Pilot study on the correlation between dermoscopic patterns and fluorescence in situ hybridization findings using whole‐slide digital imaging for acral volar melanocytic lesions

Dermoscopic evaluation of acral volar skin is helpful in differentiating malignant melanomas (MM) from benign melanocytic nevi. However, histological diagnosis remains difficult because sufficient evidence of histopathological changes to establish a diagnosis of MM are not easily obtained. The aim of the present study was to evaluate the effective use of fluorescence in situ hybridization (FISH) in the diagnosis of acral volar melanocytic lesions, and to determine whether acral volar melanocytic lesions show characteristic molecular biological features of malignant melanoma via FISH. We classified acral volar melanocytic lesions showing junctional findings into three groups: (A) parallel ridge pattern (PRP) on dermoscopic examination with melanoma in situ; (B) PRP with insufficient melanocyte proliferation and atypia to diagnose malignant melanoma using hematoxylin–eosin staining; and (C) junctional nevi. We performed FISH analysis using the same tissue section that was used for hematoxylin–eosin staining. FISH positivity was seen in 80% (4/5) of the group A sections, and in 80% (4/5) of the group B sections. One case in group C was only 0.3% over the established criteria line (63.3% > 63% in RREB1). Our results suggest that FISH using whole‐slide digital imaging may be useful in the diagnosis of early in situ MM when a typical PRP is observed in an acral volar skin lesion with non‐diagnostic histopathology.     

Distinction of conjunctival melanocytic nevi from melanomas by fluorescence in situ hybridization

A conjunctival melanocytic nevus may on occasion be difficult to distinguish from melanoma both clinically and histopathologically. An unambiguous correct diagnosis is critical because of major differences in management and prognosis. We evaluated a fluorescence in situ hybridization (FISH) assay, which has previously been shown to be of value for the diagnosis of melanocytic nevi and melanomas of the skin, using probes targeting 6p25 (RREB1), 6q23 (MYB), 11q13 (CCND1) and centromere 6 (CEP6), for its potential to assist in the distinction of conjunctival melanocytic nevi from melanomas. Four melanocytic nevi and eight melanomas of the conjunctiva were analyzed. Two of the melanomas were diagnostically problematic because of suboptimal histopathology. None of the conjunctival melanocytic nevi showed a level of chromosomal aberrations that met FISH criteria for a diagnosis of melanoma. All eight conjunctival melanomas (six unequivocal and two suspicious lesions) met FISH criteria for melanoma. Thus, results from FISH assay targeting 6p25, 6q23, 11q13 and centromere 6 correlated well with the histopathologic diagnoses and supported the histopathologic suspicion in two problem cases. The findings encourage further exploration of this technique as an ancillary method for the work‐up of conjunctival melanocytic proliferations. Busam KJ, Fang Y, Jhanwar SC, Pulitzer MP, Marr B, Abramson DH. Distinction of conjunctival melanocytic nevi from melanomas by fluorescence in situ hybridization.     

Expression of CD95 ligand in melanocytic lesions as a diagnostic marker

CD95 ligand (CD95L) potently induces apoptosis by activating CD95 on target cells. It has recently been reported that melanoma cells in vivo express a significant amount of CD95L, thereby being immediately able to kill CD95-bearing immunocompetent cells specific for cancer antigens, which infiltrate the lesions. In this study, we employed immunohistochemistry using an antibody directed against CD95L to investigate at which stage the melanoma CD95L expression is turned on. Skin biopsies of 49 lesions from 46 patients were assessed. These included benign and dysplastic naevi, melanoma in situ , stage I melanomas (Clark's level 2 or 3), advance-phase melanomas (Clark's level 4 or 5) and lymph node metastases. CD95L was expressed in all of the advance-phase melanomas as well as lymph node metastases of cutaneous origin, whereas neither melanoma in situ , benign naevi nor dysplastic naevi reacted positively with the antibody. To investigate a link between positivity and tumour size, the data were analysed on the basis of Breslow thickness, and indicated that expression was observed only when tumours were thicker than 0.75 mm. We next compared expression of CD95L and HMB-45. CD95L was positive only in melanomas in a more advanced phase than stage I, whereas HMB-45 was not only expressed in melanoma cells but also in benign pigmented naevi. This indicated the advantage of CD95L staining to diagnose melanoma. The present study indicates the significant correlation between tumorigenicity and expression of CD95L, and thereby raises the possibility that CD95L may be a useful diagnostic marker for malignant melanomas.     

Distinguishing melanocytic nevi from melanoma by DNA copy number changes: comparative genomic hybridization as a research and diagnostic tool

Cancer typically results in loosened control over genomic integrity, resulting in alterations of the genome of cancer cells. Comparative genomic hybridization (CGH) is a method that can be used on DNA extracted from routinely fixed tissue to assess the entire genome for the presence of changes in DNA copy number. CGH analysis has revealed that melanoma differs from melanocytic nevi by the presence of frequent chromosomal aberrations. In contrast, melanocytic nevi typically show no chromosomal aberrations, or have a restricted set of alterations with basically no overlap to melanoma. These marked differences between aberration patterns in melanomas and melanocytic nevi can be exploited diagnostically to classify melanocytic tumors that are ambiguous based on histopathologic assessment. In addition to potential diagnostic applications, detailed analyses of recurrent aberrations can lead to the identification of genes relevant in melanocytic neoplasia.     

The anti-MAGE antibody B57 as a diagnostic marker in melanocytic lesions

The differentiation of melanoma from certain benign melanocytic lesions on histologic grounds alone may sometimes be difficult. The anti-MAGE antibody 57B was suggested to be a useful adjunct in differentiating melanoma from nevi. Our aim was to study MAGE immunoreactivity with B57 in benign melanocytic lesions that have not been investigated to this end so far. One hundred six benign melanocytic lesions were stained with the monoclonal antibody 57B. They included deep-penetrating nevus (n = 6), desmoplastic nevus (n = 9), halo nevus (n = 10), persistent melanocytic nevus (n = 12), common blue nevus (n = 17), cellular blue nevus (n = 8), cellular blue nevus with microalveolar pattern (n = 3), desmoplastic cellular blue nevus (n = 6), epithelioid blue nevus (n = 2), sclerotic blue nevus (n = 3), and clonal nevus (n = 30). Fifty-two lesions (49%) demonstrated various patterns of MAGE immunoreactivity, with clonal nevi and deep-penetrating nevi showing the most consistent staining. In conclusion, MAGE immunoreactivity detected by the monoclonal antibody 57B in formalin-fixed, paraffin-embedded tissue can be observed in benign melanocytic lesions, and therefore this antibody cannot be used in the differential diagnosis between melanoma and nevi.     

Quantitative in situ evaluation of telomeres in fluorescence in situ hybridization-processed sections of cutaneous melanocytic lesions and correlation with telomerase activity

BACKGROUND: Telomere length is correlated with cellular ageing and immortalization processes. In some human cancers telomere length measurement has proved to be of diagnostic and prognostic value. Results comparable with the traditional terminal restriction fragment length determination by Southern blotting have been obtained in metaphase and interphase cells in some studies by fluorescence in situ hybridization (FISH) analysis; FISH additionally allows for the quantification of telomeres at the cellular level. OBJECTIVES: In this study, 32 melanocytic lesions were analysed by FISH, aiming at investigating possible telomere differences among various benign and malignant lesions and correlation with telomerase activity (TA) level. METHODS: FISH was performed on paraffin sections from six common naevi, eight Spitz naevi, 12 melanomas, six melanoma metastases and nine control samples of normal skin. Telomere mean maximum diameter (Feret max), area and number per nuclear area were calculated by image analysis on fluorescent images elaborated through KS400 and in situ imaging system (ISIS) for FISH analysis programs. Mean TA level was also calculated in all lesions and correlated with telomere parameters. RESULTS: Telomere number per nuclear area was significantly lower in melanomas and metastases than in benign common and Spitz naevi and in control skin (7 small middle dot24 +/- 3.3; 6.11 +/- 3 vs. 14.46 +/- 5.6; 16.92 +/- 7.8; and 12.59 +/- 3.4, respectively; P < 0 .001). No significant differences were found for the other telomere parameters. In common and Spitz naevi, telomere number was positively correlated with Feret max (P = 0.046 and P < 0.0001, respectively). TA was significantly higher in melanomas and metastases than in the other groups (70.18 +/- 25.2; 105.07 +/- 30 vs. 2.16 +/- 2.4; 2 .99 +/- 2.1; 2 +/- 1.2, respectively; P< or = 0. 001) and it was inversely correlated with telomere number per nuclear area in melanomas (P = 0.0041). No other significant correlations were found. CONCLUSIONS: Encouraging results have been obtained from quantitative telomere evaluation in the diagnosis of melanocytic lesions, although an analysis of a larger number of cases would be necessary to provide more reliable data. An extreme shortening of some telomeres probably results in the decrease of telomeric signals and the lower mean number of detectable telomeres in melanomas and metastases. In melanomas, telomere number per nuclear area is also inversely correlated with TA levels. Quantitative FISH of melanocytic lesions could give more specific information at the cellular level in telomere and telomerase fields of investigation.     

A case of adult T-cell leukaemia/lymphoma characterized by multiplex-fluorescence in situ hybridization, comparative genomic hybridization, fluorescence in situ hybridization and cytogenetics

Adult T-cell leukaemia/lymphoma (ATLL) is a neoplasm of mature helper (CD4) T lymphocytes. Little is known, however, about the chromosome aberrations associated with the pathogenesis of this malignancy. Using molecular cytogenetic techniques we, therefore, investigated a 44-year-old man who had a 7-year history of ATLL with cutaneous involvement mimicking primary cutaneous T-cell lymphoma. Conventional cytogenetics revealed gross chromosomal changes with chromosome numbers ranging from 71 to 82. There were structural abnormalities of chromosomes 7 and 9, partial deletions of chromosomes 1, 3, 5 and 6, and loss of chromosomes 2, 4, 9, 11–14, 21 and 22. Multiplex-fluorescence in situ hybridization (M-FISH) identified two derivative chromosomes, der(6)t(6;7)(q16;q21) and der(7)t(6;7)(q16;q21)ins(6;12)(q2?;?), and a deletion of chromosome 1p. Conventional FISH confirmed the M-FISH findings. Comparative genomic hybridization of the blood revealed gains of DNA copy number at 1q12–25, 6p24–25, 9p23, 16p13–q13, 17q11–21, 19p13 and 20q13 and loss at 11p15 while lymph nodes showed gains at 3p22–24, 3q27–29, 7q36 and 15q26 and losses at 2p24–25, 2q37, 10p14–15, 11p15, 13q33–34 and 16p13.3. No DNA copy number changes were seen in a skin lesion. These results show the extent of genetic abnormalities within this malignancy.     

巢式PCR检测石蜡包埋组织中着色霉、孢子丝菌和马尔尼菲青霉

目的 评价巢式PCR对不同深部真菌感染组织石蜡包埋标本检测的价值。方法 收集着色芽生菌病、孢子丝菌病、马尔尼菲青霉病及其他深部真菌病石蜡组织标本共44份,行组织病理观察并提取石蜡组织标本中DNA。使用针对着色霉、孢子丝菌及马尔尼菲青霉核糖体DNA特定区域的特异性巢式PCR引物,分别对所提取的真菌DNA进行扩增。分析巢式PCR对这3种病原真菌扩增的敏感性和特异性,并与组织病理检查方法比较。结果 20例着色芽生菌病组织蜡块中8例扩增阳性,10例孢子丝菌病组织蜡块中7例扩增阳性,10例马尔尼菲青霉病组织蜡块均扩增阳性,其余对照深部真菌病组织蜡块扩增均为阴性,巢式PCR检测3种真菌的敏感性分别为40%、70%和100%,特异性均达到100%。组织病理检查3种真菌的阳性率分别为95%、70%、80%。结论 巢式PCR扩增石蜡包埋组织中的真菌DNA是诊断深部真菌病的一种方法,尤其适用于诊断马尔尼菲青霉病。     

Dual‐color,break‐apart fluorescence in situ hybridization probe for distinguishing clear cell sarcoma of soft tissue from malignant melanoma

Background Clear cell sarcoma (CCS) of soft tissue is a rare soft tissue sarcoma with melanocytic differentiation and shares morphologic, immunohistochemical, ultrastructural, and molecular features with malignant melanoma (MM). Because the prognosis of CCS is much different from MM, it is important to distinguish each other by selective method. CCS is well‐recognized as having the t(12;22)(q13;q12) translocation, on the other hand MM is not. Therefore, detecting Ewing sarcoma region 1 (ESWR1) gene rearrangement can serve as a crucial diagnostic determinant. Methods Biopsy was taken from a 52‐year‐old man who reported a 3‐year history of a gradually enlarging nodule on the sole of his left foot. Routine and special stains for melanocytic markers and fluorescence in situ hybridization (FISH) evaluation using dual color break‐apart rearrangement probes specific for ESWR1 was performed for formalin‐fixed tissue. Results Neoplastic cells expressed diffuse but strong positivity for HMB45 and S100 but not for Melan‐A. Dual color, break‐apart interphase FISH revealed EWS(22q12) gene rearrangements in CCS tumor cells. Conclusion Fluorescence in situ hybridization evaluation using ESWR1 gene probe for CCS sharing clinical and histopathological characteristics with MM is a valuable tool to distinguish each other.     

FUS (16p11) gene rearrangement as detected by fluorescence in-situ hybridization in cutaneous low-grade fibromyxoid sarcoma: a potential diagnostic tool

Low-grade fibromyxoid sarcoma (LGFMS) is a rare, typically deep-seated soft tissue neoplasm with deceptively bland cytology and metastatic potential. A t(7;16)(q34;p11) translocation, yielding a FUS/CREB3L2 fusion gene, has been identified in approximately 80%-90% of deep soft tissue LGFMS. Cutaneous fibromyxoid neoplasms occur not infrequently; dermatopathologists rarely consider LGFMS in the differential diagnosis, as this lesion is uncommon in the skin. We identified a group of superficial LGFMS and a spectrum of other cutaneous fibromyxoid neoplasms and performed fluorescence in situ hybridization (FISH) to assess the frequency of FUS rearrangement. FISH for the chromosomal rearrangement of FUS (16p11), using a dual-color, break-apart probe (Abbott Molecular/Vysis, Des Plaines, IL), was performed on formalin-fixed paraffin-embedded tissue sections from superficial LGFMS (n = 6), myxomas (n = 10), and myxofibrosarcoma/myxoid malignant fibrous histiocytomas (myxoid MFH) (n = 5). One hundred nonoverlapping tumor nuclei per case were evaluated for either fused (normal) or split (translocated) signals. Of the LGFMS, 4 of 6 (67%) showed a rearrangement of FUS (range: 72%-80% positive nuclei per 100 nuclei). The other neoplasms within the differential diagnosis were devoid of any rearrangement involving FUS (range: 0%-2% positive nuclei per 100 nuclei). Our observed frequency of FUS rearrangement in superficial LGFMS is consistent with those published in the literature for more deeply seated lesions. When applied to suspicious superficial myxoid or fibromyxoid neoplasms, the FUS FISH probe in formalin-fixed paraffin-embedded tissue can be a useful ancillary technique for diagnosis of this uncommon and deceptively bland tumor.     

Fluorescence in situ hybridization as an ancillary method for the distinction of desmoplastic melanomas from sclerosing melanocytic nevi

The histopathologic distinction of desmoplastic melanomas from sclerosing (desmoplastic) melanocytic nevi can be difficult, especially when evaluating a partial or superficial biopsy. In the study reported herein, we applied and explored the use of a novel ancillary method, a four-probe fluorescence in situ hybridization (FISH) assay targeting RREB1, MYB, Cep6 and CCND1, to this diagnostic problem. Fifteen sclerosing melanocytic nevi, including desmoplastic Spitz nevi, conventional nevi with prominent stromal sclerosis and sclerotic blue nevi, as well as 15 examples of desmoplastic melanoma, were examined. None of the sclerosing melanocytic nevi showed a level of chromosomal aberrations that met FISH criteria for melanoma. Seven of the 15 desmoplastic melanomas were 'positive' (had documented chromosomal aberrations) by FISH. Thus, a positive FISH test strongly supports the diagnosis of melanoma in this context. However, in this setting a negative FISH test is of limited diagnostic value. Our findings suggest that prior reports about the high sensitivity of the FISH test for melanoma diagnosis need to be adjusted according to melanoma subtype.