Histological correlates of optical coherence tomography in non-melanoma skin cancer.

BACKGROUND: Non-melanoma skin cancer (NMSC) is rarely fatal but is now the most common malignancy occurring in white populations, accounting for 70% of the cost of managing skin cancer. Optical coherence tomography (OCT) has the potential to improve diagnostic accuracy and help delineate pre-surgical margins in NMSC. Its widespread clinical acceptance awaits the accumulation of evidence from studies of direct histological comparisons. METHOD: In this study, seventy-eight subjects presenting with skin lesions, including 28 NMSCs, were imaged using the VivoSight OCT scanner and a biopsy taken. Haemotoxylin and eosin stained histology sections were compared with the OCT images. RESULTS: The depth of superficial basal cell carcinoma (BCC) lesions (<1 mm) can be measured accurately using OCT. A low-strength OCT signal at the periphery of the cell nests seen in superficial and nodular BCC is identified as corresponding to cellular palisading. A weak inverse linear correlation (r(2) = 0.3) is found between the optical attenuation coefficient measured on OCT and the nuclear-cytoplasmic ratio (N/C) of cells determined from histology. CONCLUSIONS: OCT has clinical value in providing accurate dimensional measurement of superficial BCC and in identifying the presence of peripheral palisading in nodular BCC.

A genomic and expression study of AP-1 in primary cutaneous T-cell lymphoma: evidence for dysregulated expression of JUNB and JUND in MF and SS.

Activator protein 1 (AP-1) consists of a group of transcription factors including the JUN and FOS family proteins with diverse biological functions. This study assessed the genomic and expression status of the AP-1 transcription factors in primary cutaneous T-cell lymphoma (CTCL) by using immunohistochemistry (IHC), Affymetrix expression microarray, real-time reverse transcriptase-polymerase chain reaction (RT-PCR) and fluorescent in situ hybridization (FISH). IHC showed JUNB protein expression in tumor cells from 17 of 33 cases of Sezary syndrome (SS) and JUND protein expression in 16 of 23 mycosis fungoides cases. There was no correlation between JUNB and CD30 expression. However, 7 of 12 JUNB-positive SS cases expressed both phosphorylated and total extracellular signal-regulated kinase (ERK) 1/2 mitogen-activated protein kinase (MAPK) proteins. Expression microarray showed over threefold increased expression of JUNB in three of six SS patients and similar findings were also noted after re-analysis of previously published data. Real-time RT-PCR confirmed the overexpression of JUNB in four SS cases and of JUND in three of four cases. FISH showed increased JUNB copy number in four of seven SS cases. These findings suggest that deregulation of AP-1 expression in CTCL is the result of aberrant expression of JUNB and possible JUND resulting from genomic amplification and constitutive activation of ERK1/2 MAPK in this type of lymphoma.

Heterogeneous abnormalities of CCND1 and RB1 in primary cutaneous T-Cell lymphomas suggesting impaired cell cycle control in disease pathogenesis.

Upregulation of cyclin D1/B-cell leukemia/lymphoma 1 (CCND1/BCL1) is present in most mantle cell lymphomas with the t(11;14)(q13;q32) translocation. However, little is known about the abnormalities of CCND1 and its regulator RB1 in primary cutaneous T-cell lymphomas (CTCL). We analyzed CCND and RB status in CTCL using fluorescent in situ hybridization (FISH), immunohistochemistry (IHC), and Affymetrix expression microarray. FISH revealed loss of CCND1/BCL1 in five of nine Sézary syndrome (SS) cases but gain in two cases, and RB1 loss in four of seven SS cases. IHC showed absent CCND1/BCL1 expression in 18 of 30 SS, 10 of 23 mycosis fungoides (MF), and three of 10 primary cutaneous CD30+ anaplastic large-cell lymphoma (C-ALCL). Increased CCND1/BCL1 expression was seen in nine MF, seven C-ALCL, and six SS cases. Absent RB1 expression was detected in 8 of 12 MF and 7 of 9 SS cases, and raised RB1 expression in 7 of 8 C-ALCL. Affymetrix revealed increased gene expression of CCND2 in four of eight CTCL cases, CCND3 in three cases, and CDKN2C in two cases with a normal expression of CCND1 and RB1. These findings suggest heterogeneous abnormalities of CCND and RB in CTCL, in which dysregulated CCND and RB1 may lead to impaired cell cycle control.

Three cases of lymphomatoid papulosis with a CD56+ immunophenotype.

We report 3 cases of lymphomatoid papulosis (LyP) with a CD56+, cytotoxic immunophenotype. All 3 patients presented with clinical histories typical of LyP, with one patient having associated mycosis fungoides. Histologically, two cases were type A LyP and one was type B. All 3 cases demonstrated a T-cell receptor clone in lesional skin without evidence of blood involvement. The atypical lymphocytes in each of the 3 cases expressed cytotoxic granules (T-cell intracellular antigen-1+ and granzyme B+) and were CD8+ and CD56+. Expression of CD56 is associated with a poor prognosis in subcutaneous panniculitis-like T-cell lymphoma and blastic natural killer cell lymphoma. However, the two cases of CD56+ LyP previously reported and the 3 cases in this series all appear to be pursuing an indolent course with no evidence of systemic disease.

Mycosis fungoides with a CD56+ immunophenotype.

We report 3 cases of mycosis fungoides (MF) with a CD56+ cytotoxic immunophenotype. Each patient presented with a different clinical phenotype: one exhibited limited poikilodermatous patches (skin stage T1); one, widespread hypopigmented lesions (skin stage T2); and one, poikiloderma with a single cutaneous tumor (skin stage T3). MF was confirmed both histologically and by the presence of a T-cell receptor clone in lesional skin in all cases. CD56 and T-cell intracellular antigen-1 were expressed by the malignant lymphocytes in all patients and two expressed CD8. No sample demonstrated loss of the pan T-cell markers CD2 or CD3. None of the 3 developed systemic disease and T-cell receptor gene analysis of peripheral blood was polyclonal in all cases. Only 3 cases of CD56+ MF have been reported previously, none of which exhibited tumor-stage disease. Currently, the disease in our patients appears to be behaving in a manner similar to that predicted for MF with a normal immunophenotype but the prognosis has to be guarded in view of the rarity of this subtype.

Inactivation of tumor suppressor genes p15(INK4b) and p16(INK4a) in primary cutaneous B cell lymphoma.

Primary cutaneous B cell lymphomas represent a distinct group of lymphoproliferative disorders that can be distinguished from systemic lymphoma by their good response to local treatment and favorable prognosis. In systemic B cell lymphoma, inactivation of p15(INK4b) and p16(INK4a) is frequently observed and may be associated with a poor prognosis. There have been no comprehensive studies in primary cutaneous B cell lymphomas, however. Mechanisms of p15/p16 inactivation include loss of heterozygosity, homozygous deletion, promotor region hypermethylation, and point mutation. We analyzed DNA from 36 cases of primary cutaneous B cell lymphomas, four systemic B cell lymphomas, and six benign B cell lymphoproliferative infiltrates for abnormalities of p15 and p16 using microsatellite markers for 9p21, methylation specific polymerase chain reaction, and polymerase chain reaction/single stranded conformational polymorphism analysis with exon specific primers. Expression of both p15 and p16 protein was assessed by immunohistochemistry. Loss of heterozygosity at 9p21 was identified in 2 out of 36 primary cutaneous B cell lymphomas. Hypermethylation of p15 and p16 promotor regions was identified in 8 of 35 (23%) and 15 of 35 (43%) cases, respectively. In two cases p16 hypermethylation was identified in recurrent disease but not in the initial tumor. No point mutations were identified. In seven patients, however, a polymorphism was observed in exon 3 of the p16 gene. In primary cutaneous B cell lymphomas with allelic loss or promotor hypermethylation of either p15 or p16, loss of expression in tumor cells was identified in 5 of 8 and 9 of 10 cases, respectively. Our findings suggest that p15(INK4b) and p16(INK4a) biallelic gene abnormalities are common in primary cutaneous B cell lymphomas, most frequently as a result of promotor hypermethylation. The presence of abnormalities in recurrent disease in some cases suggests that inactivation of p15 and p16 may be involved in disease progression.

Microsatellite instability is associated with hypermethylation of the hMLH1 gene and reduced gene expression in mycosis fungoides.

Fifty-one mycosis fungoides samples were analyzed for microsatellite instability (MSI) using the panel of markers recommended for hereditary nonpolyposis colorectal cancer kindred and a panel we designed for cutaneous T cell lymphoma in order to compare detection rates and determine if MSI is a genome-wide phenomenon. Samples demonstrating MSI were analyzed for abnormalities of the hMLH1 gene including loss of heterozygosity, mutations, and promoter hypermethylation. MSI was detected in 16% using the hereditary nonpolyposis colorectal cancer panel and 22% with the cutaneous T cell lymphoma panel. Overall, 27% demonstrated MSI and 73% had a stable phenotype. hMLH1 gene studies did not detect loss of heterozygosity or reveal any mutations. Promoter hypermethylation was detected in nine of 14 patients with MSI, however (64%). In addition hMLH1 and hMSH2 protein expression was studied using immunohistochemical techniques. Five of nine patients with MSI and hMLH1 promoter methylation showed abnormal hMLH1 protein expression with normal hMSH2 gene expression. All other patients tested demonstrated normal hMLH1 and hMSH2 protein expression. MSI was found to be more prevalent in tumor stage mycosis fungoides (47%) than early stage disease (20%) and was associated with an older age of onset of mycosis fungoides. MSI may be a consequence of hMLH1 promoter hypermethylation in mycosis fungoides patients and may prevent transcription in a subset of patients. This suggests that the development of a mutator phenotype may contribute to disease progression in mycosis fungoides.

Reduced expression of insulin-like growth factor-binding protein-3 (IGFBP-3) in Squamous cell carcinoma complicating recessive dystrophic epidermolysis bullosa.

Squamous cell carcinoma (SCC) is a common complication in individuals with recessive dystrophic epidermolysis bullosa (RDEB). For the severe Hallopeau-Siemens subtype, the mortality rate from SCC is over 55% by the age of 40 y. Currently, little is known about the molecular pathology or cell biology of SCC in RDEB. In this study, we compared gene expression in RDEB SCC (n=3) and non-EB SCC (n=3) with corresponding RDEB and non-EB peri-tumoral skin, with microarray analysis using DermArray membranes as well as semi-quantitative and real-time RT-PCR. Both tumor sets showed downregulation of epidermal differentiation markers (e.g., profilaggrin, keratins 1 and 10) as well as certain pro-apoptotic genes (e.g., death-associated kinase-3 or ZIP kinase). Likewise, in both groups there was upregulation of matrix metalloproteinase 1 and laminin 5 in the tumors. But we found that the expression of insulin-like growth factor-binding protein-3 (IGFBP-3) was lower (mean of 5.8-fold) in RDEB SCC compared with non-EB SCC. These data were verified by immunohistochemistry. IGFBP-3 has an important role in cancer cell apoptosis mediated via the nuclear retinoid X receptor alpha (RXRalpha). Reduced expression of IGFBP-3 in RDEB SCC may provide a partial explanation for the aggressive behavior and poor prognosis of these tumors in this genodermatosis.

Frequent abnormalities of the p15 and p16 genes in mycosis fungoides and sezary syndrome.

There are few data on the molecular pathogenesis of cutaneous T cell lymphomas. A recent allelotyping study by our group identified frequent allelic loss on 9p, 10q, and 17p including losses on 9p21 in 16% of patients with mycosis fungoides and 46% with Sezary syndrome. The P15 and P16 genes are intricately linked on 9p21 and can be inactivated in melanoma and non-Hodgkin's lymphoma. We have therefore studied 76 patients with either mycosis fungoides or Sezary syndrome for abnormalities of these genes. DNA samples were analyzed for loss of heterozygosity, homozygous deletion, intragenic mutations, and promoter methylation. In addition P15 and P16 protein expression was assessed. Microsatellite analysis was informative in 73 of 76 cases: allelic loss on 9p21 was identified in 18 patients (25%), including 12 of 57 with mycosis fungoides (21%) and six of 16 with Sezary syndrome (37%). Single strand conformation polymorphism analysis of the entire coding regions of both genes did not identify any mutations, although two polymorphisms were identified including C613A, which has not previously been described. P15 and P16 gene promoter methylation was found in 45% and 29% of patients, respectively. Furthermore aberrant P15 protein expression was detected in 85% of patients analyzed with P15 gene abnormalities and abnormal P16 expression in 59% with P16 gene abnormalities. These abnormalities were not dependent on cutaneous stage of disease. This study suggests that abnormalities of the P15 and P16 genes are common in both early and advanced stages of mycosis fungoides and Sezary syndrome and that these genes may be inactivated by allelic loss and aberrant promoter methylation.

CD30(+) cutaneous lymphoma in association with atopic eczema.

BACKGROUND: Chronic atopic eczema is not regarded as a precursor of malignancy, and, to our knowledge, there has been only one previous case report of CD30(+) cutaneous lymphoma in association with atopic dermatitis. OBSERVATIONS: We report 4 cases of CD30(+) lymphoproliferative disease in young adult patients with active atopic eczema dating from early childhood. Three patients developed primary cutaneous anaplastic large cell lymphoma, of whom 2 developed systemic disease and 1 died. The other patient developed lymphomatoid papulosis type A, which resolved after withdrawal of cyclosporine therapy. No other patient had received immunosuppressive therapy. Three had been treated with phototherapy, and 2 of these patients exhibited positive immunostaining for p53 within a proportion of the tumor cell population. CONCLUSIONS: Although we have not been able to establish a causative link, we believe that the association of these 2 conditions has not occurred by chance. Biopsies of lesional skin from subjects with atopic eczema exhibit a proportion of CD30 cells, and clonal transformation of this subpopulation might account for the CD30(+) lymphomas in our patients.

Evaluation of melanocytic neoplasms: application of a pan-melanoma antibody cocktail.

Incidence of malignant melanoma (MM) is rising rapidly throughout the Western world, and the number of melanocytic lesions removed for histological assessment has increased. MM can present with a myriad of histological appearances that make diagnosis problematic, particularly when dealing with metastatic deposits. Immunohistochemical diagnosis relies on a panel of antibodies comprising polyclonal S100 protein and the monoclonal antibodies HMB 45, MART-1, tyrosinase and, to a lesser extent, NKIC3. Confirmation of problematic cases relies on the use of polyclonal S100 protein, as its sensitivity has yet to be matched by any monoclonal antibody. The introduction of a potentially valuable pan-melanoma cocktail, composed of HMB 45, MART-1 and tyrosinase, is examined in 50 primary cutaneous malignant melanomas, five desmoplastic malignant melanomas (DMM), 35 benign naevi, 20 metastatic malignant melanomas, 10 basal cell carcinomas (BCC) and 10 squamous cell carcinomas (SCC) and compared to individual immunolabelling with S100 protein, HMB 45, MART-1 and tyrosinase. All BCCs and SCCs were negative with all antibodies. S100 protein, MART-1, tyrosinase and the pan-melanoma cocktail were positive for all cases of benign naevi. HMB 45 labelled all junctional and compound naevi, five of the eight intradermal naevi and five of the seven blue naevi. All 50 primary cutaneous MMs were positive with S100 protein, 49/50 with the pan-melanoma cocktail and tyrosinase, 47/50 with MART-1 and 46/50 with HMB 45. Of the five cases of DMM, all were positive with S100 protein and three of the five were positive with HMB 45, MART-1, tyrosinase and the pan-melanoma cocktail. In the case of metastatic MM, all 20 cases were positive with S100 protein, the pan-melanoma cocktail and tyrosinase. MART-1 was positive in 19/20 cases and HMB 45 in 17/20 cases. The pan-melanoma cocktail showed a high sensitivity for all forms of MM and should be considered a complementary marker to polyclonal S100 protein. Results confirmed that currently there is no alternative antibody available to match the sensitivity of polyclonal S100 protein for immunolabelling DMM.

Human papillomavirus detection in dysplastic and malignant oral verrucous lesions.

The role of human papillomaviruses (HPV) in dysplastic and malignant oral verrucous lesions is controversial since there is a wide range in the incidence of virus detection. This study used a multi-tiered method of HPV detection using DNA in-situ hybridisation (ISH) for low- and high-risk subtypes, consensus PCR, and HPV genotype analysis in archival tissue from 20 cases of dysplastic and malignant oral verrucous lesions. The biological significance of HPV DNA detection was assessed by p16 immunohistochemistry (IHC). While 1/7 carcinomas and 5/13 dysplasias contained HPV DNA by consensus PCR and genotype analysis, all specimens were negative for low- and high-risk HPV ISH and negative for p16 IHC. Results show that although high-risk HPV DNA is detectable in a subset of these lesions, the lack of p16 overexpression suggests that the oncogenic process is not driven by HPV oncoproteins.

Amplification and overexpression of JUNB is associated with primary cutaneous T-cell lymphomas.

Primary cutaneous lymphomas (PCLs) represent a heterogeneous group of extranodal T- and B-cell malignancies. The underlying molecular pathogenesis of this malignancy remains unclear. This study aimed to characterize oncogene abnormalities in PCLs. Using genomic microarray, we detected oncogene copy number gains of RAF1 (3p25), CTSB (8p22), PAK1 (11q13), and JUNB (19p13) in 5 of 7 cases of mycosis fungoides (MF)/Sezary syndrome (SS) (71%), gains of FGFR1 (8p11), PTPN (20q13), and BCR (22q11) in 4 cases (57%), and gains of MYCL1 (1p34), PIK3CA (3q26), HRAS (11p15), MYBL2 (20q13), and ZNF217 (20q13) in 3 cases (43%). Amplification of JUNB was studied in 104 DNA samples from 78 PCL cases using real-time polymerase chain reaction. Twenty-four percent of cases, including 7 of 10 cases of primary cutaneous CD30(+) anaplastic large-cell lymphoma (C-ALCL), 4 of 14 MF, 4 of 22 SS, and 2 of 23 primary cutaneous B-cell lymphoma (PCBCL) showed amplification of JUNB, and high-level amplification of this oncogene was present in 3 C-ALCL and 2 MF cases. JUNB protein expression was analyzed in tissue sections from 69 PCL cases, and 44% of cases, consisting of 21 of 23 SS, 6 of 8 C-ALCL, 5 of 10 MF, and 9 of 21 PCBCL, demonstrated nuclear expression of JUNB by tumor cells. Overexpression of JUNB also was detected in 5 C-ALCL and 2 SS cases. These results have revealed, for the first time, amplification and expression patterns of JUNB in PCL, suggesting that JUNB may be critical in the pathogenesis of primary cutaneous T-cell lymphomas.

Genetic alterations in primary cutaneous CD30+ anaplastic large cell lymphoma.

Primary cutaneous CD30+ anaplastic large cell lymphoma (C-ALCL) represents a distinct clinical subtype of CD30+ anaplastic large cell lymphomas. The etiology and underlying molecular pathogenesis of C-ALCL remain unclear. This study aimed to investigate genetic changes in C-ALCL. Comparative genomic hybridization (CGH) analysis of 23 DNA samples from 15 C-ALCL cases identified chromosome imbalances (CI) in 10 samples from eight cases (43%). The mean number of CI per sample was 2.09 +/- 3.86, with gains (2.00 +/- 3.85) more common than losses (0.09 +/- 0.29). The most frequent CI were gains of 1/1p and 5 (50%) and 6, 7, 8/8p, and 19 (38%). Microarray-based CGH analysis of six DNA samples from five cases with CI revealed genomic imbalances (GI) in all of the cases studied. This included oncogene copy number gains of FGFR1 (8p11) in three cases, and NRAS (1p13.2), MYCN (2p24.1), RAF1 (3p25), CTSB (8p22), FES (15q26.1), and CBFA2 (21q22.3) in two cases. Real-time PCR analysis of nine DNA samples from eight cases with CI and GI detected amplifications of CTSB and RAF1 in seven cases (88%), REL (2p13p12) and JUNB (19p13.2) in six cases (75%), and MYCN and YES1 (18p11.3) in four cases (50%). Immunohistochemical staining of paraffin sections from six cases demonstrated expression of JUNB protein in five cases and BCL2 in three cases. These results reveal a consistent pattern of genetic alterations in C-ALCL and provide the molecular basis for further investigation of this disease.

Outcome in 34 patients with juvenile-onset mycosis fungoides: a clinical, immunophenotypic, and molecular study.

BACKGROUND: Mycosis fungoides (MF) is predominantly a disease of older patients, but occasionally occurs in children. The aims of the current study were to describe the clinical presentation, pathologic features, and disease progression (DP) in patients who developed MF before age 16 years. METHODS: A retrospective study was performed. Patients with juvenile-onset MF were identified from our databases. Clinical features were determined from the medical records and patient interviews. Histologic, immunohistochemical, and T-cell receptor (TCR) gene analysis was performed. RESULTS: Thirty-four patients were identified: 50% had Stage IA disease, 47% had Stage IB disease, and 3% had Stage IIA disease. The male-to-female ratio was 2:1. Clinical features included hypopigmented lesions (24%), poikiloderma (26%), pilotropic disease (9%), and disease associated with lymphomatoid papulosis (18%). Twenty-eight patients had diagnostic histology, and six patients were included on the basis of compatible histology and a TCR clone in lesional skin. A cytotoxic immunophenotype was observed in 38%, including 71% of patients with hypopigmented lesions. Overall disease-specific survival (DSS) rates at 5 and 10 years were 95% and 93%, respectively. DP rates were 5% at 5 years and 29% at 10 years. Subgroup analysis demonstrated improved DSS and reduced DP in patients with Stage IA disease, those with hypopigmented or poikilodermatous lesions, and those with associated lymphomatoid papulosis. CONCLUSIONS: The prognosis for juvenile-onset MF is similar to that of adult-onset disease. There was an overrepresentation of a cytotoxic phenotype, which was most marked in hypopigmented variants. Widespread cutaneous disease (Stage IB) indicated a less favorable outcome.

Lipoid proteinosis maps to 1q21 and is caused by mutations in the extracellular matrix protein 1 gene (ECM1).

Lipoid proteinosis (LP), also known as hyalinosis cutis et mucosae or Urbach-Wiethe disease (OMIM 247100) is a rare, autosomal recessive disorder typified by generalized thickening of skin, mucosae and certain viscera. Classical features include beaded eyelid papules and laryngeal infiltration leading to hoarseness. Histologically, there is widespread deposition of hyaline (glycoprotein) material and disruption/reduplication of basement membrane. The aetiology of LP is currently unknown. Using DNA from three affected siblings in a consanguineous Saudi Arabian family we performed genome-wide linkage and mapped the disorder to 1q21 (marker D1S498) with a two-point LOD score of 3.45 at theta = 0. A further 28 affected individuals from five other unrelated consanguineous family groups from different geographical regions also showed complete linkage and resulted in a maximum two-point LOD score of 21.85 at theta = 0. Using available markers in the interval between D1S442 and D1S305, the observed recombinants placed the gene in a 2.3 cM critical interval between D1S2344 and D1S2343 (Marshfield genetic map) corresponding to an approximately 6.5 Mb region on the UCSC physical map. Using a candidate gene approach (comparison of control versus LP gene expression in cultured fibroblasts) and subsequent direct sequencing of genomic DNA, we identified six different homozygous loss-of-function mutations in the extracellular matrix protein 1 gene (ECM1). Although the precise function of ECM1 is not known, our findings provide the first clinical indication of its relevance to skin adhesion, epidermal differentiation, wound healing, scarring, angiogenesis/angiopathy and basement membrane physiology, as well as defining the molecular basis of this inherited disorder.