Atypical fibroxanthoma: DNA ploidy analysis of 14 cases with possible histogenetic implications

The histogenesis of atypical fibroxanthoma (AFX) and its relationship to malignant fibrous histiocytoma (MFH) arc a subject of controversy. Many investigators have proposed that AFX may represent a reactive process, while others contend that it is a true fibrohistiocytic neoplasm, closely related to MFH. In an attempt to determine whether biologic differences between AFX and MFH may be accounted for at the cellular DNA level, we performed ploidy analysis on 14 cases of AFX by flow cytometry and compared our results with previously reported DNA analyses of MFH. Thirteen of the 14 lesions demonstrated diploid distribution of nuclear DNA, and only 1 case had an aneuploid population. This contrasts with prior data on MFH, the vast majority of which are aneuploid. Our results suggest that, despite histologic similarities, AFX may be distinguished from MFH on the basis of DNA content. These findings may be significant in understanding the biologic behavior of AFX.     

Immunohistochemical analysis of platelet-derived growth factor and its receptors in fibrohistiocytic tumors

Platelet-derived growth factor (PDGF) is known to stimulate the proliferation of fibroblasts, although the role of PDGF and its receptors in the development of fibrohistiocytic tumors has not been clarified. In this study, we investigated this role by immunohistochemically staining PDGF and PDGF β-receptors in paraffin-embedded dermatofibroma (DF), dermatofibrosarcoma protuberans (DFSP) and malignant fibrous histiocytoma (MFH) tissue. We also immunohistochemically investigated the relationship between PDGF β-receptors and CD34, which is a known immunohistochemical marker for DFSP. Immunohistochemical studies using anti-PDGF-AA or BB antibodies showed that PDGF-AA and BB was found in 20 to 40% of the tumor cells in DF, DFSP, and MFH. No definite relationship for each tumor type was found. The expression of PDGF β-receptors in DFSP and that of MFH tissue was significantly greater in comparison to DF and scar tissue. The expression of CD34 and PDGF β-receptors in DFSP was observed in identical areas. These findings suggest that autocrine or paracrine growth stimulation, through PDGF β-receptors, is related to the tumorous proliferation of fibrohistiocytic tumors, and the expression of PDGF β-receptors might play a role in the proliferation of CD34 positive tumor cells.     

Nucleolar organizer regions in fibrohistiocytic tumors of the skin

Nucleolar organizer regions (NORs) are loops of DNA which are present within the nucleoli of cells that possess ribosomal RNA (rRNA) genes. NORs are associated with proteins which can be visualized by a simple silver staining technique. As the number of NORs appears to reflect cell and nuclear activity its determination in benign, intermediate and malignant conditions could be of help in their differential diagnosis. In this study, we investigated 35 fibrohistiocytic tumors of the skin of benign, intermediate and malignant potential from 32 patients: 15 fibrous histiocytomas (FH), 5 FH with atypia (AFH), 7 atypical fibroxanthomas (AFX), 5 dermatofibrosarcoma protuberans (DFSP) and 3 malignant FH (MFH). A one-step silver technique was used on formalin or Bouin fixed specimens. Ag-NOR counts from benign conditions (FH, AFH) significantly differed from that of their intermediate-risk (AFX, DFSP) and malignant (MFH) counterparts. Furthermore, in 2 cases (one AFX; one MFH) which twice recurred, Ag-NOR counts steadily increased with time. However, a distinction could not be achieved between AFX and MFH. Although Ag-NOR is a simple and valuable technique, it does not allow a definite distinction between malignant and intermediate processes, at least as far as fibrohistiocytic tumors of the skin are concerned.     

S100A6 expression in fibrohistiocytic lesions

BACKGROUND: S100A6, an S100 calcium-binding protein, has been found in a variety of cutaneous and extracutaneous lesions including: melanocytic nevi, melanoma, some salivary gland and epithelial tumors, and malignant fibrous histiocytoma (MFH). Dermal dendrocytes (DD) in the papillary dermis of skin also express S100A6 protein. We evaluated a variety of cutaneous fibrohistiocytic lesions to determine if the immunophenotype of S100A6 positivity can be expanded to include some or all of these lesions. METHODS: Formalin-fixed, paraffin-embedded tissues from fibrous papules (FP, 20), dermatofibromas (DF, 20), dermatofibrosarcoma protuberans (DFSP, 5), atypical fibroxanthomas (AFX, 5), oral fibromas (3), digital fibroma (1), and dermatomyofibroma (1) were evaluated with antibodies to S100A6, S100B, factor XIIIa, and MAC387 using a one-hour capillary action-based immunohistochemical procedure. RESULTS: DD in 20/20 FP, 19/20 DF, and 4/4 fibromas stained positively with anti-S100A6 in a pattern similar to anti-factor XIIIa. No DFSP cases stained with anti-S100A6. Anti-S100A6 showed superior staining to anti-factor XIIIa in 4/5 AFX cases. CONCLUSIONS: The immunophenotypes of some fibrohistiocytic lesions can be expanded to include S100A6 protein. With the exception of AFX, the use of anti-S100A6 does not appear to offer added benefit over anti-factor XIIIa in the differential diagnosis of fibrohistiocytic lesions.     

Dermatofibrosarcoma protuberans is a unique fibrohistiocytic tumour expressing CD34

Dermatofibrosarcoma protuberans (DFSP) is a slow growing, locally invasive tumour whose differentiation from other fibrohistiocytic tumours sometimes poses serious diagnostic problems. We investigated CD34 expression immunohistologically in various fibrohistiocytic tumours including dermatofibroma, DFSP, malignant fibrous histiocytoma (MFH), infantile myofibromatosis, fibrosarcoma, hypertrophic scar and keloid. Among these, DFSP was unique in that tumour cells themselves expressed CD34, whereas in other tumours. CD34 expression was observed only on vascular endothelial cells amongst the tumour cells. Until now, there have been no reports of useful immunohistological markers for DFSP. CD34 expression by the tumour cells can be an extremely useful marker in establishing a definitive diagnosis of DFSP.     

Benign cellular fibrous histiocytoma with erosion of the phalanx

Fibrohistiocytic tumors are characterized by the presence of fibroblast like spindle cells and histiocytes. The benign fibrous histiocytoma (dermatofibroma, BFH) as well as the malignant dermatofibrosarcoma protuberans (DFSP) and the malignant fibrous histiocytoma (MFH) belong to this group. A recurrent painful, hard 2 cm tumor on the left hallux of a 54-year-old woman led to an erosion of the underlying phalanx. The patient had suffered from ingrown toenails for more than 10 years. Histologically there was a deep penetrating fibrohistiocytic tumor that grew in a storiform pattern with interspersed foam cells. The tumor was CD34 negative and mitoses were scarce. The diagnosis was benign cellular fibrous histiocytoma (BZFH). BZFH belong to the group of BFH with a high recurrence rate especially after incomplete removal. Damage to the underlying bone has not been reported so far.     

Clonal analysis of cutaneous fibrous histiocytoma (dermatofibroma)

BACKGROUND: Dermatofibroma (DF) or cutaneous fibrous histiocytoma is a common benign fibrohistiocytic lesion involving the dermis and subcutis. Histologically, it is subclassified into fibroblastic and histiocytoid forms. Its histogenesis is controversial. While often referred to as a neoplastic process, definite evidence of neoplasia in DF has been lacking. Alternatively, some authorities have suggested that DF is a fibrosing inflammatory process. Diagnostically, the most important question faced is the distinction from dermatofibrosarcoma protuberans (DFSP). Misdiagnosis can occur, as the early phase of DFSP can simulate DF, particularly the deep and cellular forms of DF. METHODS: To address this issue, and to investigate whether DF is in fact a neoplasm, we evaluated 31 examples of DF of various histological types in female patients and assessed clonality by analyzing X-chromosome inactivation as indicated by the methylation status of the androgen receptor gene (HUMARA). Representative cases of DFSP were analyzed for comparison. RESULTS: Among the selected 31 cases of DF, 24 cases provided intact DNA and informative polymorphism at the AR alleles, including one case of recurrent deep fibrous histiocytoma. Among these 24 cases, randomly inactivated AR alleles were observed in 17 cases including a deep, recurrent fibroblastic DF. A non-random inactivation at AR alleles was observed in seven cases, of which six cases showed either typical histiocytoid form of DF (four cases) or mixed cell types with predominant histiocytoid cell type (two cases). One fibroblastic DF also showed a monoclonal pattern. HUMARA analysis of DFSP revealed non-random inactivation of polymorphic AR alleles. CONCLUSIONS: These findings suggest that DF is a heterogeneous process. Monoclonal genotype was found in DFs with histiocytoid or mixed type with predominant histiocytoid features, suggesting that histiocytoid cells probably represent the neoplastic component. The fibroblastic form of DF may represent a reactive fibroblastic proliferation. Alternatively, it may represent a true neoplasm whose neoplastic cell type has been obscured by prominent reactive fibroblastic component.     

Aneuploidy in atypical fibroxanthoma: DNA content quantification of 10 cases by image analysis

It has recently been reported that atypical fibroxanthoma (AFX) is a predominantly diploid lesion in contrast to malignant fibrous hystiocytoma (MFH) which is usually aneuploid. To test this hypothesis, DNA content quantification was undertaken on Feulgen-stained cytology and tissue section preparations from 10 cases of AFX by image analysis. The large-atypical cells which characterize AFX were aneuploid in each case. Smaller spindle-shaped cells found in this lesion were diploid. The results suggest that AFX is indistinguishable from MFH by DNA content estimation and highlight an advantage of image analysis over (low cytometry.     

色素痣和黑素瘤的DNA含量与病理形态学及预后的关系

应用流式细胞术（ＦＣＭ）定量研究２５例ＰＮ、２９例ＭＭ及８例皮肤的ＤＮＡ含量和细胞增殖特性。结果表明ＭＭ的ＳＰＦ和ＰＩ显著高于ＰＮ和正常皮肤（Ｐ＜０．０１），二倍体ＭＭ的ＳＰＦ和ＰＩ明显高于ＰＮ（Ｐ＜０．０５），ＤＩ、ＰＩ与ＭＭ组织类型部分有关，ＭＭ的核分裂指数和血管数目与ＤＩ、ＰＩ呈正相关（Ｐ＜０．０５）。提示黑素瘤的ＦＣＭ检测可以鉴别其良恶性，在一定程度上与其组织病理指标相关。     

The expression levels of thrombospondin-1 in dermatofibroma and dermatofibrosarcoma protuberans

Dermatofibroma (DF) and dermatofibrosarcoma protuberans (DFSP) are benign or intermediate malignant fibrotic dermal tumors. The contribution of transforming growth factor β (TGF-β) in the progression of sclerosis in fibrotic diseases has been implicated. To clarify the involvement of TGF-β signaling in the pathogenesis of DF or DFSP, we investigated the expression of thrombospondin-1 (TSP-1), which mediates TGF-β1 activation, on those fibrotic tumors. In the present study, we examined the expression of TSP-1 in DF and DFSP using immunohistochemical analysis and immunoblotting. In immunohistochemical staining, the expression of TSP-1 was detected weakly on epidermis and epidermal appendages, and hardly in fibroblasts in normal skin sections. The expression levels of TSP-1 were elevated in DFSP cells in comparison to normal dermal sections or DF cells. On the other hand, there was no significant difference in the protein levels in vitro of TSP-1 among normal fibroblasts, DF cells, and DFSP cells. Although the contribution of TGF-β signaling to DF or DFSP is still unknown, the expression patterns of TSP-1 in DF cells and DFSP cells may be helpful in differential diagnosis of these tumors.     

Immunohistochemical study of membrane type-matrix metalloproteinases (MT-MMPs) and matrix metalloproteinase-2 (MMP-2) in dermatofibroma and malignant fibrous histiocytoma

Matrix metalloproteinases (MMPs) play an important role in tumor invasion and metastasis. Enhanced expression of matrix metalloproteinase-2 (MMP-2) has been demonstrated in dermatofibroma (DF) and malignant fibrous histiocytoma (MFH). MMP-2 has been shown to be activated by membrane-type MMPs (MT-MMPs). To study the role of MT-MMP in the activation of MMP-2, skin specimens of DF (five cases) and MFH (three cases) were immunohistochemically studied using in situ zymography and the antibodies against matrix metalloproteinase-2 (MMP-2) and membrane type 1-3-MMPs (MT1-3-MMPs). Both MMP-2 activity and its expression were significantly activated in the tumor cells in DF and MFH. Anti-MT2-MMP strongly reacted with tumor cells of all cases of DF and MFH, whereas anti-MT1 or 3-MMP antibody showed a weak reaction in some cases of DF and MFH. Double immunofluorescence labeling demonstrated that the immunoreactive cells with anti-MMP-2 antibody in DF and MFH consistently reacted with anti-MT2-MMP antibody. The results suggest that the activation of MMP-2 in the benign and malignant fibrous tumors is related to the activation of MT-MMPs.     

Tissue cultures of benign and malignant fibrous histiocytomas: SEM observations

Four dermatofibromas (DF), 2 dermatofibrosarcomata protuberans (DFSP), 2 atypical fibroxanthmas of the skin (AFX), and one malignant fibrous histiocytoma (MFH) were studied by explant culture technique and scanning electron microscopy. Differences in the cellular atypism, phagocytic activity and motility were observed between histiocyte-like cells extending from a DF and DFSP group and an AFX and MFH group. Such cytological characteristics was maintained during in vitro transformation of the cells into fibroblastic cells. It was concluded that culture behaviors of the cells from each tumor group correlated well with in vivo growth and histologic features. We feel that examination of m vitro morphology of fibrous histiocytomas may prove useful in arriving at a correct diagnosis.     

Applications of DNA Flow Cytometry and Fluorescence In situ Hybridization Using a Chromosome-specific DNA Probe on Paraffin-embedded Tissue Sections of Primary Malignant Melanomas

We have applied DNA flow cytometric analysis to paraffin-embedded tissue sections of primary malignant melanomas. Conventionally, flow cytometric analysis of paraffin-embedded tissue sections has been done by the method of Hedley et al. We added ultrasound treatment to the method of Hedley et al. and a lower value of coefficient of variation was shown. Furthermore, a new technique, fluorescence in situ hybridization with a chromosome-specific repetitive DNA probe, was used for the analysis of chromosomal numerical aberrations in the same paraffin-embedded tissue sections. The DNA flow cytometric analysis showed that in 8 cases six primary malignant melanomas were of the aneuploid pattern and two cases of lentigo maligna (melamona in situ) were of the diploid pattern. By fluorescence in situ hybridization, the two cases with the diploid pattern had spots/nucleus of 1.28 and 1.12, and those with the aneuploid pattern had spots/nucleus from 2.01 to 2.27. Only one nodular melanoma in an aneuploid case showed spots/nucleus of 1.71. These data indicate that fluorescence in situ hybridization with chromosome-specific repetitive DNA probes can serve as a cytogenetic tool for the analysis of interphase nuclei of solid human tumors and may be useful for the study of tumor cell heterogeneity.     

Porokeratosis palmaris et plantaris disseminata. Report of a case with abnormal DNA ploidy in lesional epidermis.

BACKGROUND--Porokeratosis is believed to be a premalignant condition of the skin. Recent flow cytometric studies showing DNA aneuploidy in the epidermis of some types of porokeratosis support this conclusion. We describe a patient with porokeratosis palmaris et plantaris disseminata in whom abnormal DNA ploidy was found in lesional epidermis with the use of flow cytometry. OBSERVATIONS--DNA flow cytometry of lesional epidermis from the back showed two populations of cells, one with diploid and the other with aneuploid DNA content and DNA index of 1.1 (hyperdiploid). The coefficient of variation of the diploid and aneuploid peaks was 2.1% and 4.1%, respectively. CONCLUSIONS--Porokeratosis palmaris et plantaris disseminata, like other forms of porokeratosis, exhibits abnormal DNA ploidy in lesional epidermis. This finding underscores the premalignant nature of this and other forms of porokeratosis.     

Application of DNA flow cytometry from paraffin-embedded tissue to the diagnosis of mycosis fungoides

The distinction of mycosis fungoides from benign inflammatory lesions is sometimes difficult by conventional histological techniques. Aneuploidy, a feature often associated with malignant tumors, can be assessed even in tissue routinely processed in paraffin using the flow cytometric technique of Hedley and associates. In many tumor systems, there are significant diploid clones. We have evaluated the flow cytometric DNA ploidy of paraffin-embedded tissue in the diagnosis and prognosis of mycosis fungoides (MF). We studied 22 cases of MF and 10 control cases of inflammatory skin lesions with epidermal involvement. Aneuploidy was found in 27% of the MF cases, but in none of the controls (ED). Aneuploid features were seen in 23% of tissues from early stage disease. Aneuploidy did not correlate with atypia, epidermotropism, or number of mitoses. There was a trend towards showing adverse outcome in those patients with aneuploid lesions. The detection of aneuploidy might be helpful for early diagnosis of MF.     

Flow cytometric DNA analysis of classic and steroid-induced Kaposi's sarcoma

Summary Flow cytometric DNA analysis of various tumours has indicated a correlation between the degree of malignancy and ploidy; results which could have clinical significance. We analysed the ploidy of Kaposi's sarcoma (KS) tumours, and classified the results according to clinical history and histological findings. We found that patients on steroid treatment had an aneuploid pattern, and most of the patients with classic-type KS had a diploid pattern on flow cytometry.     

DNA ploidy of malignant melanoma determined by image cytometry of fresh frozen and paraffin-embedded tissue

Image analysis of DNA content was performed from single nuclei of melanoma monolayer imprints made from fresh frozen tissue of 14 patients with primary malignant melanoma and 16 patients with local recurrences at the incision site and local or distant metastases. This procedure requires fewer cells and is an advantage when the quantity of tumor available is limited, especially in thin low Breslow depth cutaneous melanomas. Image analysis allowed reproducible measurement of DNA ploidy from 100 cells. The frequency of aneuploidy was similar in primary and metastatic melanomas. Three of 3 patients with euploid primary melanomas showed no evidence of recurrences or metastases, though one died of unrelated disease with short follow-up. The 4 patients with primary melanoma who developed metastases had aneuploid primaries; two of these patients died of metastatic disease. Three of 4 patients with euploid metastatic tumors were free of disease at last follow-up, and 1 patient died with stable disease. Nine of 12 patients with aneuploid tumors died of metastatic disease. The frequency of DNA ploidy in the present image analysis study correlated with previous flow cytometry studies. In 9 patients with primary tumors with a Breslow depth greater than 0.75 mm, the DNA content was also determined in nuclei obtained from formalin-fixed paraffin-embedded tissue. The frequency of aneuploidy was higher in fresh tissue (7 of 9) as compared with paraffin-embedded tissue of the same cases (4 of 9).     

Significance of DNA ploidy in cutaneous lesions.

BACKGROUND--With increasing numbers of studies examining tumor DNA content in many organ systems, accumulating evidence indicates that DNA ploidy analysis may be useful in diagnosis and determination of prognosis. OBSERVATIONS--A review of DNA ploidy studies of cutaneous lesions within the last decade reveals that DNA ploidy may aid in the diagnosis of some lesions. Several studies show an association of DNA ploidy with prognosis. In general, tumors with a diploid DNA content have a less aggressive course than aneuploid tumors. CONCLUSIONS--DNA ploidy cannot yet reliably predict the outcome of an individual patient. This is partially due to differences in methods of specimen preparation, instrumentation, and interpretation of data.