Andrew Emley

Laser Capture Microdissection: Methods and Applications

Laser microdissection is a nonmolecular, minimally disruptive method to obtain cytologically and/or phenotypically defined cells or groups of cells from heterogeneous tissues. It is a versatile technology and allows the preparation of homogenous isolates of specific subpopulations of cells from which RNA/DNA or protein can be extracted for RT-polymerase chain reaction (PCR), quantitative PCR, Western blot analyses, and mass spectrophotometry.

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.

Mixed versus pure variants of desmoplastic melanoma: a genetic and immunohistochemical appraisal

Desmoplastic melanoma is subclassified into pure and mixed variants with a higher rate of lymph node metastasis in the latter. Given that reasons for these biological differences are not currently known, we investigated these subtypes with techniques that included genetic and immunohistochemical analyses of 43 cases of desmoplastic melanoma (24 pure, 19 mixed). Direct DNA sequencing was performed on BRAFV600E, RET gene (coding region on exon 11) and KIT (exons 11, 13 and 17). Immunohistochemical stains were performed with antibodies to markers of significance with respect to biological potential of nevomelanocytic proliferations and/or desmoplastic melanoma (Ki-67, CD117, nestin, clusterin, SOX10 and CD271/p75NTR). Polymorphism at the RET coding region (RETp) was noted in 33% of pure (8/24 cases) versus 24% of mixed (4/17 cases); BRAFV600E was absent in all cases of pure (0/24 cases) versus 6% of mixed (1/17 cases); no mutations were found in any of the cases on analyses of exons 11, 13 and 17 of the c-KIT gene (P=NS for all). For immunohistochemical analyses of pure versus mixed: mean percentage of Ki-67 nuclear positivity was 5% (s.d.=5.6) versus 28% (s.d.=12.6, P<0.001); CD117 stained 26% (6/23 cases) versus 78% (14/18 cases, P<0.01); nestin stained 83% (n=19/23 cases) versus 89% (16/18 cases, P=NS); clusterin stained 4% (1/23 cases) versus 6% (1/18 cases, P=NS); SOX10 87% (20/23 cases) versus 94% (17/18 cases, P=NS) and CD271 stained 61% (14/23 cases) versus 67% (12/18 cases, P=NS). Increased CD117 staining in the mixed variant suggests that alterations in the KIT protein may be involved in tumor progression. In addition, the proliferative index of the mixed variant was higher than that of the pure variant.

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.

Oncogenic BRAF-positive dysplastic nevi and the tumor suppressor IGFBP7—challenging the concept of dysplastic nevi as precursor lesions?

Oncogenic BRAF as an early and fundamental feature of melanocytic neoplasia has been confirmed with its identification in both melanoma and nevi. Oncogenic BRAF has been shown to induce senescence/apoptosis by up-regulating the tumor suppressor IGFBP7, which acts through autocrine/paracrine pathways to inhibit BRAF-MEK-ERK signaling. Given the putative neoplastic potential of dysplastic nevi, our aim was to ascertain in dysplastic nevi from intermittently sun-exposed skin and of varying severity the frequency of oncogenic BRAF and NRAS and to assess expression of IGFBP7 in the same. BRAF and NRAS genotyping was performed on genomic DNA (isolated using laser capture microdissection) from dysplastic nevi ranging in severity from mild (12), to moderate (11), and to severe (11). Immunohistochemical staining for IGFBP7 was performed on all. Overall, 9 (26%) of 34 cases (2 severely atypical dysplastic nevi, 2 moderately atypical dysplastic nevi, and 5 mildly atypical dysplastic nevi) exhibited the BRAFV600E mutation (P = .22), with lack of IGFBP7 expression in 4 (44.4%) of 9 cases (1 severely atypical, 1 moderately atypical, and 2 mildly atypical); and 25 (73.5%) of 34 cases (9 severely atypical, 9 moderately atypical, and 7 mildly atypical) were BRAFWT, with enhanced IGFBP7 expression in 12 (48%) of 25 cases (6 severely atypical, 3 moderately atypical, and 3 mildly atypical). All cases were NRASWT. The disparate expression of IGFBP7 in BRAFV600E-positive dysplastic nevi (enhanced in 56% and diminished/absent in 44%) indicates that the behavior of oncogenic BRAF in dysplastic nevi, unlike that in malignant melanoma, does not appear to consistently induce senescence/apoptosis through pathways mediated by IGFBP7.

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.

Somatic mutations in GNAQ in amelanotic/hypomelanotic blue nevi.

A recent study indicates that somatic mutations in codon 209 of GNAQ, a gene encoding the signaling protein G-protein α subunit q, may be present in up to 80% of blue nevi. Given that mutations in GNAQ represent dominant dark skin (Dsk) mutations caused by increased dermal melanin, the primary aim of this study was to ascertain whether amelanotic/hypomelanotic blue nevi exhibited somatic mutations in GNAQ like their melanotic counterpart. Genomic DNA was isolated for genotyping per protocol using techniques including laser capture microdissection to isolate nevus cells from amelanotic/hypomelanotic blue nevi (n = 8). The positive control group comprised regular blue nevi (n = 10, all melanotic) and cellular blue nevi (n = 9, all melanotic), whereas the negative control group comprised other dermal-based nevomelanocytic proliferations such as intradermal melanocytic nevi (n = 9, 7 of which were amelanotic) and metastatic melanoma (n = 9, 5 of which were amelanotic). DNA sequencing analysis was performed on GNAQ spanning codon 209, BRAFV600E, NRAS1, NRAS2, and KRAS genes. Mutations in GNAQ were noted in 12.5% (1/8) of amelanotic/hypomelanotic blue nevi. In the control group 40% (4/10) of blue nevi and 44% (4/9) of cellular blue nevi demonstrated the GNAQ mutation, with no cases of metastatic melanoma or intradermal melanocytic nevi exhibiting the mutation. All GNAQ mutations were A/T point mutations, and statistically significant differences were not noted among the amelanotic/hypomelanotic blue nevi, blue nevi, and cellular blue nevi subgroups. Although additional mutations were not noted in cases of amelanotic/hypomelanotic blue nevi, one blue nevus exhibited a mutation in KRAS alone; one cellular blue nevus, a concurrent NRAS2 mutation; one cellular blue nevus, a concurrent KRAS mutation; and a third cellular blue nevus, a mutation in KRAS alone. The presence of GNAQ mutations in the amelanotic/hypomelanotic blue nevus indicates that mechanisms underlying pigment homeostasis in this variant appear to be similar to those of its melanotic counterparts, although it is not clear why activation of the q class of the G-protein α subunit should cause an abundance of dermal pigment in one variant and not in another. Given that dermal melanocytes are present since birth, one possible explanation is that their melanin-synthesizing pathway is usually in a dormant state. Activation of this pathway is a consequence of multiple triggers-one of which is a mutation in the GNAQ gene, whereas the other is yet to be identified.

Oncogenic BRAF and the tumor suppressor IGFBP7 in the genesis of atypical spitzoid nevomelanocytic proliferations

Rare reports indicate that the frequency of BRAFV600E mutations is high in atypical Spitz nevi. The purpose of this study was to ascertain the utility of the RAF/RAS mutational status as a diagnostic adjunct in lesions with histologic features that deviate from a typical Spitz nevus and, to examine expression of Insulin growth factor binding protein 7 (IGFBP7), a tumor suppressor acting through autocrine/paracrine pathways to inhibit BRAF‐MEK‐ERK signaling, in the same. Genomic DNA for genotyping was isolated from 6 regular Spitz nevi and 14 atypical spitzoid nevomelanocytic proliferations (including 1 melanoma with spitzoid histomorphology). NRAS1, NRAS2 and KRAS were analyzed, in addition to BRAFV600E. A mutation in BRAFV600E was noted in only one case–that of a regular Spitz nevus. IGFBP7 expression appeared to be maintained in this case, but was absent in 7/17 cases, which included 5 atypical spitzoid nevomelanocytic proliferations. Lack of expression of IGFBP7 in atypical spitzoid nevomelanocytic proliferations with histologically concerning features but BRAF‐WT indicates that the evolutionary path in atypical spitzoid nevomelanocytic proliferations is genetically distinct from that of IGFBP7‐negative BRAF‐positive melanoma. From an oncogenic BRAF perspective, our findings suggest that the majority of ‘atypical’ spitzoid nevomelanocytic proliferations are probably no different from conventional Spitz nevi.

Lack of correlation between immunohistochemical expression of CKIT and KIT mutations in atypical acral nevi.

Background: Given the correlation between KIT mutations and immunohistochemical expression of CKIT in acral melanoma, our aim was to confirm the utility of CKIT detection as a screening tool for KIT genotyping in atypical acral nevi and to ascertain the frequency of KIT mutations in the same. Design: Immunohistochemical staining for CKIT was performed and staining criteria were the following: negative = <10%, 1 = 11%–49%, and 2 = >50% of cells. Intensity grading was as follows: negative = 0, weak = 1, moderate = 2, and strong = 3. Genomic amplification was performed on KIT exons commonly mutated in acral melanomas (11, 13, and 17) from atypical acral nevi (23) ranging in severity from mild (9), moderate (10), and severe (4). The control group included acral nevi without atypia (19). For purposes of statistical analyses, cases with 11% or more staining of cells were compared with negative cases and cases with a staining intensity of 1 or higher were compared with the negatives. Results: Immunohistochemical analyses revealed the following: positive staining with an intensity 1 or more in 18 of 22 (82%) of cases with atypia (5 mild; 9 moderate and 4 severe) and in 13 of 17 (76%) nevi without atypia with no statistically significant differences between both groups. Genomic analyses of exon regions revealed no abnormalities in “hotspots” frequently associated with point mutations in acral melanomas. Conclusions: Our findings indicate a lack of correlation between immunohistochemical expression of CKIT and KIT mutations in atypical acral nevi. Atypical acral nevi do not exhibit genetic alterations in KIT associated with acral melanomas.