J. Reifenberger

Somatic mutations in the PTCH, SMOH, SUFUH and TP53 genes in sporadic basal cell carcinomas

Background  Basal cell carcinoma (BCC) of the skin is the most common human cancer. The genetic alterations underlying BCC development are only partly understood.

Analysis of p53 mutation and epidermal growth factor receptor amplification in recurrent gliomas with malignant progression.

Genomic alterations and expression of the p53 tumor suppressor gene and the epidermal growth factor receptor gene (EOFR) were investigated in 22 patients with primary World Health Organization (WHO) grade II gliomas that on recurrence had progressed to malignant gliomas of WHO grades III or IV. Mutations of the p53 gene (exons 5 to 8) were found in 12 of 22 primary tumors (10 of 13 astrocytomas, 1 of 7 oligodendrogliomas, 1 of 2 oligoastrocytomas). In each of these cases identical p53 mutations were present in the respective malignant recurrences. In all instances in which the p53 mutation was associated with p53 protein accumulation (10 of 12 cases) the percentage of p53 immunopositive tumor cells had increased from the primary to the recurrent tumor. None of the primary low-grade and none of the recurrent high-grade tumors (7 anaplastic astrocytomas, 10 anaplastic oligodendrogliomas, 4 anaplastic oligoastrocytomas, and 5 glioblastomas) showed evidence of EGFR gene amplification. Our results thus demonstrate that p53 is mutated in a high fraction of low-grade astrocytomas with progression to anaplastic astrocytomas and glioblastomas and that progression in such cases is frequently associated with an increase in the fraction of p53 immunopositive tumor cells. The general absence of EGFR amplification in our tumor series supports the hypothesis that the significance of p53 mutation and EGFR amplification may be different in glioblastomas that developed by progression from low-grade astrocytomas (secondary glioblastomas) compared to glioblastomas that developed rapidly in a de novo manner without a history of previous low-grade tumor (primary).

Somatic mutations of WNT/wingless signaling pathway components in primitive neuroectodermal tumors

Primitive neuroectodermal tumors (PNETs) represent the most frequent malignant brain tumors in childhood. The majority of these neoplasms occur in the cerebellum and are classified as medulloblastomas (MB). Most PNETs develop sporadically; however, their incidence is highly elevated in patients carrying germline APC gene mutations. The APC gene encodes a central component of the WNT/wingless developmental signaling pathway. It regulates the levels of cytoplasmic β‐catenin protein that plays a central role in neural development and cell proliferation. We analyzed 87 sporadic PNETs and 10 PNET cell lines for mutations of the APC gene and β‐catenin (CTNNB1) gene using single strand conformational polymorphism (SSCP) and sequencing analysis. We examined the mutation cluster region of APC (codons 1255–1641) for germline variants and somatic mutations. The medulloblastoma cell line MHH‐MED‐2 carried a Glu1317Gln missense germline variant and a sporadic MB sample showed a somatic Pro1319Leu substitution. Mutational analysis of exon 3 of CTNNB1 uncovered 4 PNETs (4.8%) with somatic missense mutations. These mutations caused amino acid substitutions in 3 of 80 medulloblastomas (Ser33Phe, Ser33Cys and Ser37Cys) and 1 of 4 supratentorial PNETs (Gly34Val). All mutations affected GSK‐3β phosphorylation sites of the degradation targeting box of β‐catenin and resulted in nuclear β‐catenin protein accumulation. Deletions of CTNNB1 were not detected by PCR amplification with primers spanning exons 1–5. Our data indicate that inappropriate activation of the WNT/wingless signaling pathway by mutations of its components may contribute to the pathogenesis of a subset of PNETs.

Frequent alterations of Ras signaling pathway genes in sporadic malignant melanomas

Ras signaling is important for the intracellular transduction of mitogenic stimuli from activated growth factor receptors. We have investigated 37 sporadic malignant melanomas (15 primary cutaneous melanomas and 22 melanoma metastases) and 6 melanoma cell lines for mutations in the 3 Ras genes NRAS, KRAS and HRAS. All tumors and cell lines were additionally analyzed for mutation and expression of BRAF, which encodes a Ras‐regulated serine/threonine kinase with oncogenic properties, as well as for expression of RASSF1A, which encodes a Ras‐binding protein with tumor suppressor properties. Mutational analyses identified somatic NRAS mutations in 2 primary melanomas, 4 melanoma metastases and 2 cell lines. One melanoma metastasis showed a somatic KRAS mutation whereas HRAS mutations were not detected. Eight primary melanomas, 6 melanoma metastases and 4 melanoma cell lines carried BRAF mutations affecting the known hot‐spot codon 599. None of the tumors or cell lines with BRAF mutation demonstrated NRAS or KRAS mutations. Real‐time reverse transcription‐PCR showed that 8 melanomas (3 primary tumors, 5 melanoma metastases) had reduced RASSF1A transcript levels of ≤50% relative to benign melanocytic nevi and normal skin. Three melanoma cell lines lacked detectable RASSF1A transcripts. The RASSF1A gene promoter was hypermethylated in these 3 cell lines as well as in 6 of 8 melanomas with reduced RASSF1A mRNA levels. Treatment of the cell lines with 5‐aza‐2′‐deoxycytidine and trichostatin A resulted in demethylation of the RASSF1A promoter and re‐expression of RASSF1A transcripts. Most tumors and all cell lines with RASSF1A promoter methylation additionally carried BRAF or NRAS mutations, suggesting a synergistic effect of these aberrations on melanoma growth. Taken together, 57% of the investigated melanomas and 100% of the melanoma cell lines carried mutations in either NRAS, KRAS or BRAF. In addition, 22% of the melanomas and 50% of the cell lines showed reduced RASSF1A transcript levels. Thus, alterations of Ras pathway genes are of paramount importance in the pathogenesis of sporadic melanomas.

Mutation analysis of the Ras pathway genes NRAS, HRAS, KRAS and BRAF in glioblastomas.

Aberrant activation of Ras signaling is a common finding in human glioblastomas. To determine the contribution of Ras gene mutations to this aberration, we screened 94 glioblastomas for mutations in the three Ras family genes NRAS, KRAS and HRAS. All tumors were additionally analyzed for mutations in BRAF, which encodes a Ras-regulated serine/threonine kinase with oncogenic properties. Mutation analysis of the entire coding regions of NRAS and KRAS, as well as the known mutation hot-spot sites in HRAS, identified somatic point mutations in two glioblastomas, both affecting codon 12 of NRAS (c.35G>A, p.G12D). Three additional tumors carried BRAF mutations altering the known hot-spot codon 599 (c.1796T>A, p.V599E). None of these five glioblastomas showed amplification of the EGFR or PDGFRA genes, while three of the tumors, including two with NRAS and one with BRAF mutation, demonstrated PTEN missense mutations or loss of PTEN mRNA expression. Taken together, our data suggest activating mutations in NRAS or BRAF as a molecular alteration that contributes to aberrant Ras signaling in a small fraction of glioblastomas.

Molecular genetic analysis of malignant melanomas for aberrations of the WNT signaling pathway genes CTNNB1, APC, ICAT and BTRC

Aberrant activation of the Wnt signaling pathway has been reported in different human tumor types, including malignant melanomas. We investigated 37 malignant melanomas (15 primary tumors and 22 metastases) for alterations of 4 genes encoding members of this pathway, i.e., CTNNB1 (β‐catenin gene, 3p22.1), APC (adenomatous polyposis coli gene, 5q22.2), BTRC (β‐transducin repeat–containing protein gene, 10q24.3) and ICAT (inhibitor of β‐catenin and Tcf‐4, 1p36.2). Mutational analysis of CTNNB1 identified somatic mutations in 1 primary melanoma and 1 melanoma metastasis from 2 different patients (5%). Both mutations affected the N‐terminal degradation box of β‐catenin, which is important for the regulation of β‐catenin homeostasis. Another primary melanoma carried a somatic APC missense mutation within the known mutation cluster region in exon 15. Fourteen tumors (40%) showed LOH at microsatellite markers on 1p36. None of the tumors had lost both copies of the ICAT gene, but 1 melanoma metastasis carried a somatic point mutation altering the translation start codon of ICAT. Real‐time RT‐PCR showed markedly reduced ICAT transcript levels (≤20% relative to normal skin and benign melanocytic nevi) in 28/36 malignant melanomas (78%), including 13/14 tumors with LOH on 1p36. Allelic loss on 10q was detected in 15 tumors (44%). We found neither mutations nor complete loss of expression of the BTRC gene in our melanoma series. Taken together, our results indicate that the Wnt pathway may be altered in malignant melanomas by different mechanisms, including rare somatic mutations in CTNNB1, APC or ICAT, as well as low or absent expression of ICAT transcripts.

Tumor immune escape by the loss of homeostatic chemokine expression

The novel keratinocyte-specific chemokine CCL27 plays a critical role in the organization of skin-associated immune responses by regulating T cell homing under homeostatic and inflammatory conditions. Here we demonstrate that human keratinocyte-derived skin tumors may evade T cell-mediated antitumor immune responses by down-regulating the expression of CCL27 through the activation of epidermal growth factor receptor (EGFR)–Ras–MAPK-signaling pathways. Compared with healthy skin, CCL27 mRNA and protein expression was progressively lost in transformed keratinocytes of actinic keratoses and basal and squamous cell carcinomas. In vivo, precancerous skin lesions as well as cutaneous carcinomas showed significantly elevated levels of phosphorylated ERK compared with normal skin, suggesting the activation of EGFR–Ras signaling pathways in keratinocyte-derived malignancies. In vitro, exogenous stimulation of the EGFR–Ras signaling pathway through EGF or transfection of the dominant-active form of the Ras oncogene (H-RasV12) suppressed whereas an EGFR tyrosine kinase inhibitor increased CCL27 mRNA and protein production in keratinocytes. In mice, neutralization of CCL27 led to decreased leukocyte recruitment to cutaneous tumor sites and significantly enhanced primary tumor growth. Collectively, our data identify a mechanism of skin tumors to evade host antitumor immune responses.

Allelic losses on chromosome arm 10q and mutation of the PTEN (MMAC1) tumour suppressor gene in primary and metastatic malignant melanomas

Abstract Malignant melanomas frequently show loss of alleles on the long arm of chromosome 10. The PTEN (MMAC1) gene has been identified as a tumour suppressor gene at 10q23.3 that is mutated in various types of advanced human cancers. We have investigated a series of 40 sporadic melanomas from 37 patients (15 primary cutaneous melanomas and 25 melanoma metastases) for allelic losses on chromosome 10, as well as for deletion and mutation of the PTEN gene. Microsatellite analysis revealed loss of heterozygosity at loci located on 10q in tumours from 15 of 34 patients investigated (44%). Somatic PTEN mutations were identified in melanomas from 4 of 37 patients (11%), all of whom had metastatic disease. In two of these patients, the tumours had additionally lost one PTEN allele, indicating complete loss of wild-type PTEN in the tumour cells. Our findings corroborate that loss of heterozygosity on chromosome 10 is a frequent aberration in malignant melanomas and implicate PTEN as a tumour suppressor gene inactivated by somatic mutation in a fraction of these tumours.

MUTATION OF THE VON HIPPEL–LINDAU TUMOUR SUPPRESSOR GENE IN CAPILLARY HAEMANGIOBLASTOMAS OF THE CENTRAL NERVOUS SYSTEM

A series of 20 capillary haemangioblastomas of the central nervous system was screened for mutations of the von Hippel–Lindau (VHL) tumour suppressor gene by single strand conformational polymorphism (SSCP) and heteroduplex analysis. Aberrant polymerase chain reaction (PCR) products were detected in ten tumours. DNA sequencing of these PCR products revealed that seven tumours had frameshift mutations due either to deletions of one or more base pairs (six cases) or to insertion of one base pair (one case). The remaining three tumours had either point mutations of intron splice site sequences (two cases) or a point mutation resulting in an amino acid substitution (one case). Evidence for germline alterations of the VHL gene was found in two patients who showed identical mutations in both tumour and corresponding leukocyte DNA. The results suggest that mutation of the VHL tumour suppressor gene represents a significant event in the development of capillary haemangioblastomas.

Topical Methyl Aminolevulinate Photodynamic Therapy Using Red Light‐Emitting Diode Light for Multiple Actinic Keratoses: A Randomized Study

BACKGROUND Photodynamic therapy (PDT) is an effective treatment for actinic keratoses (AKs). Light‐emitting diodes (LEDs) offer practical advantages when treating multiple lesions. OBJECTIVE To evaluate the efficacy and tolerability of PDT using a LED and topical methyl aminolevulinate (MAL) for treatment of multiple AKs. METHODS AND MATERIALS One hundred thirty‐one patients with four to 10 non‐pigmented, previously untreated thin or moderately thick AKs on the face or scalp were enrolled in this multicenter, double‐blind, randomized, placebo‐controlled study. MAL or matching placebo cream was applied to the débrided lesion surface for 3 hours before illumination with noncoherent red light (630 nm, light dose 37 J/cm2). Treatment was repeated 1 week later. RESULTS Efficacy was evaluated in 57 patients with 418 lesions treated with MAL PDT and 58 with 414 lesions treated with placebo PDT. Sixteen patients were excluded as protocol violators (not randomized). MAL PDT was superior (p<.001) to placebo PDT in lesion complete response rates (83.3%, 95% confidence interval (CI)=79.3–86.7%, vs 28.7%, 95% CI=24.4–33.4%) and patient complete response rates (all lesions showing complete response; 68.4%, 95% CI=54.8–80.1% vs 6.9%, 95% CI=1.9–16.7%). CONCLUSIONS Topical MAL PDT using a LED is an effective treatment for multiple AKs. This study was supported by Photocure, ASA.