Genetic profile of naïve untreated primary cutaneous melanomas with a negative sentinel lymph node.

Sentinel lymph node (SLN) biopsy is typically offered to patients with primary cutaneous melanomas (PCMs) of ≥ 1 mm depth, but not all SLNs are positive using this cutoff. To ascertain whether positivity is genetically regulated, genetic analysis was performed using an augmented enrichment-based next-generation DNA and RNA sequencing assay in SLN-negative (Group 1, n = 8, mean depth 1.3 mm) and SLN-positive PCMs (controls, Group 2, n = 4, mean depth 1.4 mm). In Group 1, the mean number of mutations was 21 (range 3-48) with the most frequent mutations occurring in NF1 (75%) followed by TP53 (63%), CDKN2A and BRAF (38%), and NRAS (25%), while in Group 2, the ean number of mutations was 9.5 (range 5-18) with mutations in NRAS and BRAF being the most frequent (50%) followed by those in ATM, CDKN2A, CDKN2B, and NOTCH1 (25%). Increased frequency of NF1-inactivating mutations in Group 1 provides provocative early data that the presence of NF1 mutations might confer a less aggressive phenotype.

Neurotrophin Receptors and Perineural Invasion: Analyses in Select Lineage-Unrelated Cutaneous Malignancies With a Propensity for Perineural Invasion.

In this chapter, we parse the literature on neurotrophins that have been implicated in the pathogenesis of perineural invasion (PNI) in select lineage-unrelated malignancies. We also detail evidence linking neurotrophins and their receptors (TrkA, RET, p75NGFR, and NCAM) to the pathogenesis of PNI in desmoplastic melanoma and cutaneous squamous cell carcinoma-both malignancies with an established propensity for PNI. Lastly, the clinical potential of neurotrophins as receptors for targeted therapies is explored.

Hemostasis biomarkers and risk of sepsis: the REGARDS cohort.

Essentials Few studies have investigated the risk of sepsis by baseline hemostasis biomarkers measures. Baseline hemostasis biomarkers and risk of sepsis was examined using case-control study design. Increased fibrinogen, factor IX, and factor XI levels may be associated with risk of sepsis. Hemostasis biomarkers may provide a target for sepsis mitigation or prevention. SUMMARY: Background Sepsis is a major public health concern, responsible for more than 750 000 hospitalizations and 200 000 annual deaths in the USA. Few studies have investigated the association between baseline measurements of hemostasis biomarkers and the future risk of sepsis. Objective To determine whether hemostasis biomarkers levels measured at baseline in a cohort of community-dwelling participants are associated with the risk of future sepsis events. Methods We performed a nested case-control study within the Reasons for Geographic and Racial Differences in Stroke (REGARDS) cohort. We identified sepsis hospitalizations occurring over a 10-year period. There were 50 incident sepsis cases with baseline measurements of hemostasis (fibrinogen, factor VIII, FIX, FXI, protein C, and D-dimer). Using incidence density sampling, we matched the 50 sepsis cases with 200 controls by age, sex, and race. We used conditional logistic regression to evaluate the association between baseline hemostasis biomarkers and future sepsis events. Results Comparison of 50 sepsis cases with 200 non-sepsis controls showed that sepsis cases had lower education and income, were more likely to live in the stroke belt, had chronic lung disease, and had higher albumin level/creatinine level ratios (ACRs). Individuals with higher baseline fibrinogen levels (adjusted odds ratio [OR] per standard deviation: 1.40, 95% confidence interval [CI] 1.01-1.94), FIX levels ([OR] 1.46, 95% [CI] 1.03-2.07) and FXI levels ([OR]1.52, 95% [CI] 1.04-2.23) were more likely to experience a sepsis event. Conclusion Baseline fibrinogen, FIX and FXI levels are associated with future episodes of sepsis. Hemostasis biomarkers may provide targets for sepsis mitigation or prevention.

The CXCR4/CXCL12 axis in cutaneous malignancies with an emphasis on melanoma.

The highly metastatic and variable behavior of melanoma has accentuated the need for early detection and targeted therapy. Putative targets identified include those belonging to the extensive network of chemokines and their receptors. One such target is the chemokine receptor CXCR4, a G protein-coupled receptor with a 34 amino acid extracellular N-terminus, the primary ligand of which is CXCL12 (SDF-1, stromal derived factor-1). The ligand uniquely utilizes the N-terminus of CXCR4 for signal transduction and stimulates the protein kinase B (AKT)/mitogen activated protein kinase (MAPK) pathway. Functionally, the CXCR4/CXCL12 axis is believed to play a key role in cell migration and proliferation. Upregulation of CXCR4 and consequently dysregulation of the CXCR4/CXCL12 axis has been implicated in the progression of several lineage-unrelated malignancies including melanoma. The contributions of the CXCR4/CXCL12 axis in melanomagenesis are well documented. More recently, the potential cooperativity between the mutational status of BRAF and the CXCR4/CXCL12 axis has been shown, lending credence to the concept that both CXCR4 and CXCL12 may be putative targets for therapy in melanoma. In this review, we summarize the role of the CXCR4/CXCL12 axis in cancer progression and metastasis, with an emphasis on cutaneous malignancy, melanoma in particular. Furthermore, we discuss the effects of CXCL12 on CXCR4 expressing malignant cells in vitro and the potential prognostic utility of both CXCR4 and CXCL12 expressions. Lastly, we highlight the therapeutic potential of targeting this axis and the unique response of CXCR4 expression to anti-cancer treatments with an emphasis on melanoma.

c-myc in Kaposi's sarcoma: analyses by fluorescent in situ hybridization and immunohistochemistry.

BACKGROUND: The c-myc proto-oncogene plays a central role in the regulation of cellular transcription, differentiation, and apoptosis, and has been shown to be deregulated in many types of human cancer. Recent findings have demonstrated its amplification in select vascular neoplasms, such as secondary angiosarcomas, suggesting a role in angiogenesis as well. In vitro studies have shown that the c-Myc protein is an important regulatory molecule of spindle cell proliferation and migration in Kaposi's sarcoma (KS). OBJECTIVES: In light of these findings, our primary aim was to ascertain whether c-myc, by promoting proliferation and angiogenesis, is an essential co-factor in the aetiopathogenesis of KS. We also attempted to determine a correlation between immunohistochemical expression of the c-Myc protein and c-myc gene copy amplification using fluorescent in situ hybridization (FISH). METHODS: Samples analyzed included archival tissue of KS (n = 24). PCR for detection of Kaposi's sarcoma-associated herpesvirus DNA was performed on all samples of KS. For FISH analyses, a dual-labelled technique was employed and probes for the c-myc gene and chromosome 8 were used. The monoclonal anti-c-myc antibody, 9E10, was used for immunohistochemical analyses. RESULTS: While FISH analyses revealed no amplification of c-myc in any of the cases of KS, immunohistochemical analyses revealed positive staining for c-Myc in 13/24 cases (54%). CONCLUSIONS: Amplification of the c-myc gene was not witnessed in this preliminary study of 24 cases and thus cannot be correlated with the expression of the c-Myc protein.

S100 proteins and the skin: a review.

The structurally related, low-molecular weight S100 proteins constitute a family of proteins that possess a common basic structure allowing them to carry out a range of intracellular and extracellular functions. Unifying intracellular functions relate to regulation of proliferation, energy metabolism, calcium homeostasis, enzyme activities, cell growth and differentiation. Extracellular tasks, however, appear somewhat specific to select S100 members and include participation in innate and adaptive immune responses, tissue development and repair, and/or cell migration and chemotaxis. This review is an attempt to comprehensively summarize the function and expression of S100 proteins selectively expressed in normal skin and/or involved in diseased skin.

PTEN functions as a melanoma tumor suppressor by promoting host immune response.

Cancer cells acquire several traits that allow for their survival and progression, including the ability to evade the host immune response. However, the mechanisms by which cancer cells evade host immune responses remain largely elusive. Here we study the phenomena of immune evasion in malignant melanoma cells. We find that the tumor suppressor phosphatase and tensin homolog (PTEN) is an important regulator of the host immune response against melanoma cells. Mechanistically, PTEN represses the expression of immunosuppressive cytokines by blocking the phosphatidylinositide 3-kinase (PI3K) pathway. In melanoma cells lacking PTEN, signal transducer and activator of transcription 3 activates the transcription of immunosuppressive cytokines in a PI3K-dependent manner. Furthermore, conditioned media from PTEN-deficient, patient-derived short-term melanoma cultures and established melanoma cell lines blocked the production of the interleukin-12 (IL-12) in human monocyte-derived dendritic cells. Inhibition of IL-12 production was rescued by restoring PTEN or using neutralizing antibodies against the immunosuppressive cytokines. Furthermore, we report that PTEN, as an alternative mechanism to promote the host immune response against cancer cells, represses the expression of programmed cell death 1 ligand, a known repressor of the host immune response. Finally, to establish the clinical significance of our results, we analyzed malignant melanoma patient samples with or without brisk host responses. These analyses confirmed that PTEN loss is associated with a higher percentage of malignant melanoma samples with non-brisk host responses compared with samples with brisk host responses. Collectively, these results establish that PTEN functions as a melanoma tumor suppressor in part by regulating the host immune response against melanoma cells and highlight the importance of assessing PTEN status before recruiting melanoma patients for immunotherapies.

Neuropilin-2 gene expression correlates with malignant progression in cutaneous melanoma.

BACKGROUND: It is currently not possible to predict the metastatic potential of early-stage melanoma lesions by histological examination alone; however, a significant number of thin melanomas will progress over time to advanced disease. Molecular biomarkers that could identify patients with melanoma at high risk at the time of original diagnosis would contribute significantly to improved patient outcomes and increased survival. Neuropilin-2 (NRP2), a cell surface receptor involved in tumour-associated angiogenesis and lymphangiogenesis, has recently been shown to be expressed in melanoma. OBJECTIVES: To evaluate the potential value of NRP2 gene transcript levels as biomarkers for malignant melanoma progression. METHODS: We measured NRP2 gene expression in a panel of formalin-fixed paraffin-embedded tissue specimens consisting of naevi, primary melanomas and metastatic melanomas using quantitative reverse transcriptase-polymerase chain reaction technique. RESULTS: NRP2 levels are clearly segregated among the groups of naevi, primary and metastatic melanoma samples with a statistical trend towards increasing NRP2 gene expression correlating with disease progression. Logistic regression analysis reveals that the probability of malignant progression increases with elevated levels of NRP2 (odds ratio of 2·60 with confidence interval 1·29-5·21). Within the group of primary melanomas, there is a positive correlation (r = 0·823) between NRP2 expression and Breslow depth. This correlation was validated in an independent sample set of patients with melanoma. CONCLUSIONS: This preliminary study strongly supports the significance of NRP2 as a useful biomarker for malignant progression of melanoma, which may be useful for early identification of patients with melanoma at high risk.

Pagetoid reticulosis in a prepubescent boy successfully treated with photodynamic therapy.

Pagetoid reticulosis or Woringer-Kolopp disease (WKD) is a rare variant of mycosis fungoides, consisting of localized patches or plaques containing intraepidermal proliferations of neoplastic T cells in a pagetoid distribution (similar to that of the adenocarcinomatous cells found in Paget disease of the nipple), which typically affects middle-aged and elderly men. We report a trial of photodynamic therapy (PDT) with topical aminolaevulinic acid (ALA), carried out on a 10-year-old boy with a solitary lesion of WKD on his foot, to avoid the long-term problems associated with the typical treatments for WKD of surgery and/or local irradiation. The plaque progressively flattened during treatment, and after nine PDT sessions over 13 months, the patient was clinically free of disease. PDT may be a viable alternative to surgery and local irradiation for localized cutaneous T-cell lymphoma, including WKD, especially in younger patients.

Primary cutaneous follicle center lymphoma in the setting of chronic lymphocytic leukemia.

Primary cutaneous malignancies arising in association with chronic lymphocytic leukemia (CLL) are notable for their atypical clinical and histological presentation. We report a 69-year-old man with a 17-year history of CLL who presented for evaluation of a well-defined red to violaceous nodule with a central depressed scar on the left lower extremity. Microscopic examination of a punch biopsy revealed an infiltrate of predominantly small lymphocytes with scattered large, atypical epithelioid cells. Immunohistochemical stains revealed diffuse positive staining of the lesional cells with CD20+ and bcl-6+ and focal positive staining with bcl-2+ (negative CD10 and CD23), findings which, in conjunction with the histology, were most compatible with a diagnosis of primary cutaneous follicle center lymphoma (PCFCL). A review of the clinical charts revealed several prior biopsies with varied diagnoses. In light of the most recent biopsy findings, all previous biopsies were re-reviewed and interpreted as PCFCL arising in the setting of CLL. Features contributing to the diagnostic conundrum in this case included an atypical clinical and histological presentation, lack of pertinent clinical history and multiple presentations at different institutions.

BRAF V600E mutation and the tumour suppressor IGFBP7 in atypical genital naevi.

BACKGROUND: Atypical genital naevi (AGN) are naevi of special sites with atypical histological features that overlap with those of malignant melanoma. Activating BRAF mutations, identified in the majority of banal melanocytic naevi and cutaneous melanomas, are reportedly uncommon in naevomelanocytic proliferations in nonsun-exposed sites. We have recently shown that constitutive activation of the BRAF-MEK-ERK signalling pathway in oncogenic BRAF-positive naevi increases expression and secretion of IGFBP7, which induces senescence and apoptosis. OBJECTIVES: To ascertain the frequency of BRAF V600E mutations in AGN compared with banal naevi without atypia. An additional aim was to assess the expression of IGFBP7 in oncogenic BRAF-positive AGN. METHODS: Genomic DNA was isolated per protocol from seven genital naevi without atypia and 13 AGN for BRAF genotyping. Immunohistochemical staining for IGFBP7 was performed on all cases. RESULTS: The BRAF V600E mutation was identified in 43% of genital naevi without atypia and 23% of AGN (P = 0.61). In both groups, IGFBP7 expression was maintained in 67% of BRAF V600E-positive cases. CONCLUSIONS: The prevalence of BRAF V600E in AGN suggests that ultraviolet exposure is not essential for generating the mutation. The BRAF V600E mutational status appears to be of limited diagnostic utility in distinguishing genital naevi that exhibit atypia from those that do not. Similar to oncogenic BRAF-positive common naevi without atypia, enhanced expression of the tumour suppressor IGFBP7 in oncogenic BRAF-positive AGN supports that they are biologically inert.

Follicular porokeratosis: distinct clinical entity or histologic variant?

Various clinical subtypes of porokeratosis, clinically characterized by annular plaques with a normal or atrophic center and a distinctive keratotic ridge, are described based on the age of onset, size, number and distribution of the lesions. Follicular involvement, identified by follicular localization of cornoid lamellae, is uncommon and has only been reported in association with other subtypes such as disseminated superficial actinic porokeratosis and porokeratosis of Mibelli. We present a case of follicular porokeratosis in a 40-year-old male who presented initially with scaly red "papules" in a follicular distribution on the upper extremity. Microscopic examination of a punch biopsy specimen revealed parakeratosis confined to the follicle and mild interface change. A repeat biopsy performed in 2008 revealed identical histologic features. In terms of etiopathogenesis, a clone of cells at the base of the follicle demonstrating abnormal keratinization is not a novel concept and has been demonstrated in other porokeratotic dermatoses. However, the presence of lesions that are solely follicular based, in terms of clinical presentation and histologic findings, and static over a 3-year period favors the concept that follicular porokeratosis is a distinct clinical entity and not merely a histologic variant of the porokeratotic dermatoses described in the literature thus far.

Epidermal stem cells: practical perspectives and potential uses.

Throughout adult life, the epidermis and the hair follicle undergo a perpetual cycle of growth, regression and rest. Stem cells in the epidermis not only ensure the maintenance of epidermal homeostasis and hair regeneration, but also contribute to repair of the epidermis after injury. These stem cells lie within specific niches in the hair follicle and the epidermis. The availability of monoclonal antibodies that can be used on formalin-fixed paraffin-embedded tissue has greatly facilitated the use of this methodology as an adjunct to uncovering stem cell niches. In this review, we attempt to provide an overview of the potential markers available to identify and study stem cells in an effort to providing a better understanding of the pathogenesis of skin diseases including disorders of hair loss and malignancies. The potential uses of these markers in prognosis and in expanding the therapeutic options in several disorders will also be addressed.

Stem cell markers (cytokeratin 15, CD34 and nestin) in primary scarring and nonscarring alopecia.

BACKGROUND: Although the pathogenesis of most primary scarring alopecias is poorly understood, recent studies implicate the bulge region as a possible target. OBJECTIVES: To corroborate these results, we ascertained involvement of follicular bulge stem cells using a panel of antibodies that putatively targeted the same. METHODS: Antibodies used included anticytokeratin (CK) 15, CD34 and nestin on vertical and horizontal tissue sections of 50 cases of scarring and 34 cases of nonscarring alopecia. RESULTS: Comparing expression of these markers in scarring vs. nonscarring alopecia, CK15 was noted in the follicular bulge region in 23 of 43 (53%) vs. 27 of 27 (100%) cases and in the peripheral layer of the outer root sheath (ORS) (upper two-thirds of the follicle) in 50 of 50 (100%) vs. 34 of 34 (100%) cases; CD34 was noted in the peripheral layer of the ORS (below pilar muscle attachment) in 24 of 35 (69%) vs. 18 of 18 (100%) cases; and nestin was noted in the infundibular region in 18 of 46 (39%) vs. seven of 32 (22%) cases and in the inner aspect of the ORS (below pilar muscle attachment) in eight of 31 (26%) vs. 23 of 23 (100%) cases. CONCLUSIONS: Our findings of differential follicular localization of stem cells underscore follicular progenitor cell heterogeneity and suggest the target in scarring alopecia is not merely follicular bulge stem cells but involves stem cells in the inner and outer aspect of the ORS. Enhanced expression of nestin in the infundibular region in scarring alopecia indicates availability of an accessible, in vivo niche of potential utility as an autologous source of stem cells for therapeutic application.

RAS and RAF mutations in banal melanocytic aggregates contiguous with primary cutaneous melanoma: clues to melanomagenesis.

BACKGROUND: Distinguishing banal melanocytic aggregates contiguous with malignant melanoma can be a histological challenge but is essential because of the potential for a spurious Breslow measurement. OBJECTIVES: Our aim was to ascertain whether the histological distinction between the two relates to differences in the prevalence of mutations in genes significant in melanomagenesis. METHODS: Mutations in BRAF codon 600, NRAS1 codons 12/13, NRAS2 codons 60/61 and KRAS codons 12/13 were ascertained in 18 cases of primary cutaneous malignant melanoma contiguous with banal melanocytic aggregates using laser capture microdissection. RESULTS: Overall, 12 of 18 cases (67%) exhibited a mutation in at least one gene. BRAF V600E appeared to be the most commonly mutated gene in both the melanocytic aggregate (seven of 18, 39%) and the melanoma (four of 18, 22%). Both populations demonstrated a similar BRAF genomic profile in 11 of 18 cases (61%) (two BRAF V600E, nine BRAF-WT), a similar KRAS genomic profile in 14 of 18 cases (78%) (one KRAS G12V, 13 KRAS-WT) and a similar NRAS2 genomic profile in 14 of 18 cases (all WT). Of interest, we noted a relatively high prevalence of KRAS mutations (five of 18, 28%). The frequency of KRAS mutations in the melanocytic aggregate (five of 18, 28%) was second to BRAF V600E, while in melanoma, the frequency was also second to BRAF V600E but equalled that of NRAS2 (1 of 18, 6%). No NRAS1 mutations were observed. BRAF and RAS mutations appeared to be mutually exclusive with only three of 18 cases (17%) demonstrating a mutation in both genes (melanocytic aggregate only). CONCLUSIONS: Our findings hint towards the interpretation of banal melanocytic aggregates serving as precursor lesions.

Cutaneous neuroendocrine adenoma: an uncommon neoplasm.

BACKGROUND: Cutaneous neuroendocrine neoplasms are typically malignant. Benign cutaneous neuroendocrine tumors are uncommon. METHODS: We report the case of a 32-year-old female who presented with a granular mass in the right external auditory canal. RESULTS: Microscopic examination of a shave biopsy revealed a poorly circumscribed neoplasm with glandular differentiation. While cytologic atypia and mitotic activity were not evident, pagetoid spread was observed. Immunohistochemistry was indicative of neuroendocrine origin. CONCLUSIONS: This case report of neuroendocrine adenoma indicates that this entity should be entertained in the repertoire of conditions affecting the external ear.

In vitro fixator rod loading after transforaminal compared to anterior lumbar interbody fusion.

BACKGROUND: Cages are commonly used to assist lumbar interbody fusion. They are implanted from various approaches. In many cases internal fixators are added to provide sufficient stability. However, how the rods of these fixators are loaded and whether the kind of approach affects these loads is still unknown. The aim of this in vitro study therefore was to determine the loads acting on fixator rods and cages after anterior compared to transforaminal lumbar interbody fusion. METHODS: Six intact human lumbar spine specimens (L1-5) were loaded in a spine tester with pure moments (+/-7.5 N m) in the frontal, sagittal and transverse plane. Loading was repeated, first, after the segments L2-3 and L4-5 were instrumented either with an anterior or a transforaminal lumbar interbody fusion cage "stand alone" and, second, after additional stabilisation with an internal fixator. The rods of the fixator and the four "corners" of the cages were instrumented with strain gauges. FINDINGS: The loads transmitted through the rods were highest in lateral bending. In this loading direction an axial distraction force of in median up to 140 N, an axial compression force of up to 100 N, and a resultant bending moment of up to 1.1 N m were measured in each rod. These loads tended to be lower for the anterior compared to the transforaminal approach. For comparison, the load applied was +/-7.5 N m. The axial strains recorded in the four "corners" of the cages considerably varied from one specimen to the other. Differences in cage strain between the two approaches could not be detected. INTERPRETATION: The loads acting on the rods of the fixator were small compared to the load that was applied. Thus, other structures such as the cages or the facet joints still play an important role in load transfer. The type of approach (anterior or transforaminal) had only little effect on the loading of the rods. This also applies to the local loading of the cages, which probably more depends on the fit between cage and endplates and on the local stiffness properties of the adjacent vertebral bodies.

Multiple cellular neurothekeomas--a case report and review on the role of immunohistochemistry as a histologic adjunct.

BACKGROUND: Cellular neurothekeoma is a relatively rare, benign cutaneous neoplasm, which usually presents as a solitary papule or nodule involving the head and neck area of young adults. Multiple neurothekeomas have not, to date, been known to occur. METHODS: We report a 30-year-old, otherwise healthy, male who presented with multiple neurothekeomas (15) in the head and neck area over a period of 12 years. RESULTS: While the unifying feature of all biopsied (10 of 15) lesions was the presence of epithelioid cells--the lesions differed in their cellularity and the degree of sclerosis of the stromal component. Antigenic profiling of the lesional cells revealed expression of vimentin, NKI/C3, PGP 9.5, factor XIIIa and CD68 but not S100, HMB45, MelanA, EMA, MSA, desmin, CD57 or NGF-R. CONCLUSIONS: This case report is the first to document the occurrence of multiple cellular neurothekeomas. An unusual histologic feature of some of the biopsied lesions was the presence of a markedly sclerotic stroma.

Follicular acantholysis: a subtle clue to the early diagnosis of pemphigus vulgaris.

The histologic hallmark of fully established pemphigus vulgaris (PV), a chronic autoimmune disease of the skin and mucosa, is the presence of acantholysis induced at the suprabasal level. This case report of acantholysis restricted to the follicular epithelium as a subtle histologic manifestation of the disease, draws attention to the pitfalls encountered in the histologic diagnosis of early cases of pemphigus vulgaris.