Acral melanoma (AM) is commonly distinguished from superficial spreading melanoma (SSM), the most common type of melanoma, by its clinical presentation as well as its ethnic distribution. However, justification for such a distinction is controversial because of histological overlap and lack of prognostic significance. We analyzed chromosomal aberrations of 15 AMs and 15 SSMs that were comparable for tumor thickness and patient age, using comparative genomic hybridization. All AMs had at least one (mean, 2.0) gene amplification, significantly more than the SSMs, in which only 2 of 15 (13%) had one amplification each(P < 0.0001). At least 15 different genomic regions were amplified in AM. These involved small portions of chromosomal arms, sometimes including known oncogenes implicated in melanoma. The most frequently amplified regions in AMs occurred at 11q13 (47%), 22q11–13 (40%), and 5p15 (20%). Comparison of the amplification levels of invasive and noninvasive portions of the tumors using fluorescence in situ hybridization suggested that amplifications occurred before the formation of the invasive portion. The finding of amplifications of 11q13 in three of five additional cases of AM in situ further supports the notion that amplifications arise early in the progression of AM. Very significantly, we found isolated melanocytes with amplifications in the epidermis up to 3 mm beyond the histologically recognizable extent of the melanomas in 5 of 15 invasive AMs. In conclusion, our data show that AM is a distinct type of melanoma characterized by focused gene amplifications occurring early in tumorigenesis, and that malignant cells are present beyond the histologically detectable boundary,thereby revealing one mechanism of local recurrence.

The clinical and histological presentations of cutaneous melanoma are associated with anatomical location, sun exposure, age, and ethnicity. These patterns led to a classification of cutaneous melanoma into superficial spreading, nodular, lentigo maligna, and acral types(1). However, justification for such a classification is controversial because of histological overlap (2, 3) and lack of prognostic significance (4, 5, 6).

AM3exhibits several distinct clinical and epidemiological features. It develops on palmar, plantar, and subungual skin (7), sites that have little exposure to sunlight and are protected from UV radiation by a thick stratum corneum. Therefore, it is unlikely that UV radiation plays an important role in the pathogenesis of AM. The overall incidence is similar across all racial groups (8),with AM being the most common type of melanoma in dark-skinned peoples(9, 10).

Most melanomas begin within the epidermis (in situ melanoma)and progress through a period of lateral expansion to an invasive lesion that has metastatic capacity (11). Melanoma has a potential to recur locally if not excised with a safety margin of clinically and histologically uninvolved skin (12).

In a previous study of 32 randomly selected primary cutaneous melanomas by CGH, we noted that gene amplifications [>3-fold increase in copy number (13)] were infrequent in melanomas in general but found multiple amplifications in a single AM that we analyzed(14). Here we show that AM universally demonstrates a unique type of genomic instability that is characterized by amplifications of small genomic regions. Some of these regions contain genes of known significance in melanoma progression. This is consistent with the general view that amplified regions in tumors frequently contain oncogenes or drug-resistance genes, the increased dosage of which confers a selective advantage to the cells (13). We show that these amplifications are detectable in the in situstage of AM and are found in isolated intraepidermal melanocytes in histologically normal-appearing skin surrounding the melanomas.

Study Populations

Fifteen cases that had been archived under the diagnosis of invasive AM were retrieved from the archival material of the Department of Dermatology, University of Würzburg, the Dermatopathology Section, and the Melanoma Center of the University of California, San Francisco. Two AMs were from the toe, 10 from the sole, and 3 were from the foot without further specification. By histology, 12 AMs were of the acral lentiginous type, two were unclassifiable because the radial portions of the tumors were not represented in the specimen, and one had overlapping features with SSM. As controls, 15 cases comparable for patient age (±5 years) and tumor thickness (<1.5 mm, 1.5–4.0 mm, and>4.0 mm) that had been archived as SSMs were retrieved. Histological reassessment showed typical features of SSM in all 15 controls. For the analysis of AM in situ, five cases were retrieved from the database of the Melanoma Center at the University of California, San Francisco.

CGH

DNA Extraction.

Tumor-bearing tissue was microdissected from 30-μm sections (2–20 per tumor) using H&E-stained sections as guidance. DNA extraction and labeling was performed as published by Isola et al.(15). The amount of DNA obtained ranged from 2 to 12μg/specimen.

DNA Labeling.

All measurements were performed in duplicates: once with 1 μg of tumor DNA labeled with fluorescein-12-dUTP (DuPont, Inc., Boston, MA)and 200 ng of Texas red-5-dUTP-labeled reference DNA (“standard”labeling); and a second time with the labeling reversed, as described earlier (14).

Controls and Threshold Definitions.

Normal DNA and DNA from tumor cell lines with known aberrations were used as negative and positive controls for CGH, respectively. We regarded a region as aberrant when: (a) either the standard labeling or the reverse labeling resulted in a tumor:reference fluorescent ratios <0.80 or > 1.2; or (b)both the standard and the reverse labeling resulted in tumor:reference fluorescent ratios of <0.85 or >1.15 (14).

Dual-color FISH was carried out on tissue sections of the tumors that showed amplification by CGH. Probes mapping to amplified regions and reference probes for regions that were unchanged by CGH analysis were selected from the laboratories resource. Probes were labeled with Cy3 (Amersham, Arlington Heights, IL) or with digoxigenin (Boehringer Mannheim, Indianapolis IN), and hybridization on 6-μm paraffin sections was carried out as described earlier (16).

On the basis of CGH measurements, regions were called amplified if the tumor:reference ratio of a distinct segment of a chromosomal arm exceeded 1.5 or if the ratio elevation involved a sharply demarcated segment of a chromosomal arm. In most cases, both criteria were met,but in some tumors, the amplified chromosomal segment was too small to yield a ratio>1.5. Investigation of these regions by FISH indicated copy numbers >3-fold the reference probe signal counts. Several tumors of the AM group had copy number increases exceeding a tumor:reference ratio of 1.5 that involved the entire chromosome. These changes were not considered as amplifications. On the basis of the FISH experiments,regions exhibiting at least three times the average signal number of the reference probe were called amplified.

HRAS codon 12 primers were 5′-AGGAGACCCTGTAGGAG-GA-3′ (forward) and 5′-CGCTAGGCTCACCTCTATAGTG-3′ (reverse), and codon 61 primers were 5′-CTGCAGGATTCCTACCGGA-3′ and 5′-ACTTGGTGTTGTTGATGGCA-3′. PCR was carried out in 25-μl reaction volumes for 15 min at 95°C, followed by 35 cycles of 95°C for 15 s, 55°C for 30 s, and 72°C for 60 s, and a final hold at 72°C for 10 min in a Gene Amp PCR System 9700 Thermal Cycler (Perkin-Elmer). DNA sequencing was carried out using dye terminator sequencing chemistry (PE Applied Biosystems), according to the manufacturer’s instructions.

We used a χ2 statistic to compare patterns of aberrations in AMs with those of SSMs where expected numbers were determined by marginal frequencies, taking into account the total numbers of gains and losses for each type of melanoma. This was accomplished by estimating a global factor k (for each type of aberration) such that if π is the rate of aberration for the chromosome arm in SSM, then k*π is the rate in AM.

Genomic Aberrations in AM and SSM.

To investigate a potential difference in the pattern of chromosomal aberrations between AM and SSM, we analyzed a total of 15 pairs of these two types of melanoma. Both groups were comparable with regard to patient age and tumor thickness, factors that we had found previously to affect the total number of aberrations (14). The mean age and tumor thickness in the AMs was 72.2 years (range, 44–87 years)and 4.6 mm (range, 1.3–14 mm), respectively, compared with 70.8 years(range, 40–84) and 5.0 mm (range, 1.2–20 mm) in the SSMs.

The aberrations detected by CGH are summarized in Fig. 1. The most frequent changes in both groups match our earlier findings(14). Losses of chromosomes 9p and 10q occurred in 10 of 15 (67%) and 7 of 15 (47%) of the AMs and 9 of 15 (60%) and 7 of 15(47%) of the SSMs, respectively. AMs demonstrated a significantly higher rate of aberrations than SSM, with 2.0 times as many gains and 1.6 times as many losses (Table 1). Although gains of chromosomes 7p, 5p, and losses of chromosome 6q were considerably more frequent in the AMs (53% versus13%, 33% versus 0%, and 47% versus 7%,respectively), these changes were not statistically significant when an adjustment was made for the overall greater frequency of aberrations in AMs. No difference in the overall pattern of regions that were affected by losses or gains was found between the two types of melanoma when this adjustment was applied to all of the data. However, this finding may be a result of weak statistical power inherent in this method of analysis.

In contrast, a clear qualitative difference between AM and SSM was found when amplification frequencies were analyzed (see “Materials and Methods” for definition of amplifications). All (15 of 15) AMs had at least one amplification; 13 had multiple amplifications (mean,2.0). In contrast, amplifications were found in 2 of 15 SSMs, one in each of the two tumors. This difference in amplification frequency was highly significant (P < 0.0001). The AMs contained a total of 30 amplifications involving at least 15 separate loci. Most frequently, amplifications occurred on chromosomes 11q13 and 22q. However, as shown in Fig. 1, chromosomal regions 5p15, 5p13,12q13–22, and 16q21–22 were also amplified in more than one tumor. Amplified regions mostly involved only a small fraction of a chromosomal arm.

In Situ Detection of Amplifications by FISH.

To obtain information on the copy number and tissue distribution of the amplifications, we performed dual-color FISH on tissue sections of the tumors that showed amplifications by CGH. One probe was targeted to the amplified region and the other to a region where CGH showed average copy number. A total of 63 FISH measurements encompassing 18 different loci were performed in the 17 tumors that showed amplifications by CGH(Table 2).

FISH analysis of the tumors showed amplification levels(i.e., a ratio between the two probes) of 3–20. In some cases, FISH probes chosen based on the apparent position of a CGH peak missed the peak of the amplified region (e.g., AM98, AM104,and AM105 in Table 2), indicating that the amplified regions were smaller than several megabases. The fact that amplicons in AM are relatively small is significant, because it will simplify the future identification of the target genes of the amplifications.

Amplifications Arise Early in the Progression of AM.

To determine when during tumor progression the amplifications arose, we used FISH to detect amplification in the in situ portion of tumors for which CGH found amplifications in the invasive portion(Table 2). In all cases where there was a copy number increase in the invasive portion, a corresponding increase was found in the in situ portion. In most cases, the amplification levels of in situ and invasive portion were similar. However, in four cases,the signal count of at least one marker was higher in the invasive portion than in the in situ portion, suggesting a selection advantage during progression to invasive melanoma. The finding of amplifications in the in situ portions of the tumors suggest that amplifications in AM occur early in tumor progression, prior to the formation of the invasive phase.

To address this question directly, we studied five biopsies of AM in situ using FISH with markers for the two regions (11q13 and 22q12) that were most commonly amplified in the invasive AMs. Melanoma in situ represents the earliest level of progression that is histologically recognizable and does not permit the dissection of tumor cell populations pure enough to perform CGH. Three of the five in situ AMs showed amplifications of 11q13, and one of the three had an additional amplification of 22q12. The amplification level ranged from three in one case to more than five in the two other cases (Fig. 2 A). These results show that high-level amplifications are already present at the in situ stage of AM, indicating an early event in progression.

Detection of Cells with Amplifications in Histologically Normal Skin.

In five cases of invasive AM, isolated cells with amplifications could be detected by FISH in the basal layer of the epidermis up to 1 cm away from the invasive portion of the tumor and up to 3 mm beyond the histologically detectable extent of the in situ portion. This is illustrated by case AM94 in Fig. 2,B. The tumor cells in both the in situ portion (Fig. 2,B, middle panel) and the invasive portion (Fig. 2,B, left panel)of the lesion show >20 signals with the probe targeted to chromosome 11q13.2. Single melanocytes with a similar amplification level could be detected up to 9 mm from the invasive portion of the tumor (Fig. 2,B, right panel). These cells were found in the basal layer of the epidermis and were spaced equidistantly, similar to normal melanocytes. Histologically, the melanocytes in the area most distant to the invasive portion showed only slightly enlarged, hyperchromatic nuclei or no signs of atypia (Fig. 2 B, right panel). We refer to the single cells with gene amplifications in histologically normal-appearing skin as field cells. All cases in which field cells were found had been excised with 2–3-cm safety margins, and in none of these cases the field cells extended to the excision margin.

We reasoned that the field cells could either represent micrometastatic cells derived from the invasive portion of the tumor or a residuum of the in situ melanoma that preceded the invasive portion. The evidence favors the latter hypothesis: (a) the distribution of the field cells was clearly asymmetrical relative to the invasive portion of the tumor. If these cells had spread out centrifugally from the invasive portion, one would expect a more symmetrical distribution;(b) in several cases, we observed higher amplification levels in the invasive portion than in the in situ portion of the tumors (Table 2), which would be consistent with amplification levels increasing with progression. The finding of cells with amplifications in histologically normal skin indicates that amplifications arise even before the histologically defined stage of melanoma in situ.

Amplifications Target Oncogenes Relevant in Melanoma Pathogenesis.

The most frequently amplified region 11q13 contains the known oncogenes FGF3 and FGF4 and cyclin D1. Basic FGF is a well known and highly effective mitogen for melanocytes (17)and can serve as an autocrine growth factor in human melanoma(18). This suggests that at least some of the amplifications target genes involved in the development of the tumor.

To support the notion that amplicons in AM target oncogenes, we studied the small amplification at the tip of chromosome 11p of case AM59 in more detail (Fig. 2,C, left panel). This amplicon was in the vicinity of HRAS. An activated ras pathway has been shown to be important in melanoma formation and maintenance (19). FISH using a bacterial artificial chromosome clone containing the HRAS gene showed 10–15 copies/tumor cell. The amplification level was similar throughout the tumor, and an extended in situ portion and field cells were present (Fig. 2,C, middle panel). Sequence analysis of HRAS in this case revealed a G→T mutation of codon 12 at position 34 leading to a G12C substitution. No wild-type sequence was detected (Fig. 2 C, right panel), indicating that the mutation occurred before the amplification. AM59 was the only case with an amplification of HRAS. We checked for HRAS mutations in the other 29 melanomas, and informative HRAS sequence data were obtained from 22 tumors (12 AMs and 10 SSMs). Only case AM61 had a heterozygous A→G mutation at codon 61 of HRAS, leading to a Q61R transition (data not shown). The remaining 21 cases had wild-type sequences.

Our data demonstrate a significantly higher frequency of gene amplifications in AMs than in SSM. Other human cancers such as glioblastoma, neuroblastoma, and breast cancer have been shown to have amplifications in up to 50% of cases (13). Because amplifications in other cancers can indicate a poor prognosis(20), it is currently thought that they represent a late event in human cancer (21). In contrast, the presence of at least one amplification of a chromosomal subregion in all 16 invasive AMs studied to date (including the index case), as well as the finding of amplifications in in situ AMs, suggests that AM develops a specific amplification-permissive defect early in tumorigenesis. This might indicate a unique type of chromosomal instability that leads to increased gene dosage, analogous to the aneuploidy attributable to inactivation of mitotic spindle checkpoints in colorectal cancer (22). This view is supported by the fact that some amplicons contain genes thought to play a role in melanocyte proliferation and migration.

Gene amplifications are thought to arise through chromosomal breaks and fusions that lead to unequal gene dosage in daughter cells(23). Cells that have acquired an increased dosage of genes that convey a growth advantage are selected for and can undergo further cycles of breaks and fusions. This mechanism makes it likely that the other amplicons found in AM contain additional currently unknown genes important in melanoma pathogenesis. We are currently using CGH to microarrays of mapped clones (24) to obtain a higher resolution picture of the structure of these amplicons.

Controversy exists whether AM is part of a spectrum of cutaneous melanoma, with its features being secondary to its anatomical localization, or whether it truly represents a distinct type of melanoma. Our observation that AM uniquely exhibits frequent amplifications lends support to the view that it is a distinct entity. The exact clinicopathological criteria that define AM need to be explored further. Except for the index case (14), a subungual melanoma from the finger, all AMs of the present series were located on the foot. One of our cases showed a histological growth pattern that had overlapping features with SSM and had one amplification, possibly indicating that the amplifier phenotype is independent of histological growth pattern. The occurrence on glabrous skin and nail apparatus may be the common denominator of AM.

The standard treatment of melanoma is wide excision with a margin of clinically uninvolved skin to reduce the risk of local recurrence. The efficacy of wide excision in reducing local recurrences in melanoma can only be explained by the presence of occult tumor cells in the skin surrounding the tumor. Our finding of isolated melanocytes with amplifications up to 3 mm beyond the histologically recognizable extent of the tumors is the first direct demonstration of the presence of such cells. Although these field cells might represent micrometastatic cells derived from the invasive portion of the tumors, our data suggest that they do not. The asymmetry of their distribution relative to the invasive portion and lower amplification levels in some cases suggest that they represent a lateral expansion of the noninvasive portion. It seems likely that insufficient removal of field cells could lead to local recurrences. However, the biological potential of these cells needs to be studied further by assessing the association of their presence at the excision margins with later recurrences. We have begun to collect tissues of recurrent AMs for this purpose. The first case we examined, which recurred three times over a period of 3 years, showed field cells at the margins of all but the final excision (data not shown). Further studies are also required to determine whether they are present in other types of melanoma. This could lead to a more accurate detection of residual disease and perhaps guide the individualized determination of resection margins (25).

We thank Drs. John Ziegler, Ira Herskowitz, and Andreas Trumpp for helpful comments on the manuscript and Susan Charzan for excellent technical assistance.

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.

      
1

Supported by the Deutsche Forschungsgemeinschaft Grant Ba 794/2-1 (to B. C. B.). M. K. S. is supported by a Leaders Society Clinical Career Development Award of the Dermatology Foundation.

            
3

The abbreviations used are: AM, acral melanoma;SSM, superficial spreading melanoma; FISH, fluorescence in situ hybridization; CGH, comparative genomic hybridization;FGF, fibroblast growth factor.

Fig. 1.

Chromosomal localization of DNA sequence copy number changes in 15 AM (red lines) and 15 SSM (green lines) detected by CGH. Lines to the right of the chromosome ideograms represent gains, lines to the left represent losses. Thick lines highlighted by arrowsindicate amplifications.

Fig. 1.

Chromosomal localization of DNA sequence copy number changes in 15 AM (red lines) and 15 SSM (green lines) detected by CGH. Lines to the right of the chromosome ideograms represent gains, lines to the left represent losses. Thick lines highlighted by arrowsindicate amplifications.

Close modal
Table 1

Aberration frequencies in AM and SSM

Total losses (Average losses/case)Total gains (Average gains/case)Total amplifications (Average amplifications/case)All aberrationsa (Average aberrations/case)
AM (n=15) 76 (5.1) 67 (4.5) 30 (2.0) 162 (10.8) 
SSM (n=15) 49 (3.2) 33 (2.2) 2 (0.13) 84 (5.6) 
P (Fisher) 0.01 <0.001 <0.0001 <0.001 
Total losses (Average losses/case)Total gains (Average gains/case)Total amplifications (Average amplifications/case)All aberrationsa (Average aberrations/case)
AM (n=15) 76 (5.1) 67 (4.5) 30 (2.0) 162 (10.8) 
SSM (n=15) 49 (3.2) 33 (2.2) 2 (0.13) 84 (5.6) 
P (Fisher) 0.01 <0.001 <0.0001 <0.001 
a

Comparison of the sum of all aberrations in both groups.

Table 2

FISH amd CGH analysis of tumors with amplifications

CaseTypeCGHaFISH probebRegioncInvasive portiondIn situ portion
51 AM dim (9, 10, 14q23–qter, 15q11–14, 16q23–qter,21q), enh (3p21–pter, 6p24–pter, 8p, 17q23, 20q) amp (7p21–pter, 12q14–21) RMC12P007 RMC12B014 RMC07B3078A 12.q13.2 12q14 (MDM2) 7p21 n+++++ NA NA NA 
58 AM dim (3q21–qter, 6q, 9p22–pter, 10, 11p14–pter, 11q23–qter) enh (6p22–pter, 7, 14q31–qter) amp (3q11–13.3, 11q14–22) RMC03P005 RMC11P005 3q13 11q13.2 ++ n + n 
59 AM dim (16q24) enh (1q) amp (5q11.2–pter, 11p15, 16q22–qter) RMC05P003 RMC11B022 RMC16P003 RMC11P005 5p15.2 11p15 (HRAS) 16q21–23 11q13.2 +++++ n +++++ 
60 AM dim (3p12–qter, 5p, 6q, 9p22–pter, 10q22–qter) enh (17q, 19, 20q) amp (1p31–pter,12q14–21) RMC01P070 RMC12P007 RMC12B014 1p36.2 12q13.2 12q14 +−+++ NA NA NA 
61 AM dim (2q31–qter,6q, 9, 10) enh (1q, 4, 6p23–pter) amp (11q13) RMC06B2156 RMC11P005 6p22 11q13.2 n++ n++ 
63 AM dim (9p22–qter, 12q22–qter, 13, 14, 15) enh (1p32–34, 1q, 6p21–6q21, 8q) amp (11q13,22q11) RMC01P070 RMC11P005 RMC22P011 RMC22P004 1p36.2 11q13.2 22q12 22q13.2 (S/Sn+++++ NA NA NA NA 
68 AM dim (3q26.3–qter, 6q, 9, 10, 15q11–15,21q) enh (1q22–31, 5p14–pter, 6p, 7q,8,12pter–q15) amp (7p15–pter,22q11–12) RMC07P030 RMC07B3078A RMC11B022 RMC22P011 RMC22P004 7p21 7p21 11p15 (HRAS) 22q12 22q13.2 (S/S++++ n+++ ++++ n++ 
92 AM dim (1p35–pter, 3q11–13.3, 6q22–qter, 7q36–qter, 9p21–pter, 11q14–qter, 12q21–23, 20p) enh (5q31–qter,6p, 7p 15–pter, 17q22–qter, 18q21.2–qter) amp (11q13,22q11–13.1) RMC11B022 RMC11P005 RMC22P011 RMC22P004 11p15 11q13.2 22q12 22q13.2 +++ n++ n++ n++ 
93 AM enh (6p,20p) amp (11q13,22q11–13.1) RMC11B022 RMC11P005 RMC22P011 RMC22P004 11p15 11q13.2 22q12 22q13.2 n++ n+ n++ n+ 
94 AM dim (1p12–31.1,2q11–24,6q, 11q22–qter, 12q22–qter, 15q13–22, 16q24–qter) enh (1q32–qter, 5p, 6p, 18q, 19q13.1,20) amp (11q12–13,20q) RMC01P070 RMC11B022 RMC11P005 RMC06B2156 RMC19P006 1p36.2 11p15 11q13.2 6p22 19q12 + n+++++ n + n++++ n 
95 AM dim (9, 11q21–qter, 14q11–24.1, 18q,20p) enh (1q,6p,7,20q) amp (11q13, 16q21–22) RMC06B2156 RMC11B022 RMC11P005 RMC16P003 6p22 11p15 11q13.2 16q22–23 ++ n++++ ++ n++++ 
98 AM dim (6q) enh (1p21–qter, 6p, 7, 8q,20) amp (5p12–13,22q13) RMC01P008 RMC05B3075 RMC05B3323 RMC06B2156 RMC10B036 RMC22P011 RMC22P004 1q36 5p14 5p13 6p22 10q23 22q12 22q13.2 n n n+ n++ n n n n+ n++ n 
99 AM dim (21q11–12) enh (7,20q12–qter) amp (22q13) RMC01P070 RMC22P011 RMC22P004 1p36.2 22q12 22q13.2 n n+ n n+ 
104 AM dim (9,10q) enh (1q,6p) amp (5p22–pter, 6p23–pter) RMC05B3326 RMC06B005 RMC06B2156 5p15.3 6p25–pter 6p22 ++ n+ ++ n+ 
105 AM dim (1p21–31.2, 2p23–pter, 3p21.3–pter, 9, 10, 16q, 18) enh (1q, 6p, 7, 8q22–qter, 16p) amp (1p13, 1q32–qter,21q21–22) RMC01P070 RMC01P008 RMC06B2156 RMC11P005 RMC16B2157 RMC21P025 1p36.2 1q32.2 6p22 11q13.2 16p13 21q22 n++ n++ n++ n++ 
78 SSM dim (10q, 12q22–qter) amp (6p12–21, 6p22–24) RMC06B2156 RMC11P005 6p22 11q13.2 + n + n 
106 SSM dim (9, 11q14.2–qter), enh (1q22–qter, 13) amp (11q13) RMC01P008 RMC11P005 1q32.2 11q13.2 ++++ ++++ 
CaseTypeCGHaFISH probebRegioncInvasive portiondIn situ portion
51 AM dim (9, 10, 14q23–qter, 15q11–14, 16q23–qter,21q), enh (3p21–pter, 6p24–pter, 8p, 17q23, 20q) amp (7p21–pter, 12q14–21) RMC12P007 RMC12B014 RMC07B3078A 12.q13.2 12q14 (MDM2) 7p21 n+++++ NA NA NA 
58 AM dim (3q21–qter, 6q, 9p22–pter, 10, 11p14–pter, 11q23–qter) enh (6p22–pter, 7, 14q31–qter) amp (3q11–13.3, 11q14–22) RMC03P005 RMC11P005 3q13 11q13.2 ++ n + n 
59 AM dim (16q24) enh (1q) amp (5q11.2–pter, 11p15, 16q22–qter) RMC05P003 RMC11B022 RMC16P003 RMC11P005 5p15.2 11p15 (HRAS) 16q21–23 11q13.2 +++++ n +++++ 
60 AM dim (3p12–qter, 5p, 6q, 9p22–pter, 10q22–qter) enh (17q, 19, 20q) amp (1p31–pter,12q14–21) RMC01P070 RMC12P007 RMC12B014 1p36.2 12q13.2 12q14 +−+++ NA NA NA 
61 AM dim (2q31–qter,6q, 9, 10) enh (1q, 4, 6p23–pter) amp (11q13) RMC06B2156 RMC11P005 6p22 11q13.2 n++ n++ 
63 AM dim (9p22–qter, 12q22–qter, 13, 14, 15) enh (1p32–34, 1q, 6p21–6q21, 8q) amp (11q13,22q11) RMC01P070 RMC11P005 RMC22P011 RMC22P004 1p36.2 11q13.2 22q12 22q13.2 (S/Sn+++++ NA NA NA NA 
68 AM dim (3q26.3–qter, 6q, 9, 10, 15q11–15,21q) enh (1q22–31, 5p14–pter, 6p, 7q,8,12pter–q15) amp (7p15–pter,22q11–12) RMC07P030 RMC07B3078A RMC11B022 RMC22P011 RMC22P004 7p21 7p21 11p15 (HRAS) 22q12 22q13.2 (S/S++++ n+++ ++++ n++ 
92 AM dim (1p35–pter, 3q11–13.3, 6q22–qter, 7q36–qter, 9p21–pter, 11q14–qter, 12q21–23, 20p) enh (5q31–qter,6p, 7p 15–pter, 17q22–qter, 18q21.2–qter) amp (11q13,22q11–13.1) RMC11B022 RMC11P005 RMC22P011 RMC22P004 11p15 11q13.2 22q12 22q13.2 +++ n++ n++ n++ 
93 AM enh (6p,20p) amp (11q13,22q11–13.1) RMC11B022 RMC11P005 RMC22P011 RMC22P004 11p15 11q13.2 22q12 22q13.2 n++ n+ n++ n+ 
94 AM dim (1p12–31.1,2q11–24,6q, 11q22–qter, 12q22–qter, 15q13–22, 16q24–qter) enh (1q32–qter, 5p, 6p, 18q, 19q13.1,20) amp (11q12–13,20q) RMC01P070 RMC11B022 RMC11P005 RMC06B2156 RMC19P006 1p36.2 11p15 11q13.2 6p22 19q12 + n+++++ n + n++++ n 
95 AM dim (9, 11q21–qter, 14q11–24.1, 18q,20p) enh (1q,6p,7,20q) amp (11q13, 16q21–22) RMC06B2156 RMC11B022 RMC11P005 RMC16P003 6p22 11p15 11q13.2 16q22–23 ++ n++++ ++ n++++ 
98 AM dim (6q) enh (1p21–qter, 6p, 7, 8q,20) amp (5p12–13,22q13) RMC01P008 RMC05B3075 RMC05B3323 RMC06B2156 RMC10B036 RMC22P011 RMC22P004 1q36 5p14 5p13 6p22 10q23 22q12 22q13.2 n n n+ n++ n n n n+ n++ n 
99 AM dim (21q11–12) enh (7,20q12–qter) amp (22q13) RMC01P070 RMC22P011 RMC22P004 1p36.2 22q12 22q13.2 n n+ n n+ 
104 AM dim (9,10q) enh (1q,6p) amp (5p22–pter, 6p23–pter) RMC05B3326 RMC06B005 RMC06B2156 5p15.3 6p25–pter 6p22 ++ n+ ++ n+ 
105 AM dim (1p21–31.2, 2p23–pter, 3p21.3–pter, 9, 10, 16q, 18) enh (1q, 6p, 7, 8q22–qter, 16p) amp (1p13, 1q32–qter,21q21–22) RMC01P070 RMC01P008 RMC06B2156 RMC11P005 RMC16B2157 RMC21P025 1p36.2 1q32.2 6p22 11q13.2 16p13 21q22 n++ n++ n++ n++ 
78 SSM dim (10q, 12q22–qter) amp (6p12–21, 6p22–24) RMC06B2156 RMC11P005 6p22 11q13.2 + n + n 
106 SSM dim (9, 11q14.2–qter), enh (1q22–qter, 13) amp (11q13) RMC01P008 RMC11P005 1q32.2 11q13.2 ++++ ++++ 
a

Dim indicates losses, enh gains, and amp amplification(26).

b

The clones were selected from the laboratories resource. P at the 6th position in the probe name indicates a P1-clone, B a BAC-clone.

c

Chromosomal location of the clones, with relevant containing genes indicated in parentheses.

d

The amplification level is the ratio of the copy number of the amplified region to the copy number of the reference region. n(ratio = 1); + (ratio <3); ++ (3 ≤ ratio <5); +++ (ratio ≥5); − (ratio <1); NA (not available for study).

Fig. 2.

Analysis of three separate cases of AM. A, FISH and H&E of an in situ AM (case AM2). Left, a red-labeled clone(RMC11P005) mapping to 11q13.2 and a green-labeled reference probe for 11p15.5 (RMC11B022) show amplification of 11q13.2 in basal melanocytes with an average copy number >10(inset). FISH images represent one focal plane so that not all signals of a nucleus are visible. Right,histology shows melanoma cells confined to the basal epidermis and a dense lymphocytic infiltrate in the papillary dermis. B,FISH and H&E of case AM94. The low magnification image shows the invasive portion on the left, with in situ portion and the adjacent skin on the right. Bar, 1 cm. The panels above show higher magnification views (×16) of areas indicated by arrows. FISH was performed with the same probe combination as described in Fig. 2 A. The left top is a high magnification of the invasive portion with spindled tumor cells showing over 15 red signals and two to three green signals per nucleus by FISH. The middle top shows the in situportion with scattered basal melanocytes with large hyperchromatic nuclei of atypical shape (black arrowheads and inset in H&E panel). FISH shows multiple cells in the basal epidermis with amplifications of the red signal(white arrowheads and inset). The white line indicates the dermo-epidermal boundary. The right panel shows an area of marginal skin most distant from the invasive portion where FISH could detect cells with amplifications (field cells) indicated by white arrowheads. Histologically, basal melanocytes show no clear signs of atypia (black arrowheads and inset). C, CGH, FISH, and sequence analysis of case AM59. The left panel shows the fluorescence ratio profile of chromosome 11 as determined by CGH,indicating a focused amplification of chromosome 11p15. The green and red bars on the chromosome ideogram indicate the location of the probes used for FISH, depicted in the middle panel. The green-labeled probe for chromosome 11p15 contained the HRAS gene. FISH analysis of an area 5 mm distant from the invasive part of the tumor shows isolated basal melanocytes (field cells) with amplifications of HRAS (arrows and inset). The right panel shows the sequence profile of a portion of exon 1 of HRAS for this case with a T→G mutation of codon 12 (boxed).

Fig. 2.

Analysis of three separate cases of AM. A, FISH and H&E of an in situ AM (case AM2). Left, a red-labeled clone(RMC11P005) mapping to 11q13.2 and a green-labeled reference probe for 11p15.5 (RMC11B022) show amplification of 11q13.2 in basal melanocytes with an average copy number >10(inset). FISH images represent one focal plane so that not all signals of a nucleus are visible. Right,histology shows melanoma cells confined to the basal epidermis and a dense lymphocytic infiltrate in the papillary dermis. B,FISH and H&E of case AM94. The low magnification image shows the invasive portion on the left, with in situ portion and the adjacent skin on the right. Bar, 1 cm. The panels above show higher magnification views (×16) of areas indicated by arrows. FISH was performed with the same probe combination as described in Fig. 2 A. The left top is a high magnification of the invasive portion with spindled tumor cells showing over 15 red signals and two to three green signals per nucleus by FISH. The middle top shows the in situportion with scattered basal melanocytes with large hyperchromatic nuclei of atypical shape (black arrowheads and inset in H&E panel). FISH shows multiple cells in the basal epidermis with amplifications of the red signal(white arrowheads and inset). The white line indicates the dermo-epidermal boundary. The right panel shows an area of marginal skin most distant from the invasive portion where FISH could detect cells with amplifications (field cells) indicated by white arrowheads. Histologically, basal melanocytes show no clear signs of atypia (black arrowheads and inset). C, CGH, FISH, and sequence analysis of case AM59. The left panel shows the fluorescence ratio profile of chromosome 11 as determined by CGH,indicating a focused amplification of chromosome 11p15. The green and red bars on the chromosome ideogram indicate the location of the probes used for FISH, depicted in the middle panel. The green-labeled probe for chromosome 11p15 contained the HRAS gene. FISH analysis of an area 5 mm distant from the invasive part of the tumor shows isolated basal melanocytes (field cells) with amplifications of HRAS (arrows and inset). The right panel shows the sequence profile of a portion of exon 1 of HRAS for this case with a T→G mutation of codon 12 (boxed).

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