Shivers SC, Wang X, Li W, Joseph E, Messina J, Glass LF, DeConti R, Cruse CW, Berman C, Fenske NA, Lyman GH, Reintgen DS. Molecular Staging of Malignant MelanomaCorrelation With Clinical Outcome. JAMA. 1998;280(16):1410-1415. doi:10.1001/jama.280.16.1410
From the Cutaneous Oncology Program, H. Lee Moffitt Cancer Center and Research Institute (Drs Shivers, Wang, Li, Joseph, Messina, Glass, DeConti, Cruse, Berman, Fenske, and Reintgen), and the Departments of Surgery (Drs Shivers, Wang, Li, Joseph, Cruse, and Reintgen), Pathology and Laboratory Medicine (Drs Messina and Glass), Internal Medicine (Drs DeConti, Fenske, and Lyman), and Radiology (Dr Berman), University of South Florida, Tampa.
Context.— For most solid tumors, the metastatic status of regional lymph nodes
is the strongest predictor of relapse and survival. However, routine pathological
examination of lymph nodes may underestimate the number of patients with melanoma
who have nodal metastases.
Objective.— To determine the clinical significance of a highly sensitive molecular
assay for occult nodal metastases for the staging of patients with melanoma.
Design.— A prospective cohort study of consecutive patients in which lymphatic
mapping and sentinel lymph node (SLN) biopsy were performed on 114 melanoma
patients with clinical stage I and stage II disease. The SLNs were bivalved,
and half of each specimen was submitted for routine pathological examination.
The other half was submitted for molecular detection of submicroscopic metastases
using a reverse transcriptase–polymerase chain reaction (RT-PCR) assay
for tyrosinase messenger RNA as a marker for the presence of melanoma cells.
Patient follow-up averaged 28 months.
Setting.— A major university-based melanoma referral center at a National Cancer
Institute–designated cancer center.
Patients.— A total of 114 patients with newly diagnosed cutaneous malignant melanoma
who were at risk for regional nodal metastases.
Main Outcome Measure.— Melanoma recurrence and overall survival.
Results.— Twenty-three patients (20%) had pathologically positive SLNs, and all
of these patients were also RT-PCR positive. Of the 91 pathologically negative
patients, 44 were RT-PCR negative and 47 were RT-PCR positive. There was a
recurrence rate among 14 (61%) of the 23 patients who were both pathologically
and RT-PCR positive and a recurrence rate among 1 (2%) of 44 patients who
were both pathologically and RT-PCR negative. For patients who were upstaged
by the molecular assay (pathologically negative, RT-PCR positive), there was
a recurrence rate among 6 (13%) of 47 patients. The differences in recurrence
rates and overall survival between the pathologically negative, RT-PCR–negative
and pathologically negative, RT-PCR–positive patient groups were statistically
significant (P=.02 for disease-free survival and
for overall survival). In both univariate and multivariate regression analyses,
the histological and RT-PCR status of the SLNs were the best predictors of
Conclusions.— The use of an RT-PCR assay for detection of submicroscopic melanoma
metastases in SLNs improved the prediction of melanoma recurrence and overall
survival over routine pathological examination.
FOR MOST solid tumors, including colon, lung, and breast carcinoma,
the most powerful predictor of patient survival is the status of the regional
lymph nodes. Once nodal metastases develop, prognostic factors based on the
primary tumor contribute little to the prediction of recurrence and overall
survival. For patients with malignant melanoma, the 5-year survival decreases
40% with the development of nodal metastases. Therefore, appropriate emphasis
should be placed on accurate nodal staging for melanoma patients.
Preoperative lymphoscintigraphy, intraoperative lymphatic mapping, and
sentinel lymph node (SLN) biopsy have revolutionized the surgical treatment
and staging of patients with malignant melanoma. Originally proposed by Morton
and colleagues,1,2 the SLN is
defined as the first node in the regional basin that receives a cutaneous
afferent lymphatic from the primary tumor. Morton et al hypothesized that
if the SLN is negative for metastases, then the remainder of the nodes in
the basin should also be negative. Therefore, full nodal staging can be obtained
with a conservative lymph node biopsy that is less invasive, less morbid,
and lower in cost than the previous standard, the elective lymph node dissection.3 The validity of the SLN hypothesis has now been confirmed
by 4 other reports in the literature.3- 7
Routine pathological analysis of regional lymph nodes usually consists
of the preparation of 1 to 2 sections from the central area of the node, followed
by staining with hematoxylin and eosin (H&E) and microscopic examination.
This procedure typically studies less than 1% of the submitted nodal tissue.
Since pathologists make their diagnosis and clinicians make treatment decisions
based on this limited examination, the number of patients with nodal metastases
is often significantly underestimated. In fact, studies from multiple institutions
have demonstrated that routine pathological examination of the regional lymph
node basin can fail to detect 25% to 50% of the metastatic disease.1,8 Invasion of the regional nodes often
occurs with a low volume of metastatic cells, but the question of whether
micrometastatic disease is clinically important remains controversial.
Special stains that are relatively specific for melanoma, such as S-1009 or HMB-45,10 have been
available for years, but their use has never been incorporated into the routine
pathological examination of lymph node specimens. This is mainly because immunohistochemical
analysis of all the nodes from a complete lymph node dissection, which typically
involves 10 to 30 nodes, would be prohibitively time-consuming and expensive.
A major advantage of the lymphatic mapping techniques is that surgeons can
now submit 1 or 2 SLNs that are most likely to contain metastatic disease,
thus making a detailed pathological examination more practical.
However, sensitivity of the pathological examination of regional lymph
nodes is still an issue. Recently, a number of reports have described the
development of highly sensitive immunohistochemical and molecular biology
assays for the detection of metastatic cells in peripheral blood,11 bone marrow,12- 15
and lymph nodes.16- 20
These assays, based on special immunohistochemical stains or the reverse transcriptase–polymerase
chain reaction (RT-PCR), are designed to detect markers that are commonly
expressed by metastatic cells but not by other cells in the target tissue.
However, the biological and clinical importance of finding submicroscopic
evidence of cancer cells in tissues at risk for metastatic disease remains
This study examines the clinical significance of a molecular assay for
the detection of submicroscopic metastases in SLNs from melanoma patients.
The RT-PCR assay detects messenger RNA (mRNA) for the tyrosinase gene, which
is uniquely expressed by normal melanocytes and a majority of melanoma cells.
Patients were recruited for the study while being evaluated for treatment
for malignant melanoma by the Cutaneous Oncology Program at the H. Lee Moffitt
Cancer Center and Research Institute at the University of South Florida (USF),
Tampa. The study was approved by the USF Institutional Review Board.
Patients were selected based on the presence of a biopsy-proven malignant
melanoma with no evidence of palpable lymph nodes or distant metastases (clinical
stage I or II). One hundred fourteen consecutive patients who signed the informed
consent were enrolled in the study.
Patients consented to a treatment plan consisting of wide local excision
of the primary tumor and nodal staging by preoperative lymphoscintigraphy,
intraoperative lymphatic mapping, and SLN biopsy. Sentinel lymph nodes were
bivalved, and half of each node was evaluated for metastases by routine histological
examination and the other half by RT-PCR for tyrosinase mRNA. Patients were
observed for melanoma recurrence and overall survival at 6-month intervals
for the first 2 years and yearly thereafter.
To define the regional lymphatic basins at risk for metastatic disease
and to identify the approximate number and locations of the SLNs, patients
first underwent preoperative lymphoscintigraphy.21,22
A filtered technetium Tc 99m sulfur colloid (mean dose, 16.67 MBq [450 µCi])
was injected intradermally at 4 quadrants around the primary melanoma or previous
biopsy scar. A dynamic imaging study was performed to visualize the lymphatics
leading away from the primary tumor toward the regional nodal basin. Delayed
images were taken to detect late drainage to a second or third basin. A handheld γ
probe (Neoprobe 1000, Neoprobe Corporation, Dublin, Ohio) was used to determine
the approximate location of each SLN, which was marked with an intradermal
Both radiocolloid and vital blue dye lymphatic mapping techniques were
used intraoperatively to locate and identify SLNs. The operative procedures
were performed 2 to 6 hours after lymphoscintigraphy so that radioactivity
in the SLNs was still readily detectable and reinjection of radiocolloid was
not required. The dermis around the primary melanoma site was injected with
1 mL of 1% isosulfan blue (Lymphazurin Blue, Zenith Parenterals, Rosemont,
Ill) per lymphatic basin at risk (as determined by preoperative lymphoscintigraphy).
If a previous biopsy scar was present at the site of the primary tumor, care
was taken to inject the dye into normal skin around the scar.
Sentinel lymph node dissection was begun approximately 10 minutes after
injection of the blue dye. Sentinel lymph nodes were defined by the presence
of at least one of the following criteria: (1) greater than a 3:1 ratio of
in vivo radioactivity compared with background, (2) greater than a 10:1 ratio
of ex vivo radioactivity in the SLN compared with a neighboring non-SLN, or
(3) presence of the blue dye either in the node or (4) in an afferent lymphatic
leading to the node.
Wide local excision of the primary tumor was usually performed after
the removal of all SLNs, except in cases where the nodal basin was close to
the primary melanoma and elimination of the tumor was necessary to reduce
interfering radioactivity. Wide local excision was performed with a 1-cm margin
for primary tumors less than 1.0 mm in tumor thickness23
or with a 2-cm margin for melanomas 1.0 mm or more in thickness.24
Interferon alfa-2b has been recently approved for the adjuvant therapy
of patients with resected stage III melanoma.25
Patients with nodal metastases identified by routine pathological examinations
were taken back to the operating room for a complete node dissection and were
offered adjuvant interferon alfa-2b therapy or other adjuvant therapy trials.
No clinical decisions for further surgical and/or medical treatment were based
on results of the RT-PCR assay.
Each SLN was bivalved and half of each specimen randomly underwent a
routine histological examination as follows. The specimen was sectioned at
2-mm to 3-mm intervals and submitted for paraffin embedding (1-8 blocks per
node). Sections of each block were taken at 3 µm (1-3 per slide) and
stained with H&E, and the slides were read by a dermatopathologist.26
The other half of each SLN was submitted for RT-PCR detection of tyrosinase
mRNA. Figure 1 shows a schematic
overview of this assay, but the technical details have been described elsewhere.17 Briefly, SLN specimens were sent immediately from
the operating room to the laboratory, where they were trimmed of any external
fat, weighed, and stored at −86°C until RNA extraction. Total cellular
RNA was extracted from the entire specimen by a phenolguanidinium thiocyanate
method (RNA STAT-60, Tel-Test "B" Inc, Friendswood, Tex). One microgram of
purified RNA was used for the generation of tyrosinase complementary DNA (cDNA)
using a specific primer, HTYR2.
The cDNA was then amplified by 2 rounds (30 cycles each) of PCR using
nested primer pairs. The first round of PCR used the outer primers HTYR1 (upstream,
sense) and HTYR2 (downstream, antisense) to generate a 284– base pair
(bp) fragment. Five microliters of a 1:100 dilution of the first-round product
was used as a template for a second round of PCR using the nested primers
HTYR3 (upstream, sense) and HTYR4 (downstream, antisense) to generate a 207-bp
To minimize contamination in the PCR laboratory, powder-free gloves,
disposable tubes, and aerosol-resistant tips were used throughout the procedure.
Sample preparation and RNA isolation were performed in a fume hood, and a
separate set of pipettes was used for PCR. To rule out the possibility of
carryover contamination, a blank control, which contained all assay components
except RNA, was run with each assay. Finally, both positive (SK-Mel-28, melanoma)
and negative (T47D, breast carcinoma) cell lines were included in each assay
to verify the integrity of the assay system.
Polymerase chain reaction products were analyzed by electrophoresis
in a 2% agarose gel containing ethidium bromide. A 100-bp ladder (Gibco BRL,
Inc, Grand Island, NY) was included as a molecular weight marker. After electrophoresis,
the gels were photographed using UV transillumination and Polaroid 667 film.
Reverse transcriptase–polymerase chain reaction positivity was
defined by visualization of a band at 207 bp, as long as a band was not present
in the blank or negative controls. For all tyrosinase-negative specimens,
a separate RT-PCR assay27 for the β-actin
housekeeping gene was performed to verify the integrity of the mRNA isolated
from the specimen.
Univariate and multivariate regression analyses were performed with
prognostic factors based on the primary tumor, including tumor thickness,
Clark level, ulceration, and primary site location. Age, sex, and the histological
and RT-PCR status of the SLN were also included in the analyses. Mean subject
ages were compared between groups using an independent sample Student t test, incorporating a pooled variance. Tumor thickness
was compared between groups using the nonparametric Wilcoxon rank sum test.
Categorical variables were compared between groups using either the Fisher
exact test or a χ2 test.
Patient survival functions were generated for overall and disease-free
survival (DFS) using the product-limit method (or Kaplan-Meier method).28 Survival was calculated from the date of diagnosis
to the date of death. Disease-free survival was calculated from the date of
diagnosis to the date of first recurrence or death. Patients not experiencing
an event were considered censored at the date of last contact. Statistical
inference on survival functions between subgroups was based on the log-rank
test (Mantel-Haenzel) for equality of the survival functions.29
Censored survival data were fit to Cox proportional hazards regression models.30 After satisfying the proportionality assumptions
of the model, potential interactions were studied. Those interaction terms
that failed to achieve statistical significance were not considered further
in the models. Variable entry in the models proceeded in a forward stepwise
fashion using an adjusted χ2 statistic
in variable selection. The likelihood ratio test was used to examine the hypothesis
that the covariates had no influence on DFS. Regression analyses were performed
for DFS only since the number of deaths in the series was limited. The adjusted
hazard ratios for SLN histological and SLN PCR analyses were estimated by
the exponential function of the point estimate and SE of the model coefficients.
An allowed false-positive rate of 0.05 and 95% confidence intervals were used
throughout the analysis.
A summary of the demographics for the patient population (114 patients)
is shown in Table 1. Sites of
the primary tumor were equally distributed between the trunk and extremities,
while 8% of the tumors were located on the head and neck. Most of the primary
melanomas were Clark level 3 and 4 lesions (35% and 59%, respectively), with
a mean Breslow thickness of 2.36 mm. One patient with a tumor thickness of
0.6 mm underwent an SLN biopsy and was included in the study because there
was evidence of regression and the original tumor was believed to be more
than 0.75 mm thick. After pathological staging of the primary tumor and SLNs,
40 patients (35%) were stage I (negative SLNs and Breslow thickness ≤1.5
mm), 51patients (45%) were stage II (negative SLNs and Breslow thickness >1.5
mm), and 23 patients (20%) were stage III (microscopic positive SLN). The
mean follow-up for the entire patient population was 28 months, a period in
which 75% of the recurrences of melanoma should have occurred.31
Patients were grouped according to SLN status as determined by routine
histological analyses and by RT-PCR (Table
2). Twenty-three (20%) of 114 patients had a positive SLN biopsy
result by routine histological analyses and all of these patients also had
positive biopsy results by RT-PCR (group 3). Thus, there were no false negatives
by the molecular assay. Forty-four patients (39%) had negative SLN biopsy
results by both assays (group 1) while 47 patients (41%) had negative SLN
biopsy results by routine histological analyses but positive biopsy results
by RT-PCR (group 2).
None of the patients whose SLN biopsy results were negative both histologically
and with the RT-PCR assay (group 1) has died, and only 1 patient had a recurrence
of melanoma. Patients who were upstaged by the molecular assay (group 2) had
an intermediate prognosis with a recurrence rate of 13%. Sixty-one percent
of the patients in group 3 had a recurrence of melanoma or died from their
Kaplan-Meier relapse-free and overall survival curves are shown in Figure 2 and Figure 3, respectively. Patients whose SLNs were histologically
negative (groups 1 and 2) had significantly greater relapse-free (P=.006) and overall (P=.02) survival
than patients with histologically positive SLNs (group 3). Furthermore, patients
with histologically negative results who were upstaged by the molecular assay
(group 2) had significantly lower relapse-free (P=.02)
and overall (P=.02) survival than patients who were
RT-PCR negative (group 1).
Increasing Breslow thickness or ulceration of the primary tumor are
factors that are correlated with decreased patient survival.32
In our patients, as the primary tumor thickness increased, the chance of finding
PCR-positive material in the SLN increased. For patients with a tumor thickness
less than 1.5 mm, 1.5 to 4.0 mm, or more than 4.0 mm, the chances of having
an RT-PCR–positive SLN were 48%, 68%, or 77%, respectively. In addition,
70% of patients whose primary tumor was ulcerated had an RT-PCR–positive
SLN, whereas only 41% of patients whose melanoma was not ulcerated were RT-PCR
positive. These data represent the percentage of patients in each subgroup
who were RT-PCR positive (data not shown).
The pathological stages (using routine histological analyses only) of
each patient group are summarized in Table
3. For patients whose melanoma recurred, the sites of the first
recurrence are summarized in Table 4.
The 1 patient in group 1 who had a recurrence of melanoma experienced a
local recurrence, and thus metastatic cells were not necessarily present
in the SLN when the assay was performed. In general, the distribution of
recurrence sites in patients who were histologically negative but RT-PCR
positive (group 2) was similar to that of the patients with positive histological
results (group 3). Fifty percent of the recurrences in these groups were
systemic. Three patients from group 3 had recurrences in the regional nodal
basin, even though they underwent complete node dissection. Two patients
from group 2 had recurrences regionally, although they had not undergone
a complete dissection, since they were histologically negative.
Univariate (Table 5) and
multivariate (Table 6) regression
analyses were performed for DFS considering variables based on the primary
tumor (Breslow thickness, Clark level, ulceration, and cutaneous site), clinical
variables (sex and age), and SLN status (defined by either routine histological
analyses or RT-PCR). In a univariate analysis, with each variable acting
independently in the model, the histological (P=.04)
and RT-PCR (P=.04) status of the SLN were the only
factors that significantly predicted DFS. In a multivariate analysis, the
histological (P=.04) and RT-PCR (P=.03) status of the SLN were the only variables that predicted DFS.
Therefore, once metastatic disease developed in the SLN, whether identified
by routine histological analyses or RT-PCR, factors based on the primary tumor
did not contribute to the prognostic model.
Morton et al,1 Balch et al,2
and others3- 7
have shown that the SLN is the node that is most likely to contain melanoma
metastases. Therefore, it is rational to focus more detailed searches for
metastatic cells on the SLN. We have adapted a highly sensitive molecular
assay for the detection of submicroscopic melanoma metastases in SLNs. This
article examines the ability of the molecular assay to provide more accurate
staging information for melanoma patients at the time of their initial surgery.
It is well established that the histological detection of micrometastatic
disease in lymph nodes by routine H&E staining, with or without immunohistochemistry,
is clinically important. The 5-year survival of all patients with pathologically
node-negative (stage I and stage II) disease is about 85%. However, the 5-year
survival of patients who do not have palpable nodes but are found to have
histologically positive nodes (pathological stage III) approaches 50%.33
More at issue is the clinical significance of "submicroscopic" disease
that has been missed by routine histological analyses. Our initial attempts
at developing more sensitive assays for occult metastases involved a tissue-culture
technique, in which the regional nodes were placed into tissue culture and
any malignant cells were allowed to grow out. We found that the lymph node–culture
technique grew metastatic melanoma cells from 21% of patients who were histologically
node negative.34 We observed a shorter DFS
in culture-positive patients compared with patients who were node negative
by both routine histological analyses and the cell culture assay, thus demonstrating
a clinical correlation for the culture technique.35
Other indirect evidence for the clinical relevance of missed micrometastases
comes from a recently reported series from our institution in collaboration
with the MD Anderson Cancer Center, Houston, Tex. This study performed lymphatic
mapping and SLN biopsy in patients with melanomas more than 0.76 mm in thickness.
Two hundred forty-three patients with a negative SLN biopsy result were observed
to define the natural history of the mapped, SLN-negative population.36 With a mean follow-up of 2 years, 15 patients (3.5%)
had melanoma recurrences in the nodal basin, even though their SLN biopsy
result had been negative by routine techniques (H&E). After nodal recurrence,
the SLN-tissue blocks were recut for more sections and immunohistochemistry
was performed. Metastatic melanoma cells were identified in 10 (66%) of the
15 patients, suggesting that two thirds (or 66%) of the nodal recurrences
were due to a failed pathological examination.
Molecular staging has the potential to revolutionize the pathological
examination and staging of cancer patients. Molecular assays for occult metastases
have now been reported for a number of both solid tumors and hematologic malignancies
based on various markers.11- 20
These initial reports have invariably shown increased sensitivity when compared
with routine histological analyses and correlation between stage of disease
and PCR positivity. Some studies have also shown a relationship between PCR
positivity and disease recurrence or survival.
The current study shows that the tyrosinase RT-PCR assay is more sensitive
than routine histological examinations for the detection of SLN micrometastases.
Routine histological examination by microscopy has been shown to identify
1 abnormal melanoma cell in a background of 104 lymphocytes. Immunohistochemistry
can increase the sensitivity of the examination by allowing the identification
of 1 melanoma cell in a background of 105 lymphocytes. Molecular
assays have been reported to achieve sensitivity 2 to 3 orders of magnitude
higher than routine pathological examinations.11,12,14- 17
An important issue is whether the small numbers of melanoma cells detected
by the molecular assay are clinically important. This article shows a statistically
significant difference in both disease-free (P=.02)
and overall (P=.02) survival between patients with
histologically negative results with a PCR-negative SLN and those with a PCR-positive
Since the patients with histologically negative results in this study
were followed up by observation only, it is interesting to compare the rate
of PCR positivity in these patients with the natural history of the disease.
For patients with thick melanomas (>4.0 mm), the expected rate of positive
nodes after an elective lymph node dissection (by routine histological examination
would be about 25% to 40%. However, the 10-year survival of patients with
thick melanomas is approximately 25%, which is more in line with our observed
rate of PCR positivity (77% in patients with thick melanomas). For intermediate
thickness melanomas (1.5-4.0 mm), the 10-year death rate is 40%, but only
20% of these patients have positive nodes with elective lymph node dissection
and routine histological examination. We found 68% of these patients have
positive SLNs by the RT-PCR assay. However, the 10-year survival of patients
with thin melanomas (0.75-1.0 mm) is approximately 85%, while we found a 50%
rate of PCR positivity in these patients. Therefore, the prevalence of SLN-PCR
positivity closely reflects the natural history of patients with thick and
intermediate thickness melanomas but overestimates the risk of death for patients
with thin melanomas.
One possible source of false-positive RT-PCR results is benign nevus
cells, which have been observed in approximately 5% of lymph nodes examined.
When present, these cells are usually found in the capsule or fibrous trabeculae
of the lymph node. Although they are positive for tyrosinase, benign nevus
cells can usually be distinguished morphologically from melanoma metastases
by routine H&E-stained sections.
The RT-PCR assay has the potential of identifying a group of patients
(those with a PCR-negative SLN) who are at a low risk of recurrence and death,
and these patients can be spared further surgery and/or adjuvant therapy.
The question of whether patients with negative histological results but with
positive PCR results should be offered more extensive therapy (complete node
dissection and/or adjuvant therapy) remains unclear. Although the chance of
relapse in this patient group is still low, the presence of a significantly
reduced disease-free and overall survival suggests that further therapeutic
intervention might be beneficial. This question is being addressed by an industry-sponsored
multicenter national trial, called the Sunbelt Melanoma Trial. In this trial,
patients whose SLN is histologically negative but RT-PCR positive are being
randomized into 1 of 3 arms: (1) observation only, (2) complete lymph node
dissection, or (3) complete lymph node dissection with adjuvant interferon
alfa therapy. The clinical relevance of RT-PCR positivity will be ascertained
and the best treatment for submicroscopic disease will be studied. If our
data are confirmed in this national trial, molecular staging should become
an important part of the care of the patient with melanoma.
This study demonstrates that, when combined with surgical lymphatic
mapping techniques, the RT-PCR assay for tyrosinase mRNA allows more accurate
staging and identifies clinically significant disease in patients with melanoma.