Maria Angelica Selim

Pump-Probe Imaging Differentiates Melanoma from Melanocytic Nevi

Multiphoton imaging reveals chemical changes in melanoma compared to benign nevi and could enhance current clinical diagnostic protocols. A Wolf in Sheep’s Clothing It is frequently difficult to distinguish whether something is dangerous or harmless. In the case of melanoma diagnosis, a misdiagnosed lesion could have deadly consequences. Rightly, doctors err on the side of caution; however, false-positive diagnoses result in unnecessary surgeries and biopsies, as well as emotional distress for the patient. Matthews et al. have developed a new imaging technique that can distinguish melanoma from benign lesions, which in concert with current techniques could improve patient diagnosis and decrease the need for unnecessary tests. The pigment melanin is the primary determinant of skin color. There are two dominant types of melanin in melanocytic lesions: eumelanin and pheomelanin. Eumelanin, which is brown/black, is the most common biological form of melanin, whereas pheomelanin is largely responsible for red hair and freckles. The authors use a multiphoton imaging technique, pump-probe spectroscopy, to determine the ratio of these different melanins in melanocytic lesions in the context of lesion architectural and cytological features. Eumelanin was found at higher levels in melanoma compared with both dysplastic and benign nevi. When combined with pathological examination, imaging-based determination of the melanin ratio decreased the number of false-positive diagnoses compared with pathological examination alone. Moreover, this imaging technique could be used on hematoxylin and eosin–stained slides, which are currently used by pathologists for melanoma diagnosis, and may even be able to be adapted for noninvasive diagnostics. In conjunction with traditional diagnostic methods, melanocytic imaging should greatly improve doctor’s ability to sort the wolves from the sheep. Melanoma diagnosis is clinically challenging: the accuracy of visual inspection by dermatologists is highly variable and heavily weighted toward false positives. Even the current gold standard of biopsy results in varying diagnoses among pathologists. We have developed a multiphoton technique (based on pump-probe spectroscopy) that directly determines the microscopic distribution of eumelanin and pheomelanin in pigmented lesions of human skin. Our initial results showed a marked difference in the chemical variety of melanin between nonmalignant nevi and melanoma, as well as a number of substantial architectural differences. We examined slices from 42 pigmented lesions and found that melanomas had an increased eumelanin content compared to nonmalignant nevi. When used as a diagnostic criterion, the ratio of eumelanin to pheomelanin captured all investigated melanomas but excluded three-quarters of dysplastic nevi and all benign dermal nevi. Additional evaluation of architectural and cytological features revealed by multiphoton imaging, including the maturation of melanocytes, presence of pigmented melanocytes in the dermis, number and location of melanocytic nests, and confluency of pigmented cells in the epidermis, further increased specificity, allowing rejection of more than half of the remaining false-positive results. We then adapted this multiphoton imaging technique to hematoxylin and eosin (H&E)–stained slides. By adding melanin chemical contrast to H&E-stained slides, pathologists will gain complementary information to increase the ease and accuracy of melanoma diagnosis.

Proliferative nodules arising within congenital melanocytic nevi: a histologic, immunohistochemical, and molecular analyses of 43 cases.

The histopathologic interpretation of proliferative nodules (PNs) in congenital melanocytic nevi can present significant challenges as some PNs may exhibit atypical features that make the distinction from melanoma difficult. We compared histologic features, Ki-67%, PHH3, and CD117% expression levels by immunohistochemistry in 18 benign and 25 atypical PNs (from 41 patients) with that of background congenital nevi (of these 43 cases), 10 congenital nevi, and 3 dermal melanomas arising in congenital melanocytic lesions. In addition, we evaluated the presence of BRAF, GNAQ, HRAS, KRAS, and NRAS mutations in all groups using the SNaPshot Multiplex System. Follow-up was available on 19 patients (9 benign and 10 atypical PNs) (range, 2 to 20 y; median, 8 y) and all were alive with no evidence of disease. The specific histologic features of atypical PNs, such as sharp demarcation (P<0.001), expansile growth (P<0.001), epidermal effacement (P<0.001), nuclear pleomorphism (P<0.001), and increased mitoses (P<0.001), differed significantly from those of benign PNs. Immunohistochemical results showed that Ki-67% and PHH3 scores, but not CD117% expression, were significantly higher (P<0.05) in atypical PNs. Molecular analyses showed that the PNs and background congenital melanocytic nevi of the giant congenital nevi possess more frequent NRAS mutations and infrequent BRAF mutations when compared with those of the remaining cases. These findings suggest that histologic features and Ki-67 and PHH3 expression levels are the strongest parameters to distinguish between benign versus atypical PNs. The immunohistochemical results suggest that atypical PNs are distinct borderline lesions residing between benign PNs and dermal melanomas. Although numerous mutations are detected in the samples, the diagnostic use of molecular analysis in this regard is limited.

A phase 1 study of systemic ADH-1 in combination with melphalan via isolated limb infusion in patients with locally advanced in-transit malignant melanoma.

Isolated limb infusion with melphalan is a well‐tolerated treatment for patients with in‐transit extremity melanoma with an approximately 30% complete response (CR) rate. ADH‐1 is a cyclic pentapeptide that disrupts N‐cadherin adhesion complexes and when given systemically in a preclinical model of regional melphalan therapy demonstrated synergistic antitumor activity. A phase 1 dose escalation study to evaluate the safety, tolerability, pharmacokinetics, and antitumor activity of systemic ADH‐1 in combination with melphalan via isolated limb infusion in patients with in‐transit extremity melanoma was performed.

Paget Disease of the Vulva: A Histologic Study of 56 Cases Correlating Pathologic Features and Disease Course

The Duke experience with 56 vulvar Paget disease patients was analyzed emphasizing pathologic features and controversial issues. Nearly all patients were Caucasian, and their mean age was 69 years. The average length of follow-up was 5.6 years. For each case, the following histologic features were evaluated and their association with disease course was examined: pseudo-invasion, adnexal involvement, signet-ring cells, cytologic atypia, glands formation, epidermal acantholysis, parakeratosis, hyperkeratosis, and chronic inflammation. The recurrence rate after surgical management was 32%, with epidermal acantholysis being the only statistically significant risk factor. Stromal invasion occurred in 10 patients (18%), and was not a statistically significant adverse prognostic indicator, although the single patient who died of the disease had the deepest stromal invasion. Recurrence was more common after resections with positive surgical margins, but this correlation was not statistically significant. Intraoperative frozen section analysis of the margins did not reduce recurrence rate, nor was it useful in attaining permanent free margins. The Paget cells were consistently reactive with cytokeratin-7 and carcinoembryonic antigen and unreactive with S-100 protein, HMB-45, and Mart-1. In addition, the tumor cells were usually positive for mucin stains. This profile helps distinguish vulvar Paget disease from its mimics, Pagetoid squamous cell carcinoma and malignant melanoma.

Clinicopathologic study of 85 cases of melanoma of the female genitalia

BACKGROUND Melanoma of the female genitalia has poor overall prognosis. OBJECTIVE AND METHODS To examine prognostic factors influencing survival, the Duke Melanoma and Tumor Registry Databases were queried for patients who had received their clinical care at Duke University Medical Center, with a diagnosis of melanoma of the female genitalia, including vulva, vagina, and cervix, between 1970 and 2009. From this group, any available histopathologic specimens were procured for further review. RESULTS Eighty-five patients were identified. The median follow-up time was 8.8 years with 60% of the patients experiencing melanoma-related mortality at last follow-up. Survival rates at 1, 5, and 10 years were 85%, 51%, and 30%, respectively. The available histopathologic specimens from 36 cases were reviewed by a dermatopathologist (M.A.S.). Fifteen of 36 cases were notable for the presence of atypical melanocytic hyperplasia adjacent to the primary melanoma. Breslow depth, lymph node status, systemic therapy, and surgery were also examined for differences in survival distributions using the log-rank test. In general, survival was inversely correlated with Breslow depth, extent of nodal involvement, and provision of systemic therapy. A higher survival rate was observed among those who received wide local excision. Log-rank test demonstrated that survival between different decades of diagnosis was not significantly different. LIMITATIONS Because of its small sample size, this study may be underpowered. CONCLUSION Despite new treatments developed and attempted, there is no evidence that survival has improved over the past 40 years. In summary, patients with thinner melanomas amenable to surgical resection had a better prognosis than those with more extensive, metastatic disease at presentation.

Melanoma immunotherapy using mature DCs expressing the constitutive proteasome

BACKGROUND Many cancers, including melanoma, exclusively express constitutive proteasomes (cPs) and are unable to express immunoproteasomes (iPs). In contrast, mature DCs used for immunotherapy exclusively express iPs. Since proteasomes generate peptides presented by HLA class I molecules, we hypothesized that mature melanoma antigen-loaded DCs engineered to process antigens through cPs would be superior inducers of antimelanoma immunity in vivo. METHODS Subjects with metastatic melanoma were vaccinated with mature DCs transfected with RNAs encoding melanoma antigens MART1, MAGE-3, gp100, and tyrosinase. These DCs were derived from monocytes that were untransfected (Arm A; n = 4), transfected with control siRNA (Arm B; n = 3), or transfected with siRNAs targeting the 3 inducible iP subunits (Arm C; n = 5). RESULTS Vaccination stimulated antigen-specific T cell responses in all subjects, which peaked after 3-4 vaccinations, but remained elevated in Arm C subjects. Also in Arm C, circulating melanoma cell levels (as detected by quantitative PCR) fell, and T cell lytic activity against autologous melanoma was induced. In HLA-A2⁺ subjects, CD8⁺ T cells that bound tetramers loaded with cP-derived melanoma antigenic peptides were found in the peripheral blood only in Arm C subjects. Of 2 subjects with active disease (both in Arm C), one had a partial clinical response, while the other, who exhibited diffuse dermal and soft tissue metastases, had a complete response. CONCLUSION These results suggest that the efficacy of melanoma DC-based immunotherapy is enhanced when tumor antigen-loaded DCs used for vaccination express cPs. TRIAL REGISTRATION Clinicaltrials.gov NCT00672542. FUNDING Duke Clinical Research Institute/Duke Translational Medicine Institute, Duke Melanoma Consortium, and Duke University Department of Surgery.

Patterns of GRP78 and MTJ1 expression in primary cutaneous malignant melanoma

Cell surface expression of glucose-regulated protein 78 (GRP78) occurs in several types of cancer; however, its role in the behavior of primary cutaneous melanoma is not well studied. The association of cell surface GRP78 with other proteins such as MTJ1 stimulates cell proliferation. In this study, we characterized the pattern of expression of GRP78 and MTJ1 in invasive primary cutaneous melanomas and analyzed the relationships between the pattern of expression and various clinicopathological parameters. We found two patterns of GRP78 expression in invasive primary cutaneous melanoma. One pattern showed a gradual fading of protein expression from superficial to deeper levels within the same tumor. The second pattern of expression showed a similar fading with an abrupt regaining of expression at the deep invasive edge of the melanoma. These two distinct patterns of GRP78 expression correlated with both patient survival and depth of tumor invasion. A moderate MTJ1 expression was found to be associated with decreased patient survival; however, no significant associations were observed between patterns of GRP78 and MTJ1 expression. Our study (1) describes two distinct patterns of GRP78 in invasive primary cutaneous melanoma, (2) inversely correlates regain of GRP78 expression with patient survival, and (3) suggests a modifying effect of MTJ1 on GRP78 in enhancing tumor aggressiveness.

Vulvar adnexal lesions: a 32-year, single-institution review from Massachusetts General Hospital.

CONTEXT Because the skin and modified mucosal surfaces of the vulvar region contain dense apocrine glands and anogenital mammary-like glands, in addition to eccrine glands and folliculosebaceous units, benign as well as malignant lesions derived from these adnexal structures are, not surprisingly, found in the vulva. However, their incidence occurring in the vulva has not been reported, to our knowledge. OBJECTIVE To determine the incidence of various vulvar adnexal lesions. DESIGN We performed a retrospective review (1978-2010) of the cases at our institution. RESULTS A total of 189 vulvar adnexal lesions were identified. Most of these lesions were benign (133 of 189; 70%), with hidradenoma papilliferum being the most common, followed by syringoma and various types of cysts. Rare cases of tubular adenoma, poroma, spiradenoma, hidradenoma, cylindroma, sebaceoma, and trichoepithelioma were identified. Malignant adnexal neoplasms comprised the remaining 30% (56 of 189) of the cases. Extramammary Paget disease was the most common (49 of 56), and 29% (14 of 49) demonstrated an invasive component. Rare cases of basal cell carcinoma, sebaceous carcinoma, apocrine carcinoma, adenoid cystic carcinoma, and spiradenocarcinoma were identified. CONCLUSIONS In this retrospective review, we identified several benign entities that have not been previously reported on the vulva, namely pilomatricoma, poroma, spiradenoma, and sebaceoma. Hidradenoma papilliferum and extramammary Paget disease were the most common benign and malignant adnexal neoplasms, respectively. The spectrum of various vulvar adnexal lesions appears to reflect the frequency of the underlying glandular elements.

Vulvar Manifestations of Crohn's Disease

Crohns disease is an inflammatory bowel disorder with several well-known extraintestinal manifestations, such as erythema nodosum, uveitis, and arthritis. Less commonly observed are vulvar manifestations, which have primarily been discussed in case reports or small case series. These cases generally highlight patients with histopathology limited to noncaseating granulomas. As these histological findings are identified in bowel biopsies from only approximately 50% of patients with gastrointestinal Crohns disease, there is likely an under-recognition and underdiagnosis of vulvar lesions as Crohns disease manifestations. We describe the largest case series to date involving patients with vulvar Crohns disease, discuss the varied clinical presentations, and describe the histopathological findings, which include noncaseating granulomas, ulcerations, lymphatic lesions, and even dysplasia and carcinoma. Our findings underscore the importance of keeping vulvar Crohns disease on the differential diagnosis when faced with a range of vulvar symptoms and suggest that regular gynecological surveillance in patients with Crohns disease may be of benefit.

Temporal spatial expression and function of non-muscle myosin II isoforms IIA and IIB in scar remodeling

Scar contracture is believed to be caused by the cell contractility during the remodeling phase of wound healing. Cell contractility is mediated by non-muscle myosin II (NMMII) and actin, but the temporal-spatial expression profile of NMMII isoforms A and B (IIA and IIB) during the remodeling phase and the role of NMMII in scar fibroblast tissue remodeling are unknown. Human scar tissue immunostained for IIA and IIB showed that both isoforms were highly expressed in scar tissue throughout the remodeling phase of repair and expression levels returned to normal after the remodeling phase. Human scar tissue immunostained for β-, γ- and α–smooth muscle actin showed that all isoforms were consistently expressed throughout the remodeling phase of repair. The β- and γ-smooth muscle actin were widely expressed throughout the dermis, but α-smooth muscle actin was only locally expressed within the dermis. In vitro, fibroblasts explanted from scar tissue were shown to express more IIA than fibroblasts explanted from normal tissue and scar fibroblasts contracted collagen lattices to a greater extent than normal fibroblasts. Blebbistatin was used to demonstrate the function of NMMII in collagen lattice contraction. In normal tissue, fibroblasts are stress-shielded from external tensile stress by the extracellular matrix. After dermal injury and during remodeling, fibroblasts are exposed to a matrix of increased stiffness. The effect of matrix stiffness on IIA and IIB expression was examined. IIA expression was greater in fibroblasts cultured in collagen lattices with increasing stiffness, and in fibroblasts cultured on glass slides compared with polyacrylamide gels with stiffness of 1 kPa. In conclusion, NMMII and actin isoform expression changes coordinately with the remodeling phase of repair, and NMMII is increased as matrix stiffness increases. As NMMII expression increases, so does the fibroblast contractility.