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Advances in Brief
Direct Visualization of the Clonal Progression of Primary Cutaneous Melanoma:
Application of Tissue Microdissection and Comparative Genomic Hybridization
Rodney N. Wiltshire, Paul Duray, Michael L. Bittner, Tapio Visakorpi, Paul S. Meltzer, Ralph J. Tuthill,
Lance A. Molla, and Jeffrey M. Trent1
Dt'iHirtitu'tu »ttinnitili Genetics. University of Michigan Medical Sellout. Ann Arhor, Michigan 4X109 ¡K.N. W.¡;NiitiontiìCenler fot- Human Cenóme Research ¡R./V. W..
M. LB.. T. V.. P. S. M.. J.M.T.Ì ami Laboratory of Pathology.
Department of Pathology. Cleveland, Ohio 44IV5 ¡K.J. T.¡
National
Cancer Institute ¡P.D.. LA. /../. NIH. Helhestlu. Man/lamÃ- 20KV2: anil The Cleveland
Abstract
Human cutaneous iii.iliuii.ini melanoma progresses through a series of
»elldefined clinical and histopathological stages. It has been assumed that
the neoplastic progression of this disease advances from a common ac
quired nevus or dysplastic nevus through the primary radial growth phase
11«.I"i.primary vertical growth phase (VGP), and finally to distant
metastasis. However, it has never been directly shown that VGP is clonally
derived from I«¡I'.
Furthermore, it has not been possible previously to
conduct a detailed genetic analysis on pure tumor cells from archival
material because the lesions are a hcterogenous mixture of normal and
ncoplastic cells, and the entire specimen must be excised and fixed for
clinical diagnosis. This report describes a new approach designed to
identify DNA copy number changes in tumor cells from a series of
progressive primary stages of cutaneous melanoma archival biopsies.
Under direct high-power visualization, cells are procured with a sterile
Clinic.
However, it is clearly important to identify genetic changes associated
with early steps in melanoma progression to further our understanding
of this disease.
The biological basis for melanoma histológica! heterogeneity has
been understudied due to a lack of in xiln isolation techniques. Current
isolation methods are inefficient for molecular analysis because the
tissue samples are contaminated with normal cells (stromal cells,
lymphocytes, and vascular endothelium; Refs. 4, 5). Furthermore, it is
necessary for the entire primary melanoma tissue sample to be for
malin fixed, paraffin embedded, and sectioned for clinical diagnosis,
which makes genetic analysis difficult by the prevailing techniques.
Therefore, cytogenetic (6-9) and molecular (3, 4, 10, 11) studies of
tumor biopsies have been primarily confined to metastatic melanoma
and established cell lines. These data indicate that chromosomes 1, 6,
7, 9, 10. and 11 are frequently altered in advanced melanoma. Due to
needle from highly specific areas of the tissue section. DNA is extracted
the focus on the advanced melanoma cell lines, it is difficult to
from microdissected cells (normal, KGI'. and VGP), PCK amplified.
distinguish primary and secondary chromosomal alterations. Corre
fluorescently labeled, and examined by comparative genomic hybridiza
lating the appearance of chromosomal alterations with the progressive
tion to determine DNA copy number changes. Data obtained from three
representative cases suggest a donai derivation of VGP cells from I«.I". stages of melanoma is an important first step in discerning primary
This approach could be useful in identifying the sequence of genetic
from secondary genetic alterations.
changes in progressive cutaneous melanoma stages.
This report describes a new approach that utilizes tissue microdis
section (12, 13) to procure highly purified samples of melanoma
Introduction
tumor cells for subsequent analysis of DNA copy number changes by
CGH (14, 15). Formalin-fixed, paraffin-embedded tissue sections with
Superficial spreading melanoma is the most common form of
concomitant RGP and VGP were selected for study. The DNA was
human cutaneous melanoma and accounts for approximately 80% of
extracted from the cells. PCR amplified, fluorescently labeled, and
all cases (1). Clinical and histopathological studies suggest that the
analyzed by CGH. Three cases are reported illustrating complex
progression of superficial spreading melanoma initiates as a putative
numerical genetic changes in primary RGP and VGP stages, which
precursor stage, common acquired nevus or dysplastic nevus, and
advances through a primary RGP,~ a primary VGP, and finally to suggest that the derivation of RGP and VGP cells are from the same
clonal precursor population. The approach and data detailed in this
distant metastasis (1-3). Common acquired nevi are well circum
report should help to identify potentially important genetic loci altered
scribed, flat, or raised hyperpigmented lesions generally regarded as a
in distinct progressive stages of this cancer.
normal phenotypic variant. Dysplastic nevi are a larger, generally flat,
atypical variant of common acquired nevi. Epidemiological studies
Materials and Methods
have suggested that an increased number of nevi are associated with
an increased risk of cutaneous melanoma. Cells of the RGP are
Tumor Samples. Formalin fixed, paraffin embedded tissue sections of
present within the epidermis and capable of local superficial microprimary human eutaneous malignant melanoma were obtained from the South
invasion but not metastasis, whereas VGP cells grow in expansile
west Oncology Group and the Eastern Cooperative Oncology Group. Data
from three cases containing RGP and VGP in the same tissue section are
groups within the dermal layer of the skin and are metastasis compe
reported in this article. The clinical and histológica! information of each case
tent. It has not been demonstrated previously that VGP is clonally
are summarized in Table 1.
derived from RGP. The chronology of genetic alterations associated
Tissue Microdissection. RGP and VGP were carefully microdissected
with these stages has not been amenable to current genetic analysis.
from the histológica! tissue sections as described previously (12). Briefly,
hematoxylin and eosin-stained tissue sections were used to identify and locate
Received 7/12/"5: uccepled H/4/95.
The costs of publication of this article were defrayed in part by the payment of page
charges. This article must therefore be hereby marked advertisement in accordance with
18 U.S.C. Section 1734 solely to indicate this fact.
1To whom requests for reprints should be addressed, at National Cenler for Human
(¡enomc Research. NIH. Building 4'). Room 4A22, 44 Convent Drive. MSC 4470.
Bcthesda. MU 2XIW2-4470.
~ The abbreviations used are: RCiP. radial growth phase; VCiP, vertical growth phase;
C'CiH. comparative genomic hybridization; DAF1. 4'6-diamidino-2-phenylimodole.
desired groups of cells. The cells of interest were microdissected from a single
5-fim, deparaffinized. eosin-stained adjacent section (Fig. 1, A and B) with a
modified 26-gauge sterile hypodermic needle. The cells were procured by
electrostatic adhesion. Between 25 and 100 cells from histologically normal
primary RGP and primary VGP tissues from each sample were collected
separately in sterile lubes containing 20 /j.1 of a DNA extraction buffer (50
fig/ml proteinase K-10 mM Tris-HCI (pH 8.0)| and incubated at 55°Cover-
3954
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n.ONAI.
>'K(XiKI-:SSION 01
Table 1 Clinical muÃ-ìiisto[hiliiol<>xu'iil
u
Case
no.
Age
Sex
Location
Macroscopic
BT"
Clark
of lesion
size (cm}
(mm)
level
Results
Cell
Cells from regions of RGP and VGP were sçlVïlivïlY
scctcdfromarchivalprimarymelanomatissuesectionsobtainedirom
2.0U1640-550l(>4()-h401690-641675«45MMFr'nreskin
RankBack
ft
Min.ANOMA
epitheloiuRound/euhokialthree
1.02.131.773.10IIIIVIIItpilhcloici/fusiformUirge
ative
" BT. Breslow thickness.
night. The mixtures were boiled for 20 min io inactivate the proleinase K, and
5 (¿Iwere used as a template to PCR amplify the genomic DNA.
PCR Amplification. The amplification of the extracted genomic DNA was
performed with a degenerate universal primer as described previously (16).
The following changes were made to the protocol. The initial PCR amplifica
tion step was carried out in a 10-jj.l reaction volume for 8-10 cycles, using 1
JAMuniversal primer. The second amplification step was done in a 100-fil
reaction volume for 35 cycles. The reaction mixture consisted of all the PCR
products (10 fil) from the initial amplification step. 1(1mM Tris-MCI (pH 8.3),
50 mM KC1, 1.5 rnw MgCU 0.001% gelatin, 0.20 HIMof each dNTP, l JJ.M
universal primers, and 0.05 unit of Taq DNA polymcrase. A 16-/il sample of
the PC'R products was loaded on a 1% agarose ethidium bromide gel to
visuali/e the efficiency of the amplification (Fig. 2A).
DNA Labeling. The amplified genomic tumor DNA (test DNA) was la
beled with FITC-conjugalcd dUTP by PCR. The reaction solution was com
prised of 5 /xl of the amplified genomic tumor DNA; 10 mM Tris-HCl (pH 8.3);
50 mM KCI; 1.5 mM MgCU 0.001% gelatin; 0.2 mM each of dATP, dCTP, and
dGTP; 0.05 mM dTTP; 0.12 mM fluorescein-12-dUTP (DuPont, Boston, MA);
4 fÃ-Muniversal primer; and 0.1 unit of Taq DNA polymerase. The PCR
reaction was carried out in a 25-/J.1 volume under the following conditions.
After an initial 5-min denaturation at 95°C,the reaction underwent 25 cycles
of 1 min at 95°C, 1 min at 56°C,and 2 min at 72°C,and a final 10-min
extension at 72°C. The PCR products were purified from unincorporated
nucleotidcs with Bio-Spin 6 chromatography columns (Bio-Rad Laboratories,
Hercules, CA), and 5 fil were loaded on a \r'< agarose, ethidium bromide gel
to visuali/e the additional amplification (Fig. 2/J). The PCR-labeling efficiency
(percentage of the FITC'-dUTP incorporation) and DNA concentration were
determined by a UV spcctrophotometer (Beckman DU f>40). Approximately
3% of FITC-dUTP incorporation (incorporated FITC-dUTP/tolal nucleotidcs)
and 100 ng/fil of DNA were consistently obtained.
The normal reference DNA was extracted from known normal male
(GMOI247B) and female (GM1(W5U) lymphoblastoid cell lines. The DNA was
labeled with Texas red 5-dUTP (DuPont) by nick translation.
Comparative Genomic Hybridization. The CGH analysis was performed
according to Kallioniemi et al. (15) with modifications detailed below. Briefly,
the normal metaphase spreads were prepared from normal peripheral blood
lymphocytes and aged for at least 2 weeks at room temperature ( 17). The slides
were denatured in 70% formamide/2x
SSC (IX SSC = 0.15M NaCI and
O.OI5M sodium citrate) al 68°C,dehydrated in a series of ethanol washes (70,
85, and 100%), followed by treatment with 0.1 /ig/ml proteinase K [20 mM
Tris-HCl (pH 7.5)-2 mM CaCI,] for 8 min, and dehydrated again before
hybridization. Approximately 400 ng of Texas red-labeled normal reference
DNA and FITC-labcled test DNA, along with 40 (xg of Cot-1 DNA, were
hyhridi/ed to normal metaphase spreads. After 3 days of hybridization, the
slides were washed as described by Pinkul ci til. (IK), cnuntcrstained with O.I
fig/ml DAPI, and examined with a Zeiss Axiophot microscope equipped with
appropriate epifluorcscence filters and a CCD camera interfaced with a com
puter. Three images of each metaphase were captured using filter wheelmounted, single band excitation DAPI, FITC, and Texas red fillers and a
cube-mounted triple hand pass dichroic and emission filter (Chroma Technol
ogy Corp., Brattleboro, VT). The image and profile analyses were carried out
using the image analysis application, SinApps (Resource for Molecular Cytogenetics, Lawrence Berkley Laboratory, University of California San Fran
cisco, Berkeley, CA), based on the SCIL-lmage program (TPD/TNO, Delft,
the Netherlands) (Io). At least six metaphases were analyzed to generate
fluorescence ratio profiles in each case. Interpretation of the profiles was
performed according to the guidelines described by Kallionemi et al. (15).
patients (Fig. 1, A and ß,Table 1). Fig. \A shows a represent
eosin-stained tissue section used to procure RGP and VGP cells.
The nest of RGP cells surrounded by normal squamous epithelial cells
in the epidermal layer is microdissected without contamination of
normal cells (Fig. Iß).The VGP has expanded throughout the dermal
layer of the skin, providing u large region for microdissecting a highly
purified sample of VGP cells (Fig. Iß).
The microdissected cells were treated with proteinase K to release
the genomic DNA, which was PCR amplified and FITC labeled with
the UN-1 universal primer. The results of the DNA amplification were
visualized on ethidium bromide-stained agarosc gels (Fig. 2). The size
range of the amplified DNA isolated from tissue sections was con
sistently smaller (150-1000 bp) than the product from genomic DNA
isolated from cell lines (200 to over 2000 bp), suggesting fragmenta
tion of tissue sectioned DNA (Fig. 14). A sample of the amplified
DNA was PCR labeled with a FITC-dUTP (Fig. 2B). The size range
of the labeled DNA is similar to that of the initially amplified DNA
(Fig. 2A).
Analysis of the entire tumor genome for gains and losses of DNA
copy number was carried out using CGH. As a control for the fidelity
of the procedure, normal female DNA isolated from an established
lymphocytic cell line (GM10959) was amplified and FITC-dUTP
labeled as described above. This FITC-labeled test DNA was hybrid
ized to normal metaphase spreads along with Texas red-labeled nor
mal female reference DNA and Cot-1-blocking DNA. The analysis
revealed no numerical deviation from the normal diploid genetic
complement, which was expected. However, chromosome 19p
showed a fluorescence ratio below the normal ratio of 1.0, suggesting
a false positive interpretation of a deletion (15). Chromosomes X and
Y also gave false positive CGH profiles. Therefore, the profiles from
chromosomes 19, X, and Y were excluded from the CGH analysis.
These results indicate that the PCR amplification and labeling of the
DNA was sufficiently representative for CGH analysis.
The results of the CGH analysis of three reported cases are sum
marized in Table 2. The results illustrate numerical alterations present
in both VGP and RGP stages. The CGH profile of case 1690-640
revealed loss of chromosome Ipter-p33 in both VGP and RGP stages.
and chromosomes 16 and 22 were lost in the RGP and VGP stages of
1690-641. In addition, DNA copy number changes were seen in the
VGP that were not observed in the corresponding RGP. Gain of
genetic loci on chromosomes 4, 11, and 13 was observed in the VGP
cells of 1690-640 and 1690-641, but no numerical changes of these
chromosomes were observed in the RGP cells. Also, gain of chromo
somes 6p, 6ql2-q24, and 7 was identified in the VGP cells of
1690-550, 1690-641, and 1690-640, respectively. An example of a
numerical genetic change in VGP cells of case 1690-641 is illustrated
in Fig. 1C. The CGH profile and a representative digitized image of
the hybridization to chromosome 7 are presented, using DNA isolated
from RGP and VGP cells. Gain of genetic material on the terminal
portion of 7q was observed with the CGH analysis of DNA from VGP
cells but not from RGP cells. The high profile ratio and intense green
hybridization signals suggest genomic amplification of this region.
Comparing the DAPI- and FITC-captured digital images localized the
highest intensity amplification signal to 7q32-34 (Fig. 1C). The high
ratio observed in the CGH profile from RGP cells of chromosome 7
was caused by insufficient blocking of the centromeric regions by
Cot-1 DNA (Fig. 1C). This was verified by visually examining the
captured digital images of each hybridization.
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CLONA!.
Fig. 1. Illustration of microdisscclion-CGH./t,
an eosin-stained tissue section
bm) and a large VGP region in the dermal layer (luwer bm). B. microdissected
7 with isolated DNA from microdissected RGP and VGP cells (arrows) of case
of genetic material from microdissected cells. The dotted lines above and below
7q32-34 identified in VGP cells.
I'R(XÕR1:SS!ON DI MLI.ANOMA
of a melanoma biopsy with a nest of RGP cells surrounded by normal squamous epithelial cells (upper
RGP and VGP cells. (X 100). C, representative digitized images and profiles of CGH to chromosome
1690-641. CGH profile is displayed below each image. A ratio of 1.0 represents neither gain nor loss
the baseline represent ratios of 1.5 and 0.5, respectively. Idiogram shows the amplified region (green)
Discussion
This article focuses on a new approach designed to analyze the
genome of tumor cells isolated from progressive stages of human
superficial spreading melanoma. Two recently developed techniques,
tissue microdissection and CGH, were combined to isolate specific
tumor cells from tissue sections and analyze the genomic DNA. It is
difficult to study primary melanoma samples using standard tech
niques because they are routinely fixed and sectioned for clinical
M
l
2
3
4
5
M 1 2 3 4
Fig. 2. Representative ethidium bromide stained-agarose gels. A, initially amplified
DNA from microdissected VGP cells. Lane I, no template; Lane 2. DNA from a normal
female lymphocytic cell line (GM10959; positive control); Lanes 3, 4, and 5, amplified
DNA from cases 1690-550. 1690-640, and 1690-641, respectively. B. secondary ampli
fication and labeling of the initially amplified VGP DNA. Lane I, no template; Lane 2,
1690-550; Lane 3, 1690-640; Lane 4, 1690-641. Lane M, 100-bp DNA ladder (Life
Technologies, GIBCO-BRL. Grand Island, NY).
diagnoses. The procedure used here takes advantage of the availability
of formalin-fixed, paraffin-embedded melanoma tissue sections to
examine genomic tumor DNA.
It is difficult to obtain pure homogeneous samples of tumor cells
from a melanoma tissue section because the cells are in close prox
imity to normal cells and other tumor cells. Tissue microdissection
overcomes this difficulty by selectively dissecting RGP and VGP cells
from the archival tissue without contamination from surrounding
normal cells (Fig. 1, A and B). PCR is then used to amplify and
fluorescently label the DNA isolated from the cells (Fig. 2). The
quality of the DNA obtained by this procedure appears sufficient for
CGH analysis, avoiding the necessity of using multiple or thick
(50-/nm) tissue sections to obtain high molecular weight DNA for
molecular genetic analysis (20-22).
CGH analysis was performed on samples of microdissected normal,
RGP, and VGP cells procured from three cases of primary malignant
melanoma. Genetic material from chromosome 17 was lost in both
RGP and VGP of cases 1690-640 and 1690-641 (Table 2). These data
are consistent with the molecular studies reporting loss of chromo
some 17 loci in metastatic and primary melanoma samples (3, 4). The
loss of Ipter-p33 observed in cases 1690-640 and 1690-641 is also
consistent with previously published studies illustrating chromosome
Ip36 being frequently lost in malignant melanoma cell lines (4, 10).
Chromosomes 16 and 22 were both lost in the RGP and VGP cells of
case 1690-641, although it has not been reported previously that these
chromosomes were frequently altered in melanoma. Taken together,
the results imply that the VGP cells were derived from the RGP cells
in these cancers.
The CGH profiles showed a disparity between RGP and VGP cells.
An example of this is the gain of chromosome 6p only in the VGP of
case 1690-550 (Table 2). This is consistent with several cytogenetic
reports documenting the presence of isochromosome 6p in metastatic
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CLONAL PROGRESSION OK MELANOMA
Table 2 Summary of CGH results
1690-550NormalRGPVGP1690-640NormalRGPVGP1690-641NormalRGPVGP"
14q31-qter,lp31-22,
8cen-ql2. 12q23-qter,
20pl2-cenNANIp31-cen,
4, 5pter-ql4, 7, llql4-23,
20q5q31, 16, 17.
17q25-qlcr15q22-qter,
17qcen-q21,
13, 15q24-qter,
pter-p33.Ipter-lp34Losses''9q33-qter.
22q,
16, 17, 20q.
7q31-qter.8pll-q24,
Iq25-31, 2q22-32, 3pl4-ql3, 4, 5p(er-ql5, 6pl2-q24,
9q34-qtcr lOq. 16, l7ql2-22, 17q24-qter.
13q21-32nor
Ilpl5-ll, Ilql4-q23, 12ql5-22,
N. neither a gainGainsN°N6pN8q2l-22,
loss of genetic material: NA. no analyzable hybridization obtained.NNNNIpter-p33.Ipter-p33.NA1
Bold type indicates same genetic alteration present in (he RGP and VGP stages.
22q''
melanoma (6). Additionally, the CGH profiles of the VGP cells in two
of three cases (1690-640 and 1690-641) illustrate a whole and partial
gain of chromosome 7, respectively (Table 2, Fig. 1C). Observations
of extra copies of chromosome 7 are frequently recognized in metastatic melanoma (6-8, 23). This report has identified an amplification
on the chromosomal region 7q32-34 in the VGP cells from case
1690-641 (Fig. 1C). This result implicates the amplified region as
potentially important in the pathogenesis of disease; the region is
currently under investigation in other tumors.
The method of microdissection-CGH offers a new approach to
correlate DNA copy number changes with different stages of super
ficial spreading melanoma in the same tissue section from a single
patient. Analysis of additional cases should provide new insights into
the sequence of genetic changes associated with disease progression
in cutaneous malignant melanoma.
Acknowledgments
We thank Audrey Van Kirk and Darryl Leja for their assistance in technical
illustration.
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3957
Downloaded from cancerres.aacrjournals.org on June 16, 2017. © 1995 American Association for Cancer Research.
Direct Visualization of the Clonal Progression of Primary
Cutaneous Melanoma: Application of Tissue Microdissection
and Comparative Genomic Hybridization
Rodney N. Wiltshire, Paul Duray, Michael L. Bittner, et al.
Cancer Res 1995;55:3954-3957.
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