NEJM
Monoclonal Origin of Multicentric Kaposi's Sarcoma Lesions
Charles S. Rabkin, M.D., Siegfried Janz, M.D., Alex Lash, M.D., Allen E. Coleman, Elizabeth Musaba, M.D., Lance Liotta, M.D., Ph.D., Robert J. Biggar, M.D., and Zhengping Zhuang, M.D., Ph.D.

ABSTRACT
Background Kaposi's sarcoma has features of both hyperplastic proliferation and neoplastic growth. Multiple lesions, in which spindle cells are prominent, often arise synchronously over widely dispersed areas. We tested the hypothesis that the spindle cells in these multicentric lesions originate from a single clone of precursor cells.

Methods To determine whether Kaposi's sarcoma is a monoclonal disorder, we assessed the methylation patterns of the androgen-receptor gene (HUMARA) in multiple lesions from women with the acquired immunodeficiency syndrome. In polyclonal tissues, about half the copies of each HUMARA allele are methylated, whereas in cells derived from a single clone all the copies of only one allele are methylated. To minimize contamination by normal DNA, we used microdissection to isolate areas composed primarily of spindle cells, the putative tumor cells.
Results Eight patients with a total of 32 tumors were studied. Of these tumors, 28 had highly unbalanced methylation patterns (i.e., predominant methylation of one HUMARA allele). In all the tumors that had unbalanced methylation from a given patient, the same allele predominated.

Conclusions These data indicate that Kaposi's sarcoma is a disseminated monoclonal cancer and that the changes that permit the clonal outgrowth of spindle cells occur before the disease spreads.

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BRIEF POINTS of the article

Kaposi's sarcoma is the most common tumor in patients with the acquired immunodeficiency syndrome (AIDS).1,2 The lesions associated with the disease have four characteristic components: thin-walled neovascular formations, extravasated red cells, inflammatory lymphocytes, and proliferating spindle cells. The spindle cells, which may be the primary abnormality, are more prominent in nodular tumors than in plaque or patch lesions. Although their origin is unknown, spindle cells have the characteristic immunohistochemical and ultrastructural features of endothelial cells.3,4 The neoplastic or hyperplastic nature of spindle cells has long been debated,5,6 but the demonstration that individual Kaposi's sarcoma lesions are clonal proliferations supports the idea that Kaposi's sarcoma is a neoplasm.7
In affected patients multiple lesions can appear synchronously in widely dispersed areas, without evidence of a primary tumor as a source of metastasis.
Therefore, a currently favored hypothesis is that the individual lesions of Kaposi's sarcoma arise in situ by neoplastic transformation of local precursors.8
We tested this idea by analyzing multiple lesions from the same patient to determine their clonal relatedness.
We assessed clonal relatedness in Kaposi's sarcoma cells from women by studying patterns of X-chromosome methylation.9,10 Etc........................................

For the marker gene, we used the X-linked androgen-receptor gene (HUMARA), which has a highly polymorphic (with a greater than 90 percent prevalence of heterozygosity) trinucleotide-repeat sequence proximal to a methylation site and is thus suitable for study by an assay of clonality based on the polymerase chain reaction (PCR).11 We used a methylation-sensitive restriction endonuclease (HpaII) to digest unmethylated HUMARA sequences before PCR amplification; both alleles of the gene are amplified in randomly methylated tissue, whereas in clonal tissue one allele is absent because all its copies are unmethylated (Figure 1).
Figure 1. Clonality Assay of the HUMARA Gene.
In women, each somatic cell contains two X chromosomes, one derived from the father (red) and the other derived from the mother (blue). One of the X chromosomes is inactivated by methylation (yellow halo), but the other is active and unmethylated. Normal somatic tissue (upper rows) is a mosaic of cells. In some cells the maternally derived X chromosome is methylated, and in others the paternally derived chromosome is methylated. In tumor tissue (lower rows) the same X chromosome is methylated in all cells (the maternally derived chromosome, in this example). DNA from methylated chromosomes resists digestion by methyl-sensitive restriction endonucleases such as Hpa II, which preferentially digest unmethylated DNA sequences. After Hpa II digestion, DNA from normal tissue contains a mixture of maternal and paternal X-chromosome sequences, whereas DNA from clonal tissue contains one or the other, but not both.
Over 90 percent of people are heterozygous for the number of trinucleotide repeats in exon 1 of the X-linked androgen-receptor gene (HUMARA). After amplification by PCR of the region containing the repeats (delineated by red lines), the maternal and paternal HUMARA alleles can be separated by polyacrylamide-gel electrophoresis and visualized as bands on autoradiography. DNA from normal tissue, which contains a mixture of two methylated alleles, generates bands of approximately equal intensity for each (upper gel). DNA from a monoclonal population of cells generates a single band that corresponds to its one methylated allele (lower gel). In practice, tumors usually contain some polyclonal stromal cells and generate a second band, of diminished intensity.   
Clonality was determined with an adaptation13 of the HUMARA methylation assay.11 In brief, 10 units of the methyl-sensitive restriction endonuclease HpaII was added to 5 µl of the DNA-preparation mixture, incubated at 37°C for one to three hours, and then inactivated by heat at 95°C for five minutes. Half this solution was then added to a 10-µl reaction mixture for PCR amplification of the HUMARA gene with the primer pair 'GCTGTGAAGGTTGCTGTTCCTCAT3' and 5'TCCAGAATCTGTTCCAGAGCGTGC3'.14etc............................................................ .
Results
Two of the 10 patients were excluded from further analysis because they were homozygous for the HUMARA gene. The eight heterozygous patients had from 7 to 20 CAG trinucleotide repeats at that locus, with a minimal difference of 3 repeats (i.e., 9 base pairs) between the two alleles (Table 1). In two cases, there was scant DNA from normal dermis that could not be amplified after digestion with a restriction endonuclease. Digested normal DNA from the other six patients contained two allelic bands of approximately equal intensity. Hence, balanced methylation of normal-dermis DNA was demonstrated in six of the eight patients etc.........................................


Discussion
We have demonstrated concordance among the patterns of methylation of the X-linked HUMARA alleles in different Kaposi's sarcoma tumors from a given female patient. This finding indicates that multiple Kaposi's sarcoma lesions in the same patient arise from a single clone of cells. This evidence argues that Kaposi's sarcoma is a disseminated monoclonal canceretc...........................................................
Our data imply that each lesion of Kaposi's sarcoma arises from a monoclonal population of circulating progenitor cells that home to multiple local sites and proliferate. The circulating cells are potentially related to the spindle-shaped cells that can be cultured from peripheral blood, whose concentration is increased in HIV-infected patients who have Kaposi's sarcoma or are at high risk for it.22 We do not know whether this process occurs in the endemic or transplantation-associated cases of Kaposi's sarcoma. In addition, it remains to be demonstrated whether the neoplastic clone persists over time in recurrent Kaposi's sarcoma, as has been demonstrated with regard to B-lymphocyte neoplasms.23 These data also warrant the examination of Kaposi's sarcoma lesions for other genetic changes, such as mutations, rearrangements, amplifications, deletions, and allelic losses, to further our understanding of this enigmatic disorder.

We are indebted to George Chibwe for the recruitment of patients; to David Waters, Ph.D., and James Watson for assistance with specimens; to Miriam Anver, D.V.M., Ph.D., for histologic diagnoses; to Ana Albuquerque and Galina Kovalchuk, M.D., for performing assays; and to James J. Goedert, M.D., for helpful discussions.