Background
Fibromatosis is a group of proliferative disorders of soft tissue characterized by infiltrative pattern of growth with repeated local recurrences, and proliferation of uniform well-differentiated spindled cells (mainly myofibroblasts) with presence of a variable amount of collagen between the proliferating cells. Although the lesions of fibromatosis are often locally aggressive, they lack the capacity of metastasis. [
1,
2]. Palmar fibromatosis, also known as Dupuytren's disease, is one kind of superficial fibromatosis. There are three distinct histological phases during the ontogeny described by Luck [
3]. The proliferative phase is characterized by nodular lesion with a proliferation of myofibroblasts which express α-smooth muscle actin (α-SMA). Mitotic figures are usually infrequent, but in this phase locally prominent mitotic figures may be observed [
1]. In evolutional stage, a majority of myofibroblasts are substituted by fibroblasts and spindled cells were separated by the collagen. And in the residual phase, the nodule is replaced by scar-like tissue and therefore, no expression of α-SMA due to the diminishing of myofibroblasts.
The pathogenesis of fibromatosis is poorly understood. Whether fibromatosis are reactive or neoplastic lesions has long been controversial. One of the essential tenets in defining a neoplastic proliferation is that the cells are originated from a single clone [
4,
5]. In contrast, normal tissue and reactive proliferation are polyclonal. Several studies [
6‐
8] indicate that desmoid fibromatosis, a subtype of fibromatosis reside in the deep soft tissue, is a true type of neoplastic process instead of a polyclonal reactive proliferation. Chansky [
9] assessed the clonality of palmar fibromatosis using lesional tissue from 2 patients and the result showed that the tissues from both patients are polyclonal. However, additional cases are needed to conclude that palmar fibromatosis is reactive proliferation process. In our study, tissues from 12 female patients with palmar fibromatosis were collected and the methylation inactivation pattern on X-chromosome were evaluated to determine clonality of palmar fibromatosis.
According to the Lyon hypothesis [
10,
11], one of the two X-chromosomes in each cell is inactivated by hypermethylation during the process of embryogenesis in females and the methylation patterns are highly conserved in subsequent somatic-cell divisions. Normal tissues from women consist cells randomly carry equal frequency of paternal and maternal methylated X-chromosome and therefore, are composed of a mosaic type in methylation patterns due to the random inactivation by methylation. In contrast, each individual cell in a clone derived from a common progenitor, maintains the same sequence methylation patterns of X-chromosome inactivation and the same allele is exclusively methylated. Methylation-sensitive restriction digestion followed by Polymerase chain reaction (PCR)-based methods are used to analyze the pattern of X-chromosome inactivation. The results are informative to tissues from only female patients who are heterozygous for a defined X-linked marker gene and carry approximately balanced methylation pattern for the given allele in normal condition. The methylation-sensitive restriction endonucleases HhaI or HpaII selectively target the unmethylated gene region derived from X-chromosome. In situation of balanced random methylation from normal tissue, both alleles of the marker gene are partial insensitive to the restriction digestion and therefore, both could be amplified utilizing flanking marker gene specific primer set under PCR reaction. On the contrary, marker gene from the same progenitor, inheriting the identical methylation patterns, only the methylated allele is insensitive to the enzyme cut and therefore, could be amplified by PCR while the other unmethylated allele could not be amplified due to the sensitivity to the enzyme. We investigated the clonality of palmar fibromatosis using the X-linked human androgen-receptor gene (HUMARA) assay. HUMARA is characterized by highly polymorphic trinucleotide-repeat (CAG) sequence proximal to methylation site with high incidence of heterozygosity [
12], and the target gene amplicon is less than 300 bp. Those distinctiveness of the approach make it applicable to archival formalin-fixed tissues, which are often not amenable to analysis by a variety of alternative approaches [
13].
Discussion
The pathogenesis of palmar fibromatosis remains uncertain. The disease contains two fibrotic structures, the nodule and the cord. As the disease progresses, the nodule, which contains proliferative fibroblasts, with a high proportion being myofibroblasts, develops into the cord, a collagen-rich and acellular structure [
15,
16]. The appearance and disappearance of myofibroblasts in the lesions are similar to those observed in other tissues in which fibroblasts are present, such as granulation tissue. Interleukin-1(IL-1), platelet-derived growth factor-BB (PDGF-BB), and transforming growth factor-β (TGF-β) stimulate the growth of fibroblasts [
17,
18]. The effect of TGF-β on the proliferation of myofibroblasts has been studied in vitro [
16]. The results indicate that TGF-β could induce fibroblast proliferation and increases the expression of α-SMA.
Desmoid fibromatosis (aggressive fibromatosis) is clonal fibroblastic proliferation. Despite the lack of the ability to metastasize, local recurrence is frequent and some desmoids prove fatal due to the madly local recurrence, especially in the significant viscera. In contrast, the rate of local recurrence of palmar fibromatosis is much lower, and the process of involution, from a cellular proliferation of nodule to the acellular scar-like tissue, is also different from desmoid fibromatosis.
The present study of fibromatosis shows that clonal chromosome changes are another feature of the disease process. Wever [
19] found that the frequency of cytogenetic abnormalities varied with the type of fibromatosis. The results demonstrated that more than a half of samples from desmoid fibromatosis had clonal chromosome aberrations, and only 3 out of 28 specimens of superficial fibromatosis had aberrations. The frequent finding of clonal chromosome changes in desmoid fibromatosis confirms the neoplastic nature of demoid fibromatosis, which is in line with molecular data showing that demoid fibromatosis is monoclonal [
6‐
8]. Our results suggested that palmar fibromatosis is a polyclonal reactive proliferation. It explains the infrequent chromosome aberrations in superficial fibromatosis, and the different clinic outcome and prognosis between palmar fibromatosis and desmoid fibromatosis. The both lesions belong to subtypes of fibromatosis, however, they are hyperplasia with completely different characters, namely one is reactive proliferation while the other is monoclonal proliferation.
Although the technique of clonality analysis based on the pattern of X-chromosome inactivation is powerful, there are still certain limitations. First, the purity of the cells we investigated could be reduced by the contamination of the normal cells, such as endothelial cells of vasculatures and fatty cells. Hence, it is unclear whether the appearance of polyclonality is genuine or due to contamination. In order to minimize any possibility of contamination, we used laser cut microdissection to gain the spindled cells, which allowed for rapid and accurate acquisition of cells that we were interested in [
20‐
22], and tried to reduce the contamination of endothelial cells and fatty cells. Only a single band was observed in the sample of rectum adenocarcinoma, which was used as positive control. The result suggested that tumor cells are accurately harvested, none normal tissue components is existed, and the amount of methylation-sensitive restriction endonuclease we used is suitable, by which DNA template was completely digested. The test samples DNA were digested in identical fashion as the positive control. Second, being based on the pattern of X-chromosome inactivation, the technique of clonal analysis is only applied to female patients. The application of the lesions that are more common in men than women is limited, such as palmar fibromatosis. The third limitation is the occurrence of nonrandom X-chromosome inactivation (also known as skewing or unequal Lyonization) in healthy females. The skewed X-chromosome inactivation pattern mimics clonal derivation of cells, and makes clonality results non-informative [
23]. In our study, eight informative samples were successful in amplification after digestion with methylation-sensitive restriction endonuclease HpaII, and presented two allelic bands of approximately equal intensity. The results not only assess palmar fibromatosis is polyclonality, but also show that none of the eight patients is the skewed X-chromosome inactivation pattern.
DNA from both frozen tissues and formalin-fixed, paraffin-embedded tissues were suitable for clonality analysis [
24,
25]. In the current study, only formalin-fixed, paraffin-embedded tissues due to the rare of palmar fibromatosis in women and the limited number of specimens suitable for analysis. This approach take the advantage of the large archival paraffin-embedded specimens that spanned many years and are easy to collect. In addition, the structure of tissues and the shapes of cells on paraffin-embedded specimens were clearer than frozen tissue, which made the diagnosis more credible and isolation of spindle cells of interest more precise. However, partial DNA template were degraded and damaged during process of formalin-fixed and paraffin-embedded, therefore, relative large amounts of DNA are needed from paraffin-embedded tissues comparing with frozen tissues. In our study, we cut fifteen 10 μm thick sections from each block, which ensured the amounts of DNA template, and we extracted DNA three times with Tris-buffer-saturated phenol-chloroform and once with chloroform, which purified crude DNA samples to ensure successful PCR amplification. Nevertheless, three of the twelve samples (cases 5, 10, and 11) fail to be amplified. We analyze the possible reasons for this failure. Both of lesions from patient 10 and patient 11 were in the residual stage, which was characterized by less cellular and increased amounts of dense collagen, therefore, there were so limited amounts of DNA template that PCR failed to amply. Surely, the damage of DNA template was not excluded. However, case 5 was in the proliferative phase, we speculated that there was scant optimal DNA template, which made a failure of PCR amplification. Because of the rupture of DNA, the PCR amplicon should be controlled within less than 500 base pairs. In our study, the DNA sequence we chosen to amplified was less than 300 base pairs, which ensured the success of PCR.
Competing interests
The author(s) declare that they have no competing interests.
Authors' contributions
W.L participated in the design of the study and carried out tissue samples collection, immunohistochemisitry, microdissection, DNA extraction, PCR, and completed the preparation of the manuscript. Z.H.G conceived of the study, participate in its design and coordination, and helped draft the manuscript.