Marie-Françoise Avril

Genome-wide association study identifies three loci associated with melanoma risk

We report a genome-wide association study of melanoma conducted by the GenoMEL consortium based on 317K tagging SNPs for 1,650 selected cases and 4,336 controls, with replication in an additional two cohorts (1,149 selected cases and 964 controls from GenoMEL, and a population-based case-control study in Leeds of 1,163 cases and 903 controls). The genome-wide screen identified five loci with genotyped or imputed SNPs reaching P < 5 × 10−7. Three of these loci were replicated: 16q24 encompassing MC1R (combined P = 2.54 × 10−27 for rs258322), 11q14-q21 encompassing TYR (P = 2.41 × 10−14 for rs1393350) and 9p21 adjacent to MTAP and flanking CDKN2A (P = 4.03 × 10−7 for rs7023329). MC1R and TYR are associated with pigmentation, freckling and cutaneous sun sensitivity, well-recognized melanoma risk factors. Common variants within the 9p21 locus have not previously been associated with melanoma. Despite wide variation in allele frequency, these genetic variants show notable homogeneity of effect across populations of European ancestry living at different latitudes and show independent association to disease risk.

High-risk melanoma susceptibility genes and pancreatic cancer, neural system tumors, and uveal melanoma across GenoMEL

GenoMEL, comprising major familial melanoma research groups from North America, Europe, Asia, and Australia has created the largest familial melanoma sample yet available to characterize mutations in the high-risk melanoma susceptibility genes CDKN2A/alternate reading frames (ARF), which encodes p16 and p14ARF, and CDK4 and to evaluate their relationship with pancreatic cancer (PC), neural system tumors (NST), and uveal melanoma (UM). This study included 466 families (2,137 patients) with at least three melanoma patients from 17 GenoMEL centers. Overall, 41% (n = 190) of families had mutations; most involved p16 (n = 178). Mutations in CDK4 (n = 5) and ARF (n = 7) occurred at similar frequencies (2-3%). There were striking differences in mutations across geographic locales. The proportion of families with the most frequent founder mutation(s) of each locale differed significantly across the seven regions (P = 0.0009). Single founder CDKN2A mutations were predominant in Sweden (p.R112_L113insR, 92% of familys mutations) and the Netherlands (c.225_243del19, 90% of familys mutations). France, Spain, and Italy had the same most frequent mutation (p.G101W). Similarly, Australia and United Kingdom had the same most common mutations (p.M53I, c.IVS2-105A>G, p.R24P, and p.L32P). As reported previously, there was a strong association between PC and CDKN2A mutations (P < 0.0001). This relationship differed by mutation. In contrast, there was little evidence for an association between CDKN2A mutations and NST (P = 0.52) or UM (P = 0.25). There was a marginally significant association between NST and ARF (P = 0.05). However, this particular evaluation had low power and requires confirmation. This GenoMEL study provides the most extensive characterization of mutations in high-risk melanoma susceptibility genes in families with three or more melanoma patients yet available.

Merkel cell carcinoma of the skin: pathological and molecular evidence for a causative role of MCV in oncogenesis†

Merkel cell carcinoma (MCC), a skin tumour with neuroendocrine features, was recently found to be associated with a new type of human polyomavirus, called Merkel cell virus (MCV). We investigated the specificity of this association as well as a causal role of MCV in oncogenesis. DNA and RNA from ten cases of MCC were analysed using PCR and RT‐PCR. DNA from 1241 specimens of a wide range of human tumours was also analysed. The DIPS technique was used to identify the integration locus of viral DNA sequences. Array CGH was performed to analyse structural alterations of the cell genome. MCV DNA sequences were found in all ten cases of MCC and in none of the 1241 specimens of other tumour types. Clonal integration of MCV into the host genome was seen in all MCC cases and was checked by FISH in one case. A recurrent pattern of conserved viral sequences which encompassed the replication origin, the small tumour (ST), and the 5′ part of the large tumour (LT) antigen DNA sequences was observed. Both ST and LT viral sequences were found to be significantly expressed in all MCCs. Neither recurrent site of integration nor alteration of cellular genes located near the viral sequences was observed. The tight association of MCV with MCC, the clonal pattern of MCV integration, and the expression of the viral oncoproteins strongly support a causative role for MCV in the tumour process. This information will help the development of novel approaches for the assessment and therapy of MCC and biologically related tumours. Copyright

Results of interleukin-2-based treatment in advanced melanoma: a case record-based analysis of 631 patients.

PURPOSE In patients with stage IV melanoma, durable responses have been reported with treatment regimens that involve high-dose interleukin-2 (IL-2). We analyze long-term results of 631 melanoma patients from 12 institutions who had received IL-2 alone, in combination with interferon alfa 2a or 2b (IFNalpha), or with cytotoxic drugs. METHODS Case records that contained pretreatment parameters, response data, and updated survival information were collected. After univariate analysis, the multivariate evaluation of the impact of pretreatment parameters on response and survival was performed by logistic regression and Coxs regression, respectively. RESULTS Patients were divided into four groups according to treatment: IL-2 alone (n=117), IL-2 and chemotherapy (n=49), IL-2 and IFNalpha (n=153), and IL-2, chemotherapy, and IFNalpha (n=312). The median survival of all patients was 10.5 months and the 2- and 5-year survival rates were 19.9% and 10.4%, respectively. Independent prognostic factors for response and survival were entirely different, treatment group being the only significant factor for response, and serum lactate dehydrogenase (LDH), metastatic site, and performance predicting survival. The addition of IFNalpha to IL-2 was associated with prolonged survival, but the effect of additional chemotherapy was less obvious. CONCLUSION Serum LDH, metastatic site, and performance status are useful stratification factors for randomized trials in metastatic melanoma. The improved long-term survival rates observed in melanoma patients treated with IL-2/IFNalpha-containing regimens are notable in contrast to the reported 5-year survival rates of 2% to 6% achieved with chemotherapy, but because selection bias cannot be ruled out, the impact of IL-2, as well as all other components of the treatment regimens, on survival needs to be confirmed in prospective randomized trials.

Phase II Study of Cetuximab As First-Line Single-Drug Therapy in Patients With Unresectable Squamous Cell Carcinoma of the Skin

PURPOSE To evaluate the efficacy and safety of cetuximab, a monoclonal antibody that inhibits the epidermal growth factor receptor (EGFR), as a first-line monotherapy in patients with unresectable squamous cell carcinoma of the skin (SCCS). PATIENTS AND METHODS Thirty-six patients received cetuximab (initial dose of 400 mg/m(2) followed by subsequent weekly doses of 250 mg/m(2)) for at least 6 weeks with a 48-week follow-up. The primary end point was the disease control rate (DCR) at 6 weeks (according to Response Evaluation Criteria in Solid Tumors [RECIST] criteria). Secondary end points included best response rate, overall survival, progression-free survival (PFS), and toxicity assessment. Association of treatment efficacy with RAS mutations or FcγR genotypes was investigated. RESULTS Median age of the study population was 79 years. DCR at 6 weeks was obtained in 25 of 36 patients (69%; 95% CI, 52% to 84%) of the intention-to-treat population. The best responses were eight partial responses and two complete responses. There were no cetuximab-related deaths. There were three related serious adverse events: two grade 4 infusion reactions and one grade 3 interstitial pneumopathy. Grade 1 to 2 acne-like rash occurred in 78% of patients and was associated with prolonged PFS. One HRAS mutation was identified. Combined FcγRIIa-131H/H and/or FcγRIIIa-158V/V polymorphisms were not associated with the clinical outcomes. CONCLUSION As a first-line treatment in patients with unresectable SCCS, cetuximab achieved 69% DCR. A randomized phase III trial is warranted to confirm that cetuximab may be considered as a therapeutic option especially in elderly patients. The low frequency of RAS mutations in SCCS makes SCCS tumors attractive for EGFR inhibition.

CD4+ CD56+ cutaneous neoplasms : A distinct hematological entity ?

We report seven cases of particular cutaneous tumors selected from the register of the French Study Group on Cutaneous Lymphomas. The patients (three men, four women) were aged 37-86 years. They initially presented with cutaneous nodules or papules. Three cases presented with regional lymph nodes. Stagings were negative, except for one patient with bone marrow involvement. Histological features were relevant with pleomorphic medium T-cell lymphoma, but these cells exhibited a distinguishing phenotype. They were positive for CD4, CD56, and also CD45, CD43, and HLA-DR. All other T-cell and B-cell markers were negative. The myelomonocytic markers (CD13, CD14, CD15, CD33, CD117, myeloperoxidase, and lysozyme) were negative excepted CD68, which was clearly positive in four cases and weakly in two cases. Others natural killer cell markers (CD16, CD57, TiA1, granzyme B), TdT, and CD34 were negative. Polymerase chain reaction studies did not detect any B or T clonal rearrangement. The cytogenetic studies, performed in five cases, showed a del(5q) in two cases. All patients were treated successfully by polychemotherapy, but relapsed quickly in the skin, between 4 and 28 months. Five patients developed bone marrow involvement, with leukemia in three cases, and they died in 5-27 months. One patient died at 17 months with skin progression. The seventh patient is alive at 33 months, with cutaneous progression. The origin of these cells is unclear. Despite expression of CD4 or CD56, we failed to demonstrate a T-cell, natural killer cell origin. However, CD4 and CD56 are not specific for T or natural killer lineages. Although these two markers are also known to be expressed by monocytic cells, classic myeloid antigens were negative. These seven cases, together with other rare similar cases already reported, seem to represent a distinct entity likely developed from hematological precursor cells.

Germline BAP1 Mutations Predispose to Renal Cell Carcinomas

The genetic cause of some familial nonsyndromic renal cell carcinomas (RCC) defined by at least two affected first-degree relatives is unknown. By combining whole-exome sequencing and tumor profiling in a family prone to cases of RCC, we identified a germline BAP1 mutation c.277A>G (p.Thr93Ala) as the probable genetic basis of RCC predisposition. This mutation segregated with all four RCC-affected relatives. Furthermore, BAP1 was found to be inactivated in RCC-affected individuals from this family. No BAP1 mutations were identified in 32 familial cases presenting with only RCC. We then screened for germline BAP1 deleterious mutations in familial aggregations of cancers within the spectrum of the recently described BAP1-associated tumor predisposition syndrome, including uveal melanoma, malignant pleural mesothelioma, and cutaneous melanoma. Among the 11 families that included individuals identified as carrying germline deleterious BAP1 mutations, 6 families presented with 9 RCC-affected individuals, demonstrating a significantly increased risk for RCC. This strongly argues that RCC belongs to the BAP1 syndrome and that BAP1 is a RCC-predisposition gene.

Phase 1/2 study of subcutaneous and intradermal immunization with a recombinant MAGE-3 protein in patients with detectable metastatic melanoma

The purpose of this phase 1/2 study was to evaluate toxicity, tumor evolution and immunologic response following administration of a fixed dose of a recombinant MAGE‐3 protein by subcutaneous and intradermal routes in the absence of immunologic adjuvant. Thirty‐two patients with detectable metastatic melanoma expressing gene MAGE‐3 were included and 30 received at least one injection with a fixed dose of a ProtD‐MAGE‐3 fusion protein. The immunization schedule included 6 intradermal and subcutaneous injections at 3‐week intervals. Afterward, patients without major tumor progression who required other treatments received additional vaccinations at increasing time intervals. The vaccine was generally well tolerated. Among the 26 patients who received at least 4 vaccinations, we observed 1 partial response and 4 mixed responses. For these 5 responding patients, time to progression varied from 3.5 to 51+ months. An anti‐MAGE‐3 CD4 T‐lymphocyte response was detected in 1 out of the 5 responding patients. The majority of patients had no anti‐MAGE‐3 antibody response. The clinical and immunologic responses generated by the vaccine are rather limited. Nevertheless, given the potential antitumor efficacy and the very mild toxicity of vaccinations, further studies combining MAGE proteins and/or peptides with potent immunologic adjuvants are warranted, not only in metastatic melanoma, but also in the adjuvant setting.

Molecular analysis of mutations in DNA polymerase η in xeroderma pigmentosum-variant patients

Xeroderma pigmentosum variant (XP-V) cells are deficient in their ability to synthesize intact daughter DNA strands after UV irradiation. This deficiency results from mutations in the gene encoding DNA polymerase η, which is required for effecting translesion synthesis (TLS) past UV photoproducts. We have developed a simple cellular procedure to identify XP-V cell strains, and have subsequently analyzed the mutations in 21 patients with XP-V. The 16 mutations that we have identified fall into three categories. Many of them result in severe truncations of the protein and are effectively null alleles. However, we have also identified five missense mutations located in the conserved catalytic domain of the protein. Extracts of cells falling into these two categories are defective in the ability to carry out TLS past sites of DNA damage. Three mutations cause truncations at the C terminus such that the catalytic domains are intact, and extracts from these cells are able to carry out TLS. From our previous work, however, we anticipate that protein in these cells will not be localized in the nucleus nor will it be relocalized into replication foci during DNA replication. The spectrum of both missense and truncating mutations is markedly skewed toward the N-terminal half of the protein. Two of the missense mutations are predicted to affect the interaction with DNA, the others are likely to disrupt the three-dimensional structure of the protein. There is a wide variability in clinical features among patients, which is not obviously related to the site or type of mutation.

High Levels of Antibodies Against Merkel Cell Polyomavirus Identify a Subset of Patients With Merkel Cell Carcinoma With Better Clinical Outcome

PURPOSE A new human polyomavirus, Merkel cell polyomavirus (MCV), was identified in 2008 in tumor tissue of patients with Merkel cell carcinoma (MCC), a relatively rare human skin cancer. In this study, we investigated patients with MCC and controls for the presence of antibodies against MCV and their association with clinical characteristics. PATIENTS AND METHODS Antibodies against MCV were investigated by enzyme-linked immunosorbent assay in 68 patients with MCC and 82 controls using VP1 virus-like particles produced in insect cells. RESULTS Antibodies against MCV were detected in all patients with MCC and in 85% of controls. However, high antibody titers (> 10,000) were rarely observed in controls (7.3%) and they were detected in 64.7% of patients with MCC (P < .001) in contrast to the absence of VP1 expression in tumor samples. In addition, the geometric mean titer of anti-MCV in patients with MCC was around 14 times higher than that observed in MCV-positive controls (P < .001) and was not correlated with tumor viral load. High antibody titers were not found to be associated with any subject or tumor characteristics, but better progression-free survival was observed in patients with high antibody titers (hazard ratio, 4.6; 95% CI, 1.7 to 12.2; P = .002). CONCLUSION High titers of MCV antibodies in a much higher proportion of patients with MCC than in controls confirmed the association between MCV infection and MCC. The findings also indicated that a better progression-free survival occurred in patients with high MCV antibody titers and suggested that there are at least two distinct etiologic causes of MCC.