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From the Departments of Dermatology* and Pathology, and the University of California at San Francisco Comprehensive Cancer Center, University of California at San Francisco, San Francisco, California; the Department of Pathology, Children’s Memorial Hospital, Northwestern University, Chicago, Illinois; and the Department of Pathology,¶ Memorial Sloan Kettering Cancer Center, New York, New York
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Abstract |
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During the neonatal period several types of melanocytic tumors can develop within CMN, many of which are thought to be distinct from melanoma.9 These range widely in size, and can grow very fast and ulcerate.11 Even the most clinically worrisome tumors arising within the neonatal period often have a benign course and tend to stabilize or regress after a period of rapid growth. Because true melanoma can occur in the neonate, the development of any secondary proliferation in a CMN is of great concern. These lesions can be extremely difficult to classify histologically. Four different histological patterns of secondary proliferations in CMN during the neonatal period have been described:11 1) simulants of superficial spreading melanoma, in which the epidermis and superficial dermis contain large epithelioid melanocytes, sometimes with pagetoid spread in the epidermis; 2) simulants of nodular melanoma with a nodular proliferation of large melanocytes with uniform nuclei in the dermis; 3) cases described as proliferative neurocristic hamartoma, characterized by a deep dermal or subcutaneous proliferation with a variety of forms of neural or mesenchymal differentiation; and 4) true melanoma, most of which show small blast-like melanocytes with hyperchromatic nuclei, scant cytoplasm, and a high mitotic rate. We studied the chromosomal aberrations in 10 atypical nodular proliferations and compared them to conventional congenital nevi, CMN with less alarming secondary proliferative changes as well as CMN in which clear-cut melanoma developed. We show that chromosomal aberrations are common in nodular proliferations, and are absent from conventional congenital nevi. However, in contrast to melanoma in which structural chromosomal aberrations are found in the vast majority of cases, the aberrations in these secondary proliferations are predominantly numerical changes. These findings point toward a qualitatively different type of genomic instability in atypical nodular proliferations in congenital nevi possibly explaining their less aggressive behavior. The different genomic characteristics may also be useful in the classification of histopathologically ambiguous cases.
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Materials and Methods |
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Formalin-fixed, paraffin-embedded tissues from CMN and benign proliferations arising therein (groups I to V) were retrieved from the archives of the Department of Pathology, University of California at San Francisco. The patients’ ages ranged from 1 day to 20 years, the average and median ages were 3.5 and 0.3 years. All nodular proliferations were from infants 2 days to 4 months old. The cases were assigned to one of five histological groups according to the predominant histological pattern (Tables 1 and 2)
(Figure 1)
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for clinical information). As a comparison we used a group of 122 primary cutaneous melanomas that had been previously analyzed by CGH, some of which have been previously published.12,13 The average tumor thickness in this group was 3.7 mm.
Comparative Genomic Hybridization (CGH)
DNA Extraction
Tissue used for CGH analysis was selectively microdissected from regions of highest cellular density that were most representative of the particular histological group. Tumor-bearing tissue was microdissected from 30-µm sections (2 to 20 per tumor) using hematoxylin and eosin-stained sections as guidance. DNA extraction and labeling was performed as published earlier.12
CGH
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.
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 either the standard labeling or the reverse labeling resulted in a tumor/reference fluorescent ratio <0.80 or >1.2, or both the standard and the reverse labeling resulted in a tumor:reference fluorescent ratio <0.85 or >1.15.12
Immunohistochemistry
Proliferation was assessed using an antibody against Ki-67 (Mib-1, dilution 1:500; Beckman Coulter, Fullerton, CA) according to the manufacturer’s instructions. Immunoreactivity was assessed using the x20 objective and only cells that showed definitive nuclear staining were counted.
Fluorescence in Situ Hybridization (FISH)
Dual-color FISH was performed on tissue sections of the array as described previously.14 The probes for chromosome 9p21 labeled with Spectrum Orange and 9q34.1 labeled with Spectrum Green were provided by Vysis Inc. (Downers Grove, IL).
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Results |
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. In a total of 122 melanomas that we have studied by CGH we found aberrations in 116 cases (95%). Of these 116 melanomas, 111 (96%) showed at least one aberration that involved a fraction of a chromosome. Only five melanomas (4%) showed gains or losses of entire chromosomes exclusively. In contrast, of seven lesions of group IV that had any aberration, six (86%) cases had changes involving entire chromosomes only. Only one case had a change involving a partial chromosome, which was gain of chromosome 6p.
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60% of all cases. In the cases of group IV, however, losses of chromosomes 9 and 10 were less frequent. Only one case (14%) had loss of 9, and two (20%) cases had loss of 10. Gains of 10 were never observed in any of the cases in group VI or the 122 melanomas. Loss of chromosome 7 was seen in three cases of group I, but this change was not observed in any of the122 melanomas. Interestingly, one of the melanomas arising in congenital nevi (group VI) showed this aberration.
The case showing features previously described as proliferative neurocristic hamartoma (group V)11
had an aberration pattern similar to melanoma. All six cases of melanomas arising in CMN (group VI) showed multiple cytogenetic aberrations in a pattern indistinguishable from that of the melanomas not associated with congenital nevus (Table 2)
. There were multiple aberrations that frequently involved chromosomal fragments. Moreover, loss of chromosome 9 was present in all three cases. In one case (CN35) there was sufficient tissue available to allow a separate CGH analysis of the nevus part. No aberrations were found. In an additional two cases we performed FISH to look for aberrations in the nevus that CGH detected in the melanoma part. Both cases showed loss of chromosome 9, which is a frequent and probably early event in melanoma.12
FISH confirmed the loss of chromosome 9p in the melanoma part, no copy number changes of chromosome 9 were detected in the nevus part (Figure 2)
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. The patient had a severe failure to thrive and deteriorated throughout the following months. She developed a pelvic mass that led to complete pelvic obstruction requiring colostomy and she died at the age of 1.5 years. Pathology specimens from the pelvic mass and pelvic lymph nodes were suggestive of melanoma. We analyzed tumor tissue from the initial punch biopsy from the nevus (CN14/1), which did not show any chromosomal aberrations. A second biopsy from a lymph node infiltrate of nevoid melanocytes (CN14/2) showed multiple aberrations, similar to melanoma (Table 2)
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Clinical follow-up information of five cases of group IV (mean follow-up time 1.6 years) did not show any progression to melanoma (Table 2)
. Two patients of group VI died of metastatic melanoma, two had subsequent metastasis, and for the remaining two no follow-up information was available.
We studied the proliferation rate by immunohistochemical stains for Ki-67 (Table 2)
. Eight of nine group IV cases (89%) had a proliferative index of 5% or more, whereas only 1 of 10 cases of groups I and II had a labeling rate >1%. The results seem to correlate the overall higher mitotic rate the cases of groups IV and V than groups I and II.
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Discussion |
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In contrast to the frequent aberrations found in cases of group IV, the congenital nevi in groups I to III did not show any chromosomal aberrations. This is similar to our findings in the majority of Spitz nevi14 and blue nevi, including cellular and atypical variants (BC Bastian and colleagues, unpublished data), which do not show any chromosomal aberrations at the level of CGH resolution. Only a subset of Spitz nevi has an isolated gain of chromosome 11p.14
The pattern of the aberrations that we found in the atypical nodular proliferations of group IV differs from those found in primary cutaneous melanoma.12,13 Six of seven (86%) of the atypical nodular proliferations in group IV with aberrations detected by CGH had involvement of whole chromosomes only, whereas this pattern was seen in only 5% of melanomas. By contrast, 95% of melanomas, including those arising in congenital nevi, showed one or several gains or losses of chromosomal fragments. These findings indicate that the type of genomic instability in atypical nodular proliferations might differ from that predominating in melanoma. The comparison of the nevus and melanoma portions demonstrates that the chromosomal alterations are acquired during the transition to melanoma.
Genomic instability is considered as a significant factor in the pathogenesis of cancer.16 There are several patterns of genomic instability, which are reflections of different functional defects in the cancer cells. Genetic alterations can affect the structure of chromosomes, their number, or their nucleotide sequence. An example of the last pattern is microsatellite instability in hereditary nonpolyposis colorectal cancer, in which defects in the mismatch repair machinery result in changes of nucleotide sequence. However, numerical or structural chromosomal aberrations are typically lacking in this cancer.17 This is in strong contrast to other solid cancers, which virtually all show chromosomal aberrations.18 Aberrations that only affect chromosome number are thought to be because of defects of the mitotic chromosome segregation apparatus,19 whereas intra- and interchromosomal aberrations seem to be related to the generation or the repair of DNA breaks.20 Recent data from mutator mutants in yeast have shown that genes involved in S-phase checkpoint functions, recombination, and telomere addition at double-strand breaks are essential for control of chromosomal integrity.21,22 Future studies are required to determine the relevant factors in mammalian systems.
It seems logical to assume that defects that permit numerical chromosomal aberrations result in a less malignant phenotype, as compared those that promote structural aberrations, because whole chromosomes are likely to harbor some genes that provide a growth advantage along with others that provide a disadvantage for growth. In contrast, cancers bearing only the fragment carrying the advantageous genes are freed from the normally present growth-inhibiting genes on the same chromosome and would be likely to grow faster and acquire a malignant phenotype. The degree of genomic instability would increase the plasticity of the genome and permit a rapid evolution of the hallmarks of cancer.23 Tumors with a more rigid genome, such as those whose defect only permits a change in chromosome numbers, would undergo a slower progression or may not be able to acquire all features of malignancy before they undergo replicative senescence and halt progression.
The numerical aberrations in the atypical nodular proliferations position them in the spectrum between benign melanocytic nevi that tend to have no chromosomal aberrations and outright melanoma in which both numerical and structural aberrations are the rule. The aberration pattern in the atypical nodular proliferations points toward a malfunction in chromosomal segregation, possibly within the mitotic spindle checkpoint.24 Future studies are required to prove this hypothesis.
In contrast, the only case with histological features described as proliferative neurocristic hamartoma showed multiple chromosomal aberrations that involved chromosome fragments, and was genomically indistinguishable from melanoma. However, this patient was alive and free of any signs of malignancy after 15 years. Our data suggests that these lesions may in fact be neoplasms rather than hamartomas. However, more cases of this extremely rare entity need to be studied to place it within the spectrum of tumors associated with congenital nevi.
In summary our data show frequent chromosomal aberrations in atypical nodular proliferations arising in congenital nevi. These aberrations differ from those seen in melanoma in the type of aberrations (numerical aberrations in atypical nodular proliferations versus structural aberrations in melanoma) and the pattern of chromosomes involved (losses of chromosome 7 in atypical nodular proliferations, and frequent losses of chromosomes 9 and 10 in melanoma). These data indicate fundamental differences compared with melanoma, consistent with the generally benign behavior of these lesions. Genomic analysis may help in the classification of ambiguous cases.
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Footnotes |
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Supported by the Marvin and Roma Auerback Melanoma Fund.
Accepted for publication June 20, 2002.
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  K. Hoek, D. L. Rimm, K. R. Williams, H. Zhao, S. Ariyan, A. Lin, H. M. Kluger, A. J. Berger, E. Cheng, E. S. Trombetta, et al. Expression Profiling Reveals Novel Pathways in the Transformation of Melanocytes to Melanomas Cancer Res., August 1, 2004; 64(15): 5270 - 5282. [Abstract] [Full Text] [PDF]
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