Variations of BRAF mutant allele percentage in melanomas.

BACKGROUND: BRAF mutations are present in 40% of human skin melanomas. Mutated tumors with an increased percentage of BRAF mutant alleles (BRAF-M%) may have a better response to RAF/MEK inhibitors. We evaluated the BRAF-M% in melanomas, and the genetic causes of its variation. METHODS: BRAF-M% was quantified by pyrosequencing, real-time PCR (rtPCR) and/or picoliter-droplet PCR (dPCR). BRAF mutant expression was detected by immunohistochemistry. Chromosomal alterations were analyzed with fluorescence in situ hybridization (FISH), and single nucleotide polymorphism (SNP) arrays. RESULTS: BRAF-M% quantification obtained with pyrosequencing was highly correlated (R = 0.94) with rtPCR, and with dPCR. BRAF-M% quantified from DNA and RNA were also highly correlated (R = 0.98). Among 368 samples with >80% tumor cells, 38.6% had a BRAF (V600E) mutation. Only 66.2% cases were heterozygous (BRAF-M% 30 to 60%). Increased BRAF-M% (>60%) was observed in 19% of cases. FISH showed a polysomy of chromosome 7 in 13.6%, 35.3% and 54.5% of BRAF wild-type, heterozygous and non-heterozygous BRAF-mutated samples, respectively (P < 0.005). Amplification (5.6%) and loss (3.2%) of BRAF locus were rare. By contrast, chromosome 7 was disomic in 27/27 BRAF-mutated nevi. CONCLUSIONS: BRAF-M% is heterogeneous and frequently increased in BRAF-mutant melanomas. Aneuploidy of chromosome 7 is more frequent in BRAF mutant melanomas, specifically in those with high BRAF-M%.

Prognostic value of BRAF(V6°°) mutations in melanoma patients after resection of metastatic lymph nodes.

PURPOSE: BRAF (V600) mutations are frequent in melanomas, and BRAF(V600)-targeted therapy have dramatic, but often transitory, efficacy in stage IV patients. Prognosis of patients with American Joint Committee on Cancer (AJCC) stage III melanoma is heterogeneous. We aimed to determine the overall survival (OS) of stage III patients with a nodal deposit of ≥2 mm according to BRAF (V600) mutations and other previously reported prognostic criteria. METHODS: This retrospective study included 105 consecutive patients with stage III cutaneous melanomas. Most patients underwent a prospective follow-up. BRAF (V600) mutations were detected by sequencing and pyrosequencing of DNA in samples containing >60 % melanoma cells. RESULTS: BRAF mutations (p.V600E and p.V600K in 83 and 14 % of cases, respectively) were detected in 40 % of the patients. For patients with and without BRAF mutations, death occurred in 83.3 and 60.3 %, with a median OS of 1.4 and 2.8 years, respectively. Patient age, primary melanoma ulceration, number of invaded lymph nodes, AJCC staging at study entry, and BRAF status were linked to OS in the univariate analysis. The only characteristics associated with OS in the multivariate analysis were number of invaded lymph nodes (P = 0.005, hazard ratio 2.2, 95 % confidence interval 1.3-3.9) and BRAF status (P = 0.005, hazard ratio 1.9, 95 % confidence interval 1.2-3.1). CONCLUSIONS: BRAF (V600) status could be used to stage melanoma patients with nodal deposits. Our results may also help to plan adjuvant trials in these patients, for whom the low tumor load may induce longer efficacy of BRAF-targeted therapies.

The contribution of high-resolution ultrasonography in preoperatively detecting sentinel-node metastases in melanoma patients.

To evaluate the ability of high-resolution ultrasonography (hrUS) to detect sentinel-node (SN) melanoma metastases preoperatively before sentinel-node biopsy (SNB), to define hrUS resolution, and to evaluate which US criteria should be used. During a 6.5-year period, 131 consecutive patients with 132 >or=1-mm thick or ulcerated cutaneous melanomas, who were followed up at a single center, were enrolled. All patients underwent preoperative regional lymph-node hrUS and SNB. We used the recently evaluated ultrasonographic stringent and nonstringent hrUS criteria to detect SN metastases. Sizes of the SN metastatic deposits were measured under light microscopy. Thirty-five (27%) patients had a positive SNB. HrUS identified only three positive SNs as being metastatic. Sensitivity and specificity using stringent criteria were 8.8% [95% confidence interval (CI, 2.3-24.8%) and 95.9% (95% CI, 89.3-98.7%)], respectively. Positive-predictive value was 42.9% (95% CI, 11.9-79.9%). The nonstringent criteria provided four additional true-positive results, but lowered specificity (89.8%; 95% CI, 81.6-94.7%) with no significant improvement in sensitivity (20.6%; 95% CI, 9.3-38.4%). Positive-predictive value using nonstringent criteria was 41.2% (95% CI, 19.3-66.4%). HrUS failed to detect all metastatic deposits <5 mm in diameter. HrUS assessment of early-stage melanomas cannot replace surgical SNB. Owing to its low positive-predictive value, hrUS was unable to identify patients who would have to proceed directly to completion lymphadenectomy.

Spontaneous regression of grade 3 vulvar intraepithelial neoplasia associated with human papillomavirus-16-specific CD4(+) and CD8(+) T-cell responses.

Cell-mediated immunity directed against human papillomavirus 16 (HPV-16) antigens was studied in six patients affected with grade 3 vulvar intraepithelial neoplasia (VIN3, also known as bowenoid papulosis). Five of the patients presented with a chronic and persistent disease that relapsed after destructive treatments. They showed no detectable anti-HPV blood T-cell responses and no T-cell intraepidermal vulvar infiltrate containing both CD4+ and CD8+ lymphocytes. The last patient had a complete clearance of viral lesions, 8 months after disease onset and 2 months after electrocoagulation of <50% of the VIN3 lesions. She showed high frequency anti-E6 and anti-E7 effector blood T cells by ex vivo ELISpot-IFNgamma assay before clinical regression. Immunohistochemical study of her vulvar biopsy revealed a marked dermal infiltrate containing a majority of CD4+ T lymphocytes and an epidermal infiltrate made up of both CD4(+) and CD8(+) T cells. This seems to be the first evidence of an association between spontaneous regression of VIN3 lesions and HPV-specific T-cell responses detectable in the blood. Hence, an increase of HPV-specific effector T lymphocyte responses by vaccine-based therapeutic strategies might be useful to clear the lesions in bowenoid papulosis disease.

Treatment of undifferentiated vulvar intraepithelial neoplasia with 5% imiquimod cream: a prospective study of 12 cases.

OBJECTIVE: To assess the efficacy of 5% imiquimod cream on undifferentiated vulvar intraepithelial neoplasia (VIN), a disease caused by high-risk human papillomavirus. DESIGN: Prospective, uncontrolled study. SETTING: University hospital vulvar clinic. Patients Twelve consecutive patients treated with 5% imiquimod cream for undifferentiated VIN between March 1, 1999, and May 31, 2001. INTERVENTION: Self-application of 5% imiquimod cream, initially 3 times a week, then adjusted according to tolerance, for up to 7 months according to clinical response. MAIN OUTCOME MEASURES: Therapeutic response, clinically assessed by successive photographs and histologically confirmed for complete responders, was scored as complete, partial (> or =50% decrease in lesion size), or failure. Tolerance was evaluated at each visit. RESULTS: A total of 3, 4, and 5 patients achieved complete response, partial response (> or =75% reduction in lesion size for all such cases), and failure, respectively. Mean duration of treatment was 3.6 months (37.3 applications), 5.0 months (50.7 applications), and 3.4 months (25.2 applications) for complete responders, partial responders, and failures, respectively. Follow-up after treatment was 5 to 18, 14 to 32, and 2 to 28 months, respectively, with 1 partial responder lost to long-term follow-up. No patient developed invasive carcinoma. All but 2 patients experienced vulvar discomfort, resulting in treatment withdrawal for 3. Two patients had flulike symptoms. CONCLUSIONS: Imiquimod cream could be a therapeutic option for undifferentiated VIN. Although poorly tolerated, this self-applied treatment could spare patients, either totally or partially, the classic painful and sometimes mutilating treatments of VIN. Controlled, randomized studies are needed to evaluate its efficacy and tolerance.

The histological aspects of fillers complications.

The histological aspects of resorbable heterologous fillers (bovine collagen, acid hyaluronique), autologous fillers (lipofilling, dermis-fat graft), biodegradable fillers (New-Fill), and permanent fillers (silicone, Artecoll, Evolution, Aquamid, DermaLive, DermaDeep, Bioplastique, Paraffin) are described. This article relates the morphological aspect of these materials, the normal tissue reaction after injection, and its chronological evolution as the morphological aspects from the different side effects, more frequently observed for the permanent fillers. They mainly consist of granulomatous reactions which may appear long after injection.

Detection of BRAF p.V600E mutations in melanomas: comparison of four methods argues for sequential use of immunohistochemistry and pyrosequencing.

BRAF p.V600 mutation detection recently became necessary to treat metastatic melanoma patients with vemurafenib. This study compares different methods of detection of BRAF mutations. Melanoma samples from 111 patients were analyzed for BRAF mutations, and for 89 of them, results were obtained with the four following methods: Sanger sequencing, real-time PCR, immunohistochemistry, and pyrosequencing. All samples contained at least 60% of tumor cells. Directional Sanger sequencing of PCR products failed to detect 3 of 40 p.V600E-mutated cases (7.5%) (sensitivity, 92.5%; 95% CI, 78.5% to 98.0%). BRAF p.V600E-specific real-time PCR identified 39 of 40 p.V600E-mutated cases (97.6%) (sensitivity, 97.5%; 95% CI, 87.1% to 99.6%) and all 39 wild-type (WT) cases and surprisingly was also positive for 6/6 p.V600K (specificity, 87.8%; 95% CI, 75.8% to 94.3%). However, other mutations, p.V600R (n = 1), p.K601E (n = 2), and p.600_601delinsE (n = 1), were not detected. Immunohistochemistry with VE1, specific for p.V600E, identified all p.V600E and WT cases (sensitivity, 100%; 95% CI, 91.2% to 100%) but was negative for all other BRAF mutations. Pyrosequencing successfully identified all WT and mutated cases. Immunohistochemistry is highly specific for p.V600E, and could be used as a first-line method, as is currently performed for HER2 amplification detection. Pyrosequencing proved to be the most efficient method to detect BRAF mutations in melanomas and could be performed on VE1-negative or uninterpretable cases.

Anti-HPV16 E2 protein T-cell responses and viral control in women with usual vulvar intraepithelial neoplasia and their healthy partners.

T-cell responses (proliferation, intracellular cytokine synthesis and IFNγ ELISPOT) against human papillomavirus 16 (HPV16) E2 peptides were tested during 18 months in a longitudinal study in eight women presenting with HPV16-related usual vulvar intraepithelial neoplasia (VIN) and their healthy male partners. In six women, anti-E2 proliferative responses and cytokine production (single IFNγ and/or dual IFNγ/IL2 and/or single IL2) by CD4+ T lymphocytes became detectable after treating and healing of the usual VIN. In the women presenting with persistent lesions despite therapy, no proliferation was observed. Anti-E2 proliferative responses were also observed with dual IFNγ/IL2 production by CD4+ T-cells in six male partners who did not exhibit any genital HPV-related diseases. Ex vivo IFNγ ELISPOT showed numerous effector T-cells producing IFNγ after stimulation by a dominant E2 peptide in all men and women. Since the E2 protein is absent from the viral particles but is required for viral DNA replication, these results suggest a recent infection with replicative HPV16 in male partners. The presence of polyfunctional anti-E2 T-cell responses in the blood of asymptomatic men unambiguously establishes HPV infection even without detectable lesions. These results, despite the small size of the studied group, provide an argument in favor of prophylactic HPV vaccination of young men in order to prevent HPV16 infection and viral transmission from men to women.

Eosinophilic fasciitis: a rare skin sclerosis.

Eosinophilic fasciitis (Schulman's syndrome) is a rare disease with specific clinical symptoms such as the groove sign which facilitate diagnosis. We report a typical case of eosinophilic fasciitis in an otherwise healthy 49-year-old man who presented with "prayer and groove signs". Histological analysis showed sclerosis and eosinophilic infiltration of the fascia. The patient was successfully treated with systemic corticotherapy and Cyclosporine. A short review of the clinicopathological features of the lesions is presented.