph C. Jones’s current address: Department of
Surgery, National Naval Medical Center, 8901 Wisconsin Ave., Bethesda, MD 20889-5600.
Address for reprints: Donald L. Morton, M.D., John
Wayne Cancer Institute, 2200 Santa Monica Blvd.,
Santa Monica, CA 90404.
The views expressed herein are solely those of the
authors and do not necessarily reflect the views of
the U.S. Navy, the U.S. Army, the Uniformed Services University of Health Sciences or the Department of Defense.
Received December 19, 1997; revision received
April 3, 1998; accepted April 3, 1998.
© 1998 American Cancer Society
BACKGROUND. TA-90 is a tumor-associated antigen first identified in the urine and
sera of patients with metastatic melanoma. In the early stages of disease, TA-90 is
present in circulating immune complexes (ICs) that may be detected with an
antigen specific enzyme-linked immunosorbent assay (ELISA). In this study, the
authors evaluated the efficacy of the TA-90 IC assay in detecting subclinical
metastasis of early stage melanoma and predicting the survival of patients with this
disease.
METHODS. Archival sera were collected preoperatively from 114 patients who
underwent wide excision with or without regional lymphadenectomy in the treatment of clinical Stage I melanoma. Sera were analyzed for TA-90 IC in a blinded
fashion, and results were correlated with the patient’s clinical course as determined by database and chart review. Subclinical metastases were considered
present at the time of surgery if the lymphadenectomy specimen was pathologically positive and/or the patient subsequently developed recurrence.
RESULTS. The TA-90 IC assay predicted subclinical metastasis in 43 of 56 patients
(P , 0.0001), with 14 false-positive and 13 false-negative results. Sensitivity and
specificity for the detection of occult metastasis were 77% and 76%, respectively.
Positive and negative predictive values were 75% and 77%, respectively. Fifteen of
18 tumor positive regional lymph node basins (83%) and 34 of 46 recurrences (74%)
were accurately predicted when considered independently (P , 0.004). Preoperative TA-90 IC status was also highly correlated with survival: 5-year overall and
disease free survival rates were 63% and 46%, respectively, for the TA-90 IC positive
group, compared with 88% and 82%, respectively, for the TA-90 IC negative group
(P 5 0.0001). A multivariate analysis with standard prognostic variables identified
preoperative TA-90 IC status as a strong, independent prognostic factor for both
overall and disease free survival.
CONCLUSIONS. To the authors’ knowledge, TA-90 is the first tumor marker that
accurately predicts subclinical metastatic disease and survival for patients with
early stage melanoma. For this reason, the TA-90 IC assay has the potential to
improve dramatically the prognostic evaluation of patients with this disease. Its
role in postoperative risk stratification and early detection of recurrence is being
evaluated in a prospective study. Cancer 1998;83:1355– 61.
© 1998 American Cancer Society.
KEYWORDS: melanoma, tumor marker, tumor-associated antigen, TA-90 immune
complexes, occult metastasis, prognostic factor.
T
umor markers may be used to detect occult metastatic disease or
recurrence, determine prognosis, and monitor response to therapy.1,2 Some of the markers described for melanoma have been correlated with disease stage and recurrence, but none have achieved
1356
CANCER October 1, 1998 / Volume 83 / Number 7
clinically useful accuracy.3–5 The need for a sensitive
and accurate marker of occult metastatic melanoma is
probably greatest when selecting patients most likely
to benefit from postoperative adjuvant therapies,6
such as melanoma vaccines7 and high dose interferon
a-2b,8 after surgical resection of clinical Stage I (CS-I)
melanoma.
Urine tumor-associated antigen (U-TAA) is a high
molecular weight glycoprotein identified in the urine
of patients with metastatic melanoma.9 It comprises
multiple subunits, including an immunogenic 90-kD
subunit designated TA-90.10 TA-90 is expressed by
71% of melanoma cell lines and tumor biopsies and
up to 70% of breast, colon, and lung carcinomas and
soft tissue sarcomas.10,11 Immunochemical analysis,
including antibody blocking studies showing lack of
cross-reactivity, indicate that TA-90 is distinct from
known tumor markers, such as carcinoembryonic antigen (CEA), prostate specific antigen (PSA), CA-15-3,
a-fetoprotein, and other oncofetal antigens (R. K.
Gupta, unpublished data).
TA-90 is also a major target for host antitumor
immune responses. Nearly all melanoma patients
whose tumors express this antigen develop anti-TA-90
immunoglobulin (Ig)M and/or IgG antibodies spontaneously.12,13 Patients who receive an allogeneic polyvalent melanoma cell vaccine (PMCV) also develop
increased humoral and delayed-type hypersensitivity
(DTH) responses to TA-90.12,13 In an analysis of patients receiving PMCV after surgical treatment for regional metastatic melanoma, a high IgM and DTH
response to TA-90 correlated directly with survival,
whereas a strong IgG response correlated inversely
with survival. These observations were the basis for a
highly predictive immunologic survival model for patients treated with PMCV.13
TA-90 is found in the serum of melanoma patients
as a free antigen and as an immune complex (IC) with
IgG antibody.14 Although TA-90 also forms an IC with
IgM antibody in melanoma patients, assays to detect
this IgM-IC are less reliable. Patients with extensive
tumor burden express high levels of free TA-90 and
low levels of TA-90 IgG-IC; the opposite is true of
patients with a low tumor burden.14 An antigen specific enzyme-linked immunosorbent assay (ELISA)
was developed to detect TA-90 IgG-IC in the sera of
cancer patients.11 In the current study, this TA-90 IC
assay was used to detect subclinical metastasis in patients with CS-I melanoma and predict their survival.
METHODS
Patient Population
The John Wayne Cancer Institute (JWCI) prospective
melanoma database was used to identify individuals
who underwent definitive surgical treatment of World
Health Organization (WHO) CS-I melanoma between
January 1, 1980, and December 31, 1990. This period
was chosen to guarantee that all patients had been
treated at least 6 years previously and therefore had
been followed long enough to allow for recurrence.15,16
The study population comprised 114 randomly
selected patients who had biopsy-proven primary
melanoma with no clinical or radiographic evidence of
metastasis, and from whom serum samples had been
obtained and cryopreserved no more than 30 days
before surgery.
All patients underwent wide excision of the primary site with a surgical margin $2 cm; 71% of primary lesions were intermediate in thickness (0.75– 4.0
mm) (Table 1). One hundred one patients also underwent elective regional lymphadenectomy, preceded in
25 cases by intraoperative lymphatic mapping and
sentinel lymph node dissection.17 Eighteen patients
who underwent lymphadenectomy had tumor in a
single lymph node (n 5 9), in 2– 4 lymph nodes (n 5
6), or in $5 lymph nodes (n 5 3). Of the 9 patients with
a single tumor-involved lymph node, 5 had only a
single micrometastasis (,2 mm in greatest dimension) in the sentinel lymph node.
Twenty-eight patients underwent postoperative
adjuvant therapy: 11 received bacillus CalmetteGuérin (BCG) and 17 received PMCV plus BCG.7 Recurrence was recorded and correlated with preoperative TA-90 IC status. The minimum postoperative
follow-up was 72 months for surviving patients, and
the median follow-up was 91 months (range, 4 –181
months) for the entire group. All patients gave informed consent to participate in a prospective study of
TA-90 antigen expression, which was approved by the
Institutional Review Boards of the University of California, Los Angeles, and Saint John’s Health Center,
Santa Monica, California.
TA-90 Immune Complex Assay
The preoperative serum sample from each patient was
assayed for TA-90 IC using an antigen specific IC
ELISA as previously described (Fig. 1).11 Briefly, sera
were diluted 1:60 in 0.025 M phosphate-buffered saline and 1% Triton X-100, pH 7.2. A 1:60 dilution was
chosen because it gave the optimal discrimination of
TA-90 IC values between normal volunteers and cancer patients when serial dilutions of sera were studied.
Diluted sera were placed in ELISA plates containing
wells coated with murine anti-TA-90 monoclonal antibody (AD1-40F4), incubated for 45 minutes, and
washed to remove unbound proteins. Goat antihuman
IgG Fab conjugated to alkaline phosphatase was add-
Tumor Marker for Early Stage Melanoma/Kelley et al.
TABLE 1
Demographic and Pathologic Data for 114 Patients
Characteristics
Age (yrs)
Median
Range
Primary site
Head/neck
Trunk
Extremity
Clark’s level
II
III
IV
V
Unknown
Ulceration
Satellitosis
Gender
Male
Female
Histologic type
SSM
Nodular
LMM/other
Breslow’s depth
,0.75 mm
0.75–1.50 mm
1.51–4.0 mm
.4.0 mm
Unknown
Tumor positive wide excision specimen
Pathologic stage
IA
IB
IIA
IIB
IIIB
1st recurrence:
None
Local
In transit
Regional lymph nodes
Distant sites
No. (%) of patients
48
20–79
17 (15%)
47 (41%)
50 (44%)
1357
was $0.410 optical density (OD) at 405 nm. This value
was generated by testing 59 normal volunteers11: their
mean OD was 0.169 6 0.080, and none had an OD
$0.410, which was .3 standard deviations above the
group mean. A cutoff of 0.410 was validated in a separate group of 250 normal controls and 419 patients
with a variety of malignancies: only 3% of controls
were positive, compared with over 50% of cancer patients.11
Clinical Correlation and Statistical Analysis
9 (8%)
48 (42%)
47 (41%)
6 (5%)
4 (4%)
9 (8%)
4 (4%)
67 (59%)
47 (41%)
55 (48%)
28 (25%)
31 (27%)
15 (13%)
33 (29%)
48 (42%)
14 (12%)
4 (4%)
16 (14%)
8 (7%)
32 (28%)
45 (39%)
11 (10%)
18 (16%)
68 (60%)
6 (5%)
7 (6%)
9 (8%)
24 (21%)
SSM: superficial spreading melanoma; LMM: lentigo maligna melanoma.
ed; the plates were incubated for 45 minutes and then
washed to remove unbound IgG Fab. The addition of
1 mM p-nitrophenyl phosphate in 10% diethanolamine produced a yellow color in the presence of the
bound alkaline phosphatase conjugate. After incubation for 1 hour, absorbance was measured at 405 nm.
Samples were assayed in duplicate, corrected for nonspecific binding, and compared with standard positive
and negative controls. All assays were performed by
laboratory personnel with no knowledge of the identity or clinical status of the patient.
Sera were considered positive if the absorbance
TA-90 IC status was correlated with lymph node status
and recurrence. Patients who had pathologically positive lymph nodes and/or later developed recurrence
were considered to have had occult metastasis at the
time of surgery. Sensitivity, specificity, and predictive
values of the TA-90 IC assay for lymph node status,
recurrence, and subclinical metastasis were calculated. The significance of the association between
TA-90 IC status and these endpoints was determined
with Fisher’s exact test.
Survival curves for TA-90 IC positive and negative
patients were constructed with the SAS statistical software program (SAS Inc., Cary, NC), and statistical significance was evaluated using the log rank test. Univariate and multivariate analyses were performed with
the Cox proportional hazards model, using TA-90 IC
status (positive vs. negative) and the following standard prognostic variables (Table 1): age (,50 vs. $50
years), gender, Breslow’s depth (,1.5 mm vs. $1.5
mm), Clark’s level (II or III vs. IV or V), regional lymph
node status (positive vs. negative), histologic type (superficial spreading vs. other), primary site (extremity
vs. other), ulceration, satellitosis, and tumor status of
the wide excision specimen (positive vs. negative).
Results were considered significant if the P value was
,0.05.
RESULTS
Correlations Among TA-90 IC Status, Lymph Node Status,
and Recurrence
The preoperative TA-90 IC assay was positive for 57
patients and negative for 57. When the TA-90 IC status
was related to the lymph node status of the 101 patients who underwent elective lymphadenectomy, 38
of 83 with tumor free regional lymph nodes were
TA-90 IC positive (Table 2). It is noteworthy that 27 of
38 TA-90 IC positive, lymph node negative patients
subsequently developed recurrence, compared with 9
of 45 TA-90 IC negative, lymph node negative patients.
This suggests that the TA-90 IC assay may have detected systemic occult disease. All 3 patients with tumor positive lymph nodes but a negative TA-90 IC
assay had only a single lymph node micrometastasis
1358
CANCER October 1, 1998 / Volume 83 / Number 7
FIGURE 1. This diagram represents the TA-90
antigen specific immune complex assay. Diluted
serum samples are added to plates coated with
murine anti-TA-90 monoclonal antibody. After
incubating for 45 minutes and washing to remove unbound protein, goat antihuman immunoglobulin Fab conjugated with alkaline phosphatase is added, and the plates are incubated
for 45 minutes and washed. Then p-nitrophenyl
phosphate is added and absorbance measured
at 405 mm after 1 hour.
(in the sentinel lymph node). Thus, the TA-90 IC assay
predicted lymph node status with a sensitivity of 83%
and a specificity of 54%; its positive and negative
predictive values were 28% and 94%, respectively.
Forty-six patients developed local (n 5 6), intransit (n 5 7), regional lymph node (n 5 9), or distant
(n 5 24) recurrence. The sensitivity and specificity of
the TA-90 IC assay for predicting recurrence were 74%
and 66%, respectively (Table 2). Positive and negative
predictive values were 60% and 79%, respectively. The
high false-positive rate of the TA-90 IC assay was explained in part by the absence of recurrence in several
patients with lymph node metastases.
Occult metastasis, defined as a tumor positive
lymphadenectomy specimen and/or postoperative recurrence of disease, was identified in 43 TA-90 IC
positive patients but only 13 TA-90 IC negative patients (Table 2). The sensitivity and specificity of the
TA-90 IC assay for occult metastasis were 77% and
76%, respectively; corresponding positive and negative predictive values were 75% and 77%.
Correlation Between TA-90 IC Status and Survival
Five-year disease free survival was 82% for TA-90 IC
negative patients but only 46% for the TA-90 IC positive group (P 5 0.0001; Fig. 2). The median disease free
survival was 53 months and .120 months for the
TA-90 IC positive and negative groups, respectively.
The 5-year rate of overall survival was 63% for TA-90
IC positive patients versus 88% for TA-90 IC negative
individuals (P 5 0.0001; Fig. 3). The median overall
survival was 91 months and .170 months in the TA-90
IC positive and negative cohorts, respectively.
Analysis of TA-90 IC Status as a Prognostic Factor for
Survival
Univariate analysis identified TA-90 IC status as a
significant prognostic factor for overall and disease
free survival (P 5 0.0001). As expected, Breslow’s
depth was also a significant predictor of both overall
and disease free survival (P 5 0.0001). The histologic
type of melanoma was significant for overall and disease free survival, whereas a tumor-positive, wide ex-
Tumor Marker for Early Stage Melanoma/Kelley et al.
1359
TABLE 2
Preoperative TA-90 Immune Complex Status as a Predictor of Lymph
Node Status and/or Recurrence
TA-90 IC
Lymph Nodes (n 5 101)
Positive
Negative
Recurrence (n 5 114)
Positive
Negative
Subclinical metastasisa
(n 5 114)
Positive
Negative
Positive
Negative
15
38
3
45
34
23
12
45
43
14
13
44
Sensitivity/
specificity
P value
83%/54%
0.004
74%/66%
,0.0001
77%/76%
,0.0001
a
Subclinical metastasis is defined as the presence of lymph node metastases and/or subsequent
recurrence.
FIGURE 3. The correlation between preoperative TA-90 immune complex (IC)
status and overall survival is shown. Individuals with a positive TA-90 IC assay
had a significantly lower median overall survival (91 vs. .170 months) and
5-year overall survival (63% vs. 88%) than those with a negative TA-90 assay.
men remained a significant prognostic factor for recurrence in multivariate analysis. No other factors
(including lymph node status) predicted recurrence or
survival in this cohort. Similar results were obtained
using the TA-90 IC OD and number of involved lymph
nodes as continuous variables (data not shown).
DISCUSSION
FIGURE 2. The correlation between preoperative TA-90 immune complex (IC)
status and disease free survival is shown. TA-90 IC positive (optical density
$0.410) individuals had a significantly lower median survival (53 vs. .120
months) and 5-year disease free survival (46% vs. 82%) than TA-90 IC negative
patients (P 5 0.0001).
cision specimen was a significant predictor of disease
free survival.
Multivariate analysis also identified TA-90 IC as an
independent prognostic factor for overall (P 5 0.0167)
and disease free (P 5 0.0084) survival. Breslow’s depth
remained an independent predictor of overall (P 5
0.0034) and disease free (P 5 0.0080) survival, but the
histologic type was no longer significant. It is noteworthy that the tumor status of the wide excision speci-
A significant proportion of patients thought to have
localized, early stage melanoma actually have subclinical metastasis.15,16 Although surgical lymph node
staging can detect regional lymph node metastases,
there is no way to detect occult disease accurately at
other sites. This distinction had only prognostic significance until a recent study demonstrated that high
dose interferon a-2b improved the survival of patients
with high risk melanoma.8 Because the survival benefit conferred by high dose interferon is only modest
and the regimen is quite toxic and expensive,18 identifying the patients with occult metastatic disease who
would be most likely to benefit from such therapy
would be of great potential utility.
In this study, the TA-90 IC assay had a sensitivity
of 77% and a specificity of 76% for occult melanoma,
which were far greater than percentages reported with
tumor markers for other malignancies.1,2 The sensitivity of the assay may be limited by the fact that not all
melanomas express TA-90, and some patients may
develop immune complexes containing IgG antibod-
1360
CANCER October 1, 1998 / Volume 83 / Number 7
ies that recognize an epitope which is spatially close to
the epitope recognized by the mouse monoclonal antibody used to capture the TA-90-IgM IC. The second
limitation could be addressed by an ELISA utilizing a
capture antibody that recognizes a spatially distinct
epitope. The specificity for detecting lymph node metastasis was somewhat lower than that for occult metastasis because several TA-90 IC positive patients
without regional lymph node metastases ultimately
developed recurrence, suggesting that the assay was
actually detecting distant metastatic disease. In addition, all three TA-90 IC negative patients with tumorinvolved regional lymph nodes had only a solitary
lymph node micrometastasis (in the sentinel lymph
node), which may have been too small a tumor burden
for the assay to detect. Although the result of the
TA-90 IC assay is positive in most patients with lymph
node metastases, it does not approach the sensitivity
and specificity of lymph node dissection and therefore
cannot be suggested as a substitute for surgical staging, particularly staging based on a focused immunohistochemical analysis of the sentinel lymph node.17,19
In the current study, the TA-90 IC assay might have
yielded false-negative results for patients whose
lymphadenectomy specimens were examined by standard histologic techniques prior to routine use of sentinel lymphadenectomy and immunohistochemistry.
Use of the TA-90 IC assay to analyze a single
preoperative blood sample distinguished 2 subgroups
with a 25% difference in 5-year survival. It may be
possible to increase the assay’s diagnostic accuracy by
eliminating confounding variables associated with
preoperative evaluation. For example, after biopsy but
prior to wide excision, the assay may detect residual
melanoma at the primary site rather than occult metastatic disease. In this study, 14 patients had microscopic tumor in the wide excision specimen; although
this was not strongly correlated with TA-90 IC status
(data not shown), it was a potential source of falsepositive results. In addition, TA-90 IC formed in response to a primary melanoma may persist after excisional biopsy and thus be a source of false-positive
results.
Multivariate analysis demonstrated that TA-90 IC
was surpassed only by Breslow’s depth as an independent predictor of recurrence and survival. Regional
lymph node status was not significant, possibly because 9 of 18 lymph node positive patients (50%) had
only a single lymph node metastasis, and 5 of these
metastases were micrometastases in the sentinel
lymph node. Patients with micrometastatic deposits in
a single lymph node have a much better prognosis
than patients with multiple or macroscopically positive lymph nodes.15,20
In summary, the TA-90 IC assay can accurately
detect subclinical metastasis and predict the survival
of patients with early stage melanoma. Further investigation is needed to define its role in postoperative
risk stratification and early detection of recurrent disease, and the authors have undertaken a large prospective study of serial TA-90 IC values obtained 1–3
months after surgical resection of early stage melanoma. Prospective validation of the results reported in
this article is underway as part of an international
multicenter trial of sentinel lymphadenectomy performed on patients with CS-I melanoma.
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