Wang L. Cheung

A quantitative proteomic analysis of FFPE melanoma.

To the Editor, Currently, melanoma is diagnosed based on microscopic features, and some of these attributes, including tumor thickness, ulceration, mitotic index, and extent of lymph node involvement, have prognostic significance (1). Patients with melanoma detected at an early stage undergo surgery to remove the primary tumor, but some patients progress to advanced stage disease despite treatment (2). Thus, there is a major need for the identification of prognostic biomarkers of melanoma. Unfortunately, biomarker studies using frozen tissue from primary human tumors are problematic, due to the inherent instability and tissue heterogeneity of the samples. In contrast, formalin-fixed paraffin embedded (FFPE) tissue is very stable and can be coupled with laser microdissection for targeted sample isolation. However, harvesting enough cells and extracting the cross-linked proteins has been challenging. We describe an approach that successfully extracts sufficient amounts of protein from FFPE tissue for mass spectrometric analysis and for free quantification of identified proteins. Similar approaches have proven successful for the analysis of other FFPE samples (3,4,5). Our approach is the first described for the comprehensive analysis of melanoma and melanocytic nevi using a coupled method with gel electrophoresis and spectral counting. For this proof-of-principle analysis, FFPE patient samples were collected from a single melanocytic nevus and single example of metastatic melanoma. Approximately 100,000 cells of melanocytic nevus and metastatic melanoma lesions were harvested with a Leica AS LMD laser microdissector. Proteins were uncross-linked and extracted with the Liquid Tissue MS Protein Prep Kit (Expression Pathology). Equal amounts of the proteins were split into 3 gel lanes and were analyzed by Coomassie/SDS-PAGE, which revealed the extraction of micrograms of protein (Figure 1). Each gel lane was sliced into 17 bands of 2 mm each and digested with trypsin. Tryptic peptides from the 102 gel bands were analyzed by LC-MS/MS with a Thermo LTQ-XL mass spectrometer coupled to an Eksigent nanoLC-2D (6). We identified a total of 888 proteins (0.45% false discovery rate using a decoy database from 56,013 spectra). Relaxing the stringency of the protein identification to a false discovery rate of 1.7% provided for the identification of 1,167 unique proteins from 88,180 spectra. Figure 1 Technical triplicate analyses of metastatic melanoma and nevus samples demonstrate the reproducibility of the quantitative mass spectrometric approach for the analysis of FFPE tissue samples In order to determine whether a protein was differentially expressed between nevus and metastatic melanoma samples, a label-free approach based on spectral counting was used (7,8,9,10). Spectral count is the number of tandem mass spectra assigned to a given protein in all bands from a single gel lane. To determine the relative amount of a protein in a given gel lane, a normalized spectral abundance factor (NSAF) was calculated (7). The NSAF was calculated as follows: (NSAF)k=(SpCL)k∑i=1N(SpCL)i where k is a given protein, SpC is the spectral count, L is the length of the protein, and N is all proteins identified in the gel lane. Plotting the frequency distribution of ln(NSAF) values clearly showed that the data followed a normal distribution as indicated by the fitting of a Gaussian curve with an R2 value of 0.99 (Figure 2A). In accordance to t-test, there were 390 proteins out of 888 total proteins that were found differentially expressed (p<0.05) between metastatic melanoma and nevus lesions. The distribution of the p-values from the t-test was then divided into bins of size 0.025 and the number of proteins for each bin plotted in a bar graph (Figure 2B). The 32 most significant proteins, according to the lowest p-value from the t-test and adjusted with Bonferroni correction, were visually inspected by hierarchical clustering (Figure 2C). Two proteins of particular interest, silver and fatty acid synthase (SILV and FASN), were found in the top 10 most significant proteins and appeared as up-regulated in metastatic melanoma as compared to melanocytic nevus. SILV and FASN both have been shown to be up-regulated in cancers, which makes them appropriate validation tools for this proof-of-principle study (11,12,13). Figure 2 Label-free quantification of proteins identified from FFPE nevus and metastatic melanoma tissues For validation, melanocytic nevus and melanoma samples were stained with either SILV or FASN antibodies and the samples were scored based on the intensity of the staining and the percentage of extent (Figures 3 & 4). The intensity of staining was scored as nil (0), low (1), medium (2), or high (3). The percentage of melanocyte or melanoma cell staining was scored as 0 (no staining), 1 ( 50% staining). The intensity was multiplied by the percentage of extent and the following product is then categorized as such: 0–1 is 0; 2–3 is 1+; 4–5 is 2+; 6–9 is 3+. In general, most of benign lesions (benign nevi or dysplastic nevi) have low or no expression (0 or 1+) of SILV or FASN. Many of the melanomas have higher expression (2+ or 3+) of SILV or FAS. Table 1 shows the number of cases with each score for SILV and FASN indicating higher staining in melanoma compared to benign. Both SILV and FASN were found to be significantly different by Chi square analysis between melanoma and benign with p-values of >0.0001 and 0.0015, respectively. Figure 3 SILV is up-regulated in melanoma Figure 4 Fatty acid synthase is up-regulated in melanoma Table 1 Scoring of SILV and FASN staining In conclusion, we present an unbiased, high throughput and quantitative approach for the identification of proteins that are differentially expressed in metastatic melanoma. Using quantitative label-free mass spectrometry of laser microdissected samples, we have identified 390 proteins differentially expressed in melanoma. Two of these proteins, silver and fatty acid synthase, were validated as being up-regulated in melanoma. Our proof-of-principle analysis lays the foundation for an extensive examination of archived human melanoma tissues for the discovery of biomarkers that will help clinicians with diagnosis, prognosis and treatment of this cancer.

Amelanotic melanoma: a detailed morphologic analysis with clinicopathologic correlation of 75 cases

Amelanotic melanoma can have a varied appearance both clinically and microscopically. Here, we present our experiences with 75 cases of amelanotic melanoma defined clinically as a non‐pigmented lesion and histopathologically as a tumor lacking significant melanization. We evaluated microscopic features such as morphology, mitotic count, nuclear atypia and presence of solar elastosis. Our amelanotic melanomas exhibited the following morphology: epitheloid (72%), spindled (18.7%) or desmoplastic (5.3%). In addition, we obtained patient information and clinical presentations on most of the cases (74/75; 98.7%) and follow‐up data on 40% (30/75) of the cases. The majority of amelanotic melanomas in men were found on the trunk (13/45; 29%), head and neck (12/45; 26.7%), and lower limb (13/45; 29%) and in women were found on the lower limb (12/30; 40%), upper limb (10/30; 33.3%) and head and neck (6/30; 20%). In addition, we found that an increase in mitotic index correlated with worse survival (p < 0.026), whereas there were no differences in survival for other pathological features, such as nuclear atypia or solar elastosis. Furthermore, in cases with available tissue, all amelanotic melanoma expressed microphthalmia‐associated transcription factor and tyrosinase, suggesting that the tumor cells retained melanocytic lineage and an enzyme in melanin formation, respectively. As the occurrence of amelanotic melanoma and the expression melanoma markers were similar to pigmented melanoma, we favor that amelanotic melanoma represents a subtype of melanoma rather than poorly differentiated or de‐differentiated melanoma.

Intraoperative sentinel lymph node analysis in melanoma

The objective of this retrospective cohort study was to evaluate the sensitivity and specificity of touch preparation cytology (TPC) and frozen section (FS) histology in the intraoperative staging of melanoma.

A case of cutaneous Scedosporium infection in an immunocompromised patient

Scedosporium apiospermum, the asexual stage of Pseudoallescheria boydii, is a fungus ubiquitous in soil as well as organically polluted areas, where nitrogen‐containing compounds are abundant. It is an emerging opportunistic pathogen that can range from cutaneous to disseminated infection and can be fatal within months of diagnosis. Here we present a case of disseminated S. apiospermum infection with cutaneous manifestations in a 59‐year‐old woman with myelodysplastic syndrome, in remission from chronic lymphocytic leukemia, presented with pneumonia and deteriorating mental status. An X‐ray computed tomography scan showed three non‐contrast‐enhancing hypodensities affecting the brain. Many erythematous, indurated skin lesions, measuring 3–5 mm in diameter, were noted on her chest, shoulders and arms. Biopsies were submitted for culture and histology. Histopathologic examination revealed superficial and deep perivascular and periadnexal inflammatory infiltrates of lymphocytes and neutrophils. Scattered collections of fungal organisms were noted near the eccrine glands. The periodic acid Schiff with diastase stain showed the presence of variable sized spores and hyphae with some acute angle branching. Both tissue and blood cultures were positive for a single Scedosporium species. Histologically, eccrine or peri‐eccrine involvement by fungi may be an important finding for Scedosporium infection of the skin.

Cutaneous ciliated cyst of the scalp: a case report of a cutaneous ciliated eccrine cyst and a brief review of the literature.

Cutaneous ciliated cysts (CCC) are rare benign cysts known to occur in the lower extremities of females of reproductive age. Currently, there are 2 theories that attempt to explain the histogenesis of this rare entity. The theory of Mullerian heterotopia provides a plausible histogenetic explanation for the vast majority of CCC. A proposed alternative theory is the ciliated metaplasia of eccrine glands. We believe that previously reported cases of CCC include 2 distinct entities. We report, herein, the first case reported in the literature of a cutaneous ciliated eccrine cyst occurring on the scalp.

Misregulation of Rad50 expression in melanoma cells.

DNA double‐strand breaks are increased in human melanoma tissue as detected by histone H2AX phosphorylation. 1–3 We investigated two of the downstream effectors of DNA double‐strand breaks, Rad50 and 53BP1 (tumor suppressor p53 binding protein 1), to determine if they are altered in human primary melanoma cells. Melanoma cases showed high Rad50 staining (81.8%; 9/11) significantly more frequently than conventional or atypical melanocytic nevi (0%; 0/18). In contrast, the staining pattern for 53BP1 appears similar between melanoma and nevi. This is the first study that shows activation and misregulation of the DNA repair pathway in human melanoma cells. The staining features of Rad50, a component of an essential DNA double‐strand break repair complex, are clearly increased in melanoma cells with regards to both staining intensity and the number of positive melanoma cells. Interestingly, among the melanoma cases with increased Rad50 staining, most demonstrated cytoplasmic rather than nuclear staining (88.9%, 8/9). Further studies are needed to determine the cause of this mislocalization and its affects, if any, on DNA double‐strand break repair in melanoma.