F. Boitier

A SUMOylation-defective MITF germline mutation predisposes to melanoma and renal carcinoma

So far, no common environmental and/or phenotypic factor has been associated with melanoma and renal cell carcinoma (RCC). The known risk factors for melanoma include sun exposure, pigmentation and nevus phenotypes; risk factors associated with RCC include smoking, obesity and hypertension. A recent study of coexisting melanoma and RCC in the same patients supports a genetic predisposition underlying the association between these two cancers. The microphthalmia-associated transcription factor (MITF) has been proposed to act as a melanoma oncogene; it also stimulates the transcription of hypoxia inducible factor (HIF1A), the pathway of which is targeted by kidney cancer susceptibility genes. We therefore proposed that MITF might have a role in conferring a genetic predisposition to co-occurring melanoma and RCC. Here we identify a germline missense substitution in MITF (Mi-E318K) that occurred at a significantly higher frequency in genetically enriched patients affected with melanoma, RCC or both cancers, when compared with controls. Overall, Mi-E318K carriers had a higher than fivefold increased risk of developing melanoma, RCC or both cancers. Codon 318 is located in a small-ubiquitin-like modifier (SUMO) consensus site (ΨKXE) and Mi-E318K severely impaired SUMOylation of MITF. Mi-E318K enhanced MITF protein binding to the HIF1A promoter and increased its transcriptional activity compared to wild-type MITF. Further, we observed a global increase in Mi-E318K-occupied loci. In an RCC cell line, gene expression profiling identified a Mi-E318K signature related to cell growth, proliferation and inflammation. Lastly, the mutant protein enhanced melanocytic and renal cell clonogenicity, migration and invasion, consistent with a gain-of-function role in tumorigenesis. Our data provide insights into the link between SUMOylation, transcription and cancer.

Features associated with germline CDKN2A mutations: a GenoMEL study of melanoma-prone families from three continents

Background: The major factors individually reported to be associated with an increased frequency of CDKN2A mutations are increased number of patients with melanoma in a family, early age at melanoma diagnosis, and family members with multiple primary melanomas (MPM) or pancreatic cancer. Methods: These four features were examined in 385 families with ⩾3 patients with melanoma pooled by 17 GenoMEL groups, and these attributes were compared across continents. Results: Overall, 39% of families had CDKN2A mutations ranging from 20% (32/162) in Australia to 45% (29/65) in North America to 57% (89/157) in Europe. All four features in each group, except pancreatic cancer in Australia (p = 0.38), individually showed significant associations with CDKN2A mutations, but the effects varied widely across continents. Multivariate examination also showed different predictors of mutation risk across continents. In Australian families, ⩾2 patients with MPM, median age at melanoma diagnosis ⩽40 years and ⩾6 patients with melanoma in a family jointly predicted the mutation risk. In European families, all four factors concurrently predicted the risk, but with less stringent criteria than in Australia. In North American families, only ⩾1 patient with MPM and age at diagnosis ⩽40 years simultaneously predicted the mutation risk. Conclusions: The variation in CDKN2A mutations for the four features across continents is consistent with the lower melanoma incidence rates in Europe and higher rates of sporadic melanoma in Australia. The lack of a pancreatic cancer–CDKN2A mutation relationship in Australia probably reflects the divergent spectrum of mutations in families from Australia versus those from North America and Europe. GenoMEL is exploring candidate host, genetic and/or environmental risk factors to better understand the variation observed.

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

Fetal Microchimeric Cells Participate in Tumour Angiogenesis in Melanomas Occurring during Pregnancy

Melanoma is a major malignancy in younger individuals that accounts for 8% of all neoplasias associated with gestation. During pregnancy, a small number of fetal cells enter the maternal circulation. These cells persist and then migrate to various maternal tissues where they may engraft and differentiate, particularly if there is organ damage, adopting the phenotype of the host organ. To understand the relationship between melanoma and pregnancy, we analyzed these tumors in both humans and mice. Fetal cells were detected in 63% of human primary melanomas versus 12% in nevi during pregnancy (P = 0.034) and in 57% of B16 melanomas in pregnant mice but never in normal skin (P = 0.000022). More than 50% of these fetal cells expressed the CD34, CD31, or von Willebrand factor endothelial cell markers. In addition, the Lyve-1 lymphatic antigen was expressed by more than 30% of fetal cells in mice. In conclusion, we show that melanomas during pregnancy frequently harbor fetal cells that have an endothelial phenotype. Further studies are needed to assess whether the fetal contribution to lymphangiogenesis may alter the prognosis of the maternal tumor.

Pregnancy Promotes Melanoma Metastasis through Enhanced Lymphangiogenesis

The relationships of pregnancy and melanoma have been debatable. Our aim was to assess the influence of gestation on the course of melanoma in a classic murine model of tumor progression and in women. B16 mouse melanoma cells were injected in nonpregnant or pregnant mice on day 5 of gestation. Animals were evaluated for tumor progression, metastases, and survival. Tumor sections were analyzed for lymphatic and blood vessel number and relative surface and expression of angiogenic growth factors. Finally, primary melanomas from pregnant and nonpregnant women, matched for age and tumor thickness, were also considered. Tumor growth, metastasis, and mortality were increased in B16-injected pregnant mice. Tumors displayed an increase in intratumoral lymphangiogenesis during gestation. This increased lymphatic angiogenesis was not observed in normal skin during gestation, showing its specificity to the tumor. An analysis of melanoma from pregnant and matched nonpregnant women showed a similar increase in lymphatic vessels. Tumors from pregnant mice had increased expression of vascular endothelial growth factor A at the RNA and protein levels. The increased vascular endothelial growth factor A production by melanoma cells could be reproduced in culture using pregnant mouse serum. In conclusion, pregnancy results in increased lymphangiogenesis and subsequent metastasis. Caution should be applied in the management of patients with advanced-stage melanoma during gestation.

Increase lymphangiogenesis in melanoma during pregnancy: correlation with the prolactin signalling pathway

Editor The evolution of melanoma during pregnancy has been a matter of debate. It is still debated whether pregnancy aggravates melanoma and increases overall death. We recently reported an increased level of metastases in pregnant mice with melanoma compared to non-pregnant controls. Our study revealed an increased level of lymphatic angiogenesis in tumours occurring during pregnancy both in mice and humans. Suspecting a hormonal factor related to gestation as a main cause of this increased lymphangiogenesis, we examined prolactin and ⁄ or growth hormone via their associated Jak ⁄ STAT5 signalling pathway in melanomas during and outside gestation. Archival paraffin embedded primary tumours were obtained for 14 pregnant women and 14 non-pregnant controls, all with melanoma, matched for sex, age, Breslow index and tumour AJCC stage at diagnosis (Table 1a). Sections were labelled withCD34 and D2-40 to stain blood and lymphatic vessels respectively and evaluation were performed as previously described. We also labelled slides with a monoclonal antiPhospho Y694 Stat-5 antibody (Epitomics, Burlingame, CA, USA) to demonstrate STAT5 activation. Computer-assisted morphometric analysis showed that lymphatic vessels (D2-40 staining) were larger by 45% in pregnant women (64.39 vs. 43.78 lm; P = 0.024) (Table 1b). In the peritumoural area, we measured an increase of 80% in the relative lymphatic vessel area in pregnant women compared to non-pregnant controls (1.194 vs. 0.65%; P = 0.0179). As expected, blood vessel relative area did not significantly differ between pregnant and non-pregnant groups. We therefore confirm here, in an independent series matched for sex, age, Breslow index but also AJCC stage our previous findings indicating that pregnant patients with melanoma have a higher lymphatic vessel relative surface area at the periphery of the tumour compared to non-pregnant controls. Among the 28 patients, 22 were analyzed for P-STAT5 staining. Among these patients, 17 had P-STAT5 in at least one cell type from the tumour sample. P-STAT5 was found mostly in inflammatory (n = 11) and endothelial cells (n = 14) although it could be detected in surrounding keratinocytes (n = 10) or rarely in melanocytes (n = 4). The cell type identification was made based on morphology (Fig. 1a–d). The P-STAT5 staining was nuclear in all cell types and only keratinocytes had some cytoplasmic staining. P-STAT5 staining was not different between pregnant and nonpregnant women in any cell type. We next investigated the association between P-STAT5 staining and both blood and lymphatic neovascularisation. P-STAT5 presence in leucocytes or melanocytes was associated with reduced diameter of lymphatic vessels in intratumoural areas (respectively 44.3 vs. 65.83 lm, P = 0.0247 and 38.15 vs. 59.2 lm, P = 0.0469; Fig. 1e). In contrast, lymphatic vessel density was significantly increased in the intratumoural area of patients with P-STAT5 in infiltrating leucocytes or keratinocytes (respectively 23.87 vs. 13.58 ⁄ mm, P = 0.0127 and 24.63 vs. 13.78 ⁄ mm, P = 0.017; Fig. 1f). Probably, as a result of the opposite effect of P-STAT5 on vessels diameter and density, no variation was observed in terms of relative lymphatic vessel area. We did not observe any variation in peritumoural areas (Fig. 1g). Moreover, we did not observe any variation in intra or peritumoural blood vessels in relation to P-STAT5 staining. Although STAT5 activity did not significantly vary between pregnant and non-pregnant patients, it clearly related to lymphangiogenesis. In conclusion, we confirm here that pregnant patients with melanoma had a higher lymphatic vessel relative surface area at the periphery of the tumour compared to non-pregnant controls. Table 1 Cohort properties

Age and clear eyes are associated with an increased risk of cutaneous squamous cell carcinomas in vemurafenib-treated melanoma patients.

Cutaneous squamous cell carcinoma (cSCC) is a frequent side-effect of vemurafenib treatment. The main aim of this study was to identify the clinical risk factors associated with the development of cSCC in melanoma patients treated with vemurafenib. We carried out a retrospective study, including 63 consecutive melanoma patients treated with vemurafenib for BRAF-mutant metastatic melanoma in an oncodermatological department. Clinical and follow-up data were collected and analysed, and a comparison of the subgroups who did and did not develop cSCC was performed. A total of 42.9% of patients (n=27) treated with vemurafenib developed one or more cSCC. Patients with cSCC were significantly older (P=0.01). Clear eyes were also associated with a higher risk of developing cSCC (odds ratio=3.50; 95% confidence interval: 1.08–12.43). Three patients developed cSCC more than 1 year after the initiation of treatment (12, 16 and 18 months, respectively). Clinicians should be vigilant in older patients undergoing vemurafenib therapy as well as patients with clear eyes as they seem to be at increased risk of developing cSCC, even late after the initiation of treatment.

Late occurrence of Histoplasma duboisii cutaneous and pulmonary infection 18 years after exposure

We report an imported case of Histoplasma capsulatum var. duboisii (H. duboisii) infection in a white French woman revealed by cutaneous lesions of the scalp, 18 years after her last stay in West and Central Africa. Asymptomatic bilateral pulmonary infiltrates were discovered on thoracic computed tomography. Skin biopsy allowed the positive diagnosis showing the typical yeasts; culture of biopsy specimens was positive for H. capsulatum. In the absence of criteria of severity, the patient was treated for one year with oral itraconazole 400mg/day. The outcome was favourable, skin and pulmonary lesions resolved slowly. The follow up is 5 years without relapse after the end of treatment. This case illustrates the possibility of late occurrence of H. duboisii infection, many years after exposure and the major importance of asking any patient for travelling or residency in tropical countries.

La régression tumorale n’est pas un facteur de risque d’atteinte du ganglion sentinelle dans les mélanomes fins (indice de Breslow ≤ 1 mm)

BACKGROUND Thin melanomas (Breslow thickness < or = 1 mm) are considered highly curable. The aim of this study was to evaluate the correlation between histological tumour regression and sentinel lymph node (SLN) involvement in thin melanomas. PATIENTS AND METHODS This was a retrospective single-centre study of 34 patients with thin melanomas undergoing SLN biopsy between April 1998 and January 2005. RESULTS The study included 14 women and 20 men of mean age 56.3 years. Melanomas were located on the neck (n=3), soles (n=4), trunk (n=13) and extremities (n=14). Pathological examination showed 25 SSM, four acral lentiginous melanomas, three in situ melanomas, one nodular melanoma and one unclassified melanoma with a mean Breslow thickness of 0.57 mm. Histological tumour regression was observed in 26 over 34 cases and ulceration was found in one case. Clark levels were as follows: I (n=3), II (n=20), III (n=9), IV (n=2). Growth phase was available in 15 cases (seven radial and eight vertical). Mitotic rates, available in 24 cases, were: 0 (n=9), 1 (n=11), 2 (n=2), 3 (n=1), 6 (n=1). One patient with histological tumour regression (2.9% of cases and 3.8% of cases with regressing tumours) had a metastatic SLN. One patient negative for SLN had a lung relapse and died of the disease. Mean follow-up was 26.2 months. CONCLUSION The results of the present study and the analysis of the literature show that histological regression of the primary tumour does not seem predictive of higher risk of SLN involvement in thin melanomas. This suggests that screening for SLN is not indicated in thin melanomas, even those with histological regression.

Skin necrosis revealing antiphospholipid syndrome during immunotherapy for melanoma.

Immunotherapeutic approaches have been developed recently for the treatment of advanced melanoma, based on the finding that the immunological response to mela-noma plays a key role in control of the disease (1). We report here a case of a patient with metastatic melanoma who developed antiphospholipid syndrome (APLS) after initiation of immunotherapy.CASE REPORTIn December 2006, a 53-year-old man presented with toe discoloration and subungueal haemorrhages. Fifteen years previously (in 1991) he had been diagnosed with a superficial spreading melanoma of the left thigh, 4 mm in thickness, Clark’s level IV. During follow-up, the patient presented with four successive involvements of regional lymph nodes, requiring lymphadenectomy. In April 2002, pre-operative blood tests had revealed a prolongation of the partial thromboplastin time test (PTT), with a 1.31 ratio (normal range <1.2) and au-toantibodies consistent with a lupic anticoagulant. The patient was negative for antiphospholipid (APL) anti-bodies, negative for rheumatoid factor, but positive for antinuclear antibodies at a titre of 1/160. In April 2006, computerized tomography (CT) revealed a lymph node of 14 mm in the external left iliac area. Histological analyses revealed the presence of metastasis. The patient received six subcutaneous injections of mature dendritic cells pulsed with lysates of three me-lanoma cell lines from 3 August to 11 October 2006, followed by subcutaneous pegylated interferon-alpha-2b (PEG-IFNα 2b) (1.5 µg/kg/week) from 5 August to 15 November 2006 (Fig. 1). In October 2006, a new CT scan revealed bilateral enlargement of lymph nodes in the iliac and inguinal areas, but lymphadenectomy showed no metastasis. Six days later, cyanotic and hype-raesthesic lesions appeared on the first left toe, followed by spreading of bilateral purpuric, necrotic areas and erythematous, purple painful macules to the other toes (Fig. 2A), and subungueal splinter haemorrhages appea-red on both hands (Fig. 2B). Doppler ultrasonography demonstrated a deep vein thrombosis at the junction between the left external iliac vein and the left common femoral vein, and occlusion of the right radial artery. Biological parameters were: white blood cell (WBC) count 10 × 10