A. Spatz

High expression of DNA repair pathways is associated with metastasis in melanoma patients.

We have identified a gene-profile signature for human primary malignant melanoma associated with metastasis to distant sites and poor prognosis. We analyse the differential gene expression by looking at whole biological pathways rather than individual genes. Among the most significant pathways associated with progression to metastasis, we found the DNA replication (P=10−14) and the DNA repair pathways (P=10−16). We concentrated our analysis on DNA repair and found that 48 genes of this category, among a list of 234 genes, are associated with metastatic progression. These genes belong essentially to the pathways allowing recovery of stalled replication forks due to spontaneous blockage or induced DNA lesions. Because almost all these differentially expressed repair genes were overexpressed in primary tumors with bad prognosis, we speculate that primary melanoma cells that will metastasize try to replicate in a fast and error-free mode. In contrast to the progression from melanocytes to primary melanoma, genetic stability appears to be necessary for a melanoma cell to give rise to distant metastasis. This overexpression of repair genes explains nicely the extraordinary resistance of metastatic melanoma to chemo- and radio-therapy. Our results may open a new avenue for the discovery of drugs active on human metastatic melanoma.

Interobserver reproducibility of ulceration assessment in primary cutaneous melanomas

In the recently revised melanoma staging system proposed by the American Joint Committee on Cancer (AJCC), ulceration assessment by the pathologist is a pivotal parameter. Patients upstaged because of ulceration might be included in adjuvant trials conducted in AJCC stage II melanoma patients. Therefore, accuracy based on interobserver reproducibility for melanoma ulceration assessment is crucial for proper clinical management. In some cases, it is extremely difficult, even for an experienced pathologist, to distinguish between trauma-induced ulceration, artifact and tumoral ulceration. Whether this difficulty may be resolved by the use of a more precise definition of ulceration has not been evaluated. Therefore, we have proposed a refined definition of melanoma ulceration and we tested whether this definition might improve the interobserver interpretative reproducibility of ulceration in primary cutaneous melanomas. The results of this study support the need for a more precise definition of melanoma ulceration that rules out biopsy trauma or processing artifact and could be incorporated into a standardised pathology worksheet for reporting primary melanomas.

Circulating melanoma cells and distant metastasis-free survival in stage III melanoma patients with or without adjuvant interferon treatment (EORTC 18991 side study)

AIM To evaluate the prognostic and predictive importance of detection of melanoma cells in peripheral blood using reverse transcriptase polymerase chain reaction (RT-PCR) in stage III cutaneous melanoma patients after sentinel or regional lymph node dissection. PATIENTS AND METHODS Serial testing for tyrosinase and Mart-1/Melan-A transcripts in peripheral blood was performed every 6 months over a maximum period of 60 months in a subset of patients enrolled in EORTC 18991 phase 3 trial, comparing pegylated interferon-alpha-2b with observation. Univariate and multivariate analyses were performed to estimate the role of RT-PCR as prognostic and predictive factor for distant metastasis-free survival (DMFS). RESULTS Among 299 patients who underwent RT-PCR analyses, 109 (36.5%) had at least one positive sample, either at time of randomisation (N=17) or subsequently (N=92). The cumulative rate of positive results was similar in the two treatment groups, as the DMFS from first RT-PCR positivity. RT-PCR result, positive versus negative, at a given time point, had no prognostic impact on subsequent DMFS. Cox time-dependent analysis indicated a significantly higher risk of developing distant metastasis in patients with a positive sample as compared to those with a negative one: hazard ratio (HR) of 2.23 (95% confidence interval (CI), 1.40-3.55; p<.001). These results were comparable in the 2 treatment groups, indicating that RT-PCR assessment was not predictive for treatment outcome. CONCLUSION Detection of circulating tumour cells by RT-PCR for tyrosinase and Mart-1/Melan-A was a time-dependent moderate prognostic factor for subsequent development of distant metastasis in stage III melanoma patients.

Human tissue research: EORTC recommendations on its practical consequences

Improvement in cancer care is possible by applying new treatment modalities, which are emerging from knowledge and discoveries coming from laboratory research. This is possible through international collaboration and the collection of tumour tissues. Creation of a European Organisation for Research and Treatment of Cancer (EORTC) Tumor Bank is a natural step in this direction, by offering tumour sample collection from patients entered in EORTC trials. The aim of such a bank is not only to improve the diagnostic review process, but also to facilitate translational research by allowing clinicians and basic scientists to enter into close collaborations. The EORTC Tissue Research Policy is developed to assure, under the EORTC legal framework, an ethical and scientific review of research projects, guarantee adequate information is given to patients, establish procedures on the use of materials, including legal aspects, and publication policies. Being part of the EU TubaFrost project, the EORTC will provide a common international platform for the use of tissues for research purposes, finding a balance between different laws and assuring scientific progress.

Microstaging in cutaneous melanoma.

Recent papers have addressed critical issues regarding the microstaging of cutaneous melanoma. They concern the new staging proposal by the American Joint Committee on Cancer (AJCC), the presentation of new prognostic models than seem applicable in daily practice, and new immunohistochemical findings than demonstrate prognostic information independently of the conventional major factors. These issues are commented on by the Pathology Committee of the EORTC Melanoma Group. Copyright

TuBaFrost: European Virtual Tumor Tissue Banking

TuBaFrost is a consortium responsible for the task to create a virtual European human frozen tumor tissue bank, composed of high quality frozen tumor tissue collections with corresponding accurate diagnosis stored in European cancer centers and universities, searchable on the Internet, providing rules for access and use and a code of conduct to comply with the various legal and ethical regulations in European countries. Such infrastructure would enlarge tissue availability and accessibility in large amounts of specified or even rare tumor samples. Design of an infrastructure for European residual tissue banking with the described characteristics, clear focus points emerge that can be broken down in dedicated subjects: (1) standardization and quality assurance (QA) to avoid inter-institute quality variation; (2) law and ethics enabling exchange of tissue samples possible between institutes in the different European countries, where law and ethics are characterized by a strong variability; (3) rules for access, with sufficient incentives for collectors; (4) central database application containing innovations on search and selection procedures; (5) support when needed with histology images; and (6) Internet access to search and upload, with in addition a solid website giving proper information on the procedures, intentions and activities not only to the scientific community, but also to the general public. One consortium decision, part of the incentives for collectors, had major impact on the infrastructure; custodianship over the tissues as well as the tissues stay with the collector institute. Resulting in specimens that are not given to an organization, taking decisions on participation of requests, but instead the local collected tissues stay very easy to access by the collector and allows autonomous negotiation between collector and requestor on cooperation, coauthorship in publication or compensation in costs. Thereby, improving availability of large amounts of high quality samples of a highly specified or rare tumor types and contact opportunities for cooperation with other institutes.

G5 Ulceration of primary melanoma and responsiveness to adjuvant interferon therapy: Analysis of the adjuvant trials EORTC18952 and EORTC18991 in 2,644 patients

9007 Background: Ulcerated (Ulc) melanomas have a worse prognosis than non-ulcerated (N-Ulc) melanomas. Ulc and N-Ulc primaries have different stromal characteristics and gene profiles reflecting differences in biology. We analyzed outcome after adjuvant interferon (IFN) therapy in the 2 largest phase III trials (EORTC18952 and 18991) ever conducted in stage IIB-III melanoma patients (pts). METHODS EORTC18952 compared IFNα-2b (10 MIU, sc, qd, 5 days/wk, for 4 wks) followed by either 10MIU,sc, tiw for 12 mts, or 5MIU for 24 mts) with observation in 1,388 stage IIB-III pts (Lancet 2005;366). EORTC18991 evaluated pegylated IFNα-2b (6 μg/Kg, 1×/wk, for 8 wks followed by 3μg/Kg, 1×/wk for up to 5 yrs) versus observation in 1,256 stage III pts (Lancet 2008;372). Using meta-analytical methods, predictive value for Ulc on the value of IFN on relapse-free survival (RFS), distant metastasis-free survival (DMFS), and overall survival (OS) was assessed, overall, and according to stage (IIB, III-N1 or N2=microscopic or macroscopic-nodal disease). RESULTS Overall, the comparison (PEG-)IFNα-2b versus observation regarding RFS, DMFS, and OS led to a reduction in the hazard ratio (HR) of -16% (SE=5%), -13%(5%), and -8%(5%). Among 2,644 pts randomized, 849 had Ulc primaries, 1,336 N-Ulc primaries, and 459 Ulc unknown. In Ulc group the impact was much greater than in N-Ulc group for RFS (Test For Interaction: p=0.02), DMFS (p<0.001), and OS (p<0.001). The greatest reductions occurred in pts with Ulc and stages IIB/III-N1 In N-Ulc pts reduction was absent. Consistency in the treatment impact was seen in both trials. CONCLUSIONS The post hoc analyses of EORTC1892 and EORTC18991 indicate strongly that pts with an Ulc primary are far more sensitive to IFN than pts with N-Ulc primaries. This hypothesis will now be tested in the EORTC18081 trial, which compares PEG-IFNα-2b versus observation in pts with Ulc primaries ≥ 1mm. [Table: see text] [Table: see text].

Association of critical losses in X chromosome with melanoma progression: An EORTC Melanoma group study.

9000 Background: Integrative studies correlating DNA changes and expression data are powerful to identify new genetic defects involved in tumor progression. In order to identify new pathways involved in melanoma progression and to better understand the gender effect on melanoma prognosis, we correlated micro-array comparative genomic hybridization (aCGH) with expression levels of several genes in frozen melanoma samples. Methods: 48 primary melanomas from 32 females and 16 males with a median follow-up of 4 years (range: 0.6–15 years), and for which DNA and RNA were co-extracted from the same frozen slices, were analysed using long oligo 244K aCGH slides (Agilent Technologies; Pao Alto). Each sample was hybridized in single versus a standard pool of DNA with matched sex. Expression data and copy number modifications were correlated and adjusted for the False Discovery Rate. The active or inactive X chromosome (chr) status was characterized by RNA FISH with a DNA probe detecting Xist RNA. The relative abun...

Changes in microRNAs spectrum during melanoma progression

gated tubules, inside which the melanoma cells were ‘floating’. Moreover, FN and PCOL-I appeared to colocalize with TN-C in these tubular channels. Further, we studied the effects of purified TN-C and FN proteins on the behaviour of primary melanoma cells (PMEL29) in three-dimensional collagen gels to clarify the functional roles these proteins may play in melanoma progression. Interestingly, both TN-C and FN, especially together, induced the migration/growth of the melanoma cells. Our data suggest that melanoma invasion is associated with formation of special channel-like structures, providing a new concept, structured tumour cell spreading. Altogether, these data may offer potential new prognostic markers and targets for the development of new therapeutic drugs/strategies to prevent melanoma invasion and dissemination.