Variation of mutant allele frequency in NRAS Q61 mutated melanomas.

BACKGROUND: Somatic mutations of BRAF or NRAS activating the MAP kinase cell signaling pathway are present in 70% of cutaneous melanomas. The mutant allele frequency of BRAF V600E (M%BRAF) was recently shown to be highly heterogeneous in melanomas. The present study focuses on the NRAS Q61 mutant allele frequency (M%NRAS). METHODS: Retrospective quantitative analyze of 104 NRAS mutated melanomas was performed using pyrosequencing. Mechanisms of M%NRAS imbalance were studied by fluorescence in situ hybridization (FISH) and microsatellite analysis. RESULTS: M%NRAS was increased in 27.9% of cases. FISH revealed that chromosome 1 instability was the predominant mechanism of M%NRAS increase, with chromosome 1 polysomy observed in 28.6% of cases and intra-tumor cellular heterogeneity with copy number variations of chromosome 1/NRAS in 23.8%. Acquired copy-neutral loss of heterozygosity (LOH) was less frequent (19%). However, most samples with high M%NRAS had only one copy of NRAS locus surrounding regions suggesting a WT allele loss. Clinical characteristics and survival of patients with either <60% or ≥60% of M%NRAS were not different. CONCLUSION: As recently shown for M%BRAF, M%NRAS is highly heterogeneous. The clinical impacts of high M%NRAS should be investigated in a larger series of patients.

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%.

Incomplete efficacy of 5-aminolevulinic acid (5 ALA) photodynamic therapy in the treatment of widespread extramammary Paget's disease.

Photodynamic therapy (PDT) using 5-aminolevulinic acid (5-ALA) is an effective treatment for several conditions such as Bowen's disease, subsets of basal cell carcinomas and actinic keratosis. Surgical resection is the first-choice therapy for extramammary Paget's disease (EMPD), but extensive resection is highly invasive and recurrences are frequent. We report two cases of genital EMPD treated by PDT with partial efficacy. The first patient, a 78-year-old male, suffered from pubic and scrotal Paget's disease for 6 years despite numerous treatments. The second patient, a 78-year-old female, had vulvar involvement for 2 years that was resistant to multiple treatments. The disease was recurrent and chronic with important pruritus and significant impact on the quality of life. Methyl 5-aminolevulinate was applied for 3 h, and irradiation was applied with red light (630 nm) using a total light dose of 37 J/cm(2) for a period of 10 min. The patients were treated every 2 to 4 weeks for a total of at least three treatments. Both patients experienced a partial transient reduction in their symptoms. One patient had a partial transient remission (< 50% reduction of the involved surface), whereas in the other patient, PDT failed to reduce the surface area of the lesions.

Impact of expert pathology review in skin adnexal carcinoma diagnosis: Analysis of 2573 patients from the French CARADERM network.

PURPOSE: To prospectively assess the impact of expert pathological review of skin adnexal carcinoma diagnosis in France. METHODS: From 2014 to 2019, 2573 samples from patients with newly diagnosed or suspected skin adnexal carcinomas were reviewed prospectively by expert pathologists through the national CARADERM (CAncers RAres DERMatologiques) network. Changes in diagnosis between referral and expert review were analysed regarding their potential impact on patient care or prognosis. RESULTS: The samples comprised 2205 newly diagnosed adnexal carcinomas, 129 benign adnexal tumours, 136 basal cell carcinomas, 74 squamous cell carcinomas, six cutaneous metastases and 13 other malignancies. There were 930 (42%) sweat gland carcinomas, of which porocarcinoma (261; 11.8%), microcystic adnexal carcinoma (125; 5.7%) and hidradenocarcinoma (109; 4.9%) were the most frequent subtypes; 778 (35%) hair follicle carcinomas, 238 (11%) sebaceous carcinomas and 212 (10%) extramammary Paget diseases/mammary-like anogenital gland adenocarcinomas. A diagnostic change between referral and expert review occurred in 503 (21.3%) patients, significantly higher for cases sent with a provisional diagnosis seeking an expert second opinion (45.7%) than for cases sent with a formal diagnosis (2.8%) (p < .0001). Sweat gland carcinomas were more prone to diagnostic discrepancies than other tumours (p < .0001), including 1.8% of patients with sweat gland carcinoma subtype misclassification with predicted clinical impact. Changes between benign and malignant conditions occurred in 117 samples (5% of patients). CONCLUSION: The study provides a unique description of the distribution of skin adnexal carcinomas and highlights the importance of expert review for these rare cancers. Optimal clinical management was impacted in a significant proportion of patients.

Adverse reactions to injectable soft tissue fillers.

In recent years, injections with filler agents are often used for wrinkle-treatment and soft tissue augmentation by dermatologists and plastic surgeons. Unfortunately, the ideal filler has not yet been discovered and all of them may induce adverse reactions. Quickly biodegradable or resorbable agents may induce severe complications, but they will normally disappear spontaneously in a few months. Slowly biodegradable or nonresorbable fillers may give rise to severe reactions that show little or no tendency to spontaneous improvement. They may appear several years after the injection, when the patient does not remember which product was injected, and treatment is often insufficient. In this review, we discuss the most commonly used fillers, their most frequent adverse reactions as well as the characteristic histopathologic findings that allow the identification of the injected filler agent. In conclusion, histopathologic study remains as the gold standard technique to identify the responsible filler.

Identification in daily practice of patients with Lynch syndrome (hereditary nonpolyposis colorectal cancer): revised Bethesda guidelines-based approach versus molecular screening.

BACKGROUND: The identification of individuals who should undergo hereditary nonpolyposis colorectal cancer (HNPCC) genetic testing remains a critical issue. The Bethesda guidelines were developed to preselect patients for microsatellite instability (MSI) testing before germline mutation screening. These criteria have been revised, and a new set of recommendations, the revised Bethesda guidelines, has been proposed. OBJECTIVE: To evaluate the performance of these revised guidelines for identifying patients with HNPCC in a series of unselected consecutive patients and compare this revised guidelines-based approach with a molecular strategy (MSI testing for all tumors, followed by exclusion of MSI-positive sporadic cases from mutational testing). PATIENTS AND METHODS: The study included 214 patients with newly diagnosed colorectal cancer. The MSI analysis was performed for all tumors. Germline testing, guided by immunohistochemical staining for mismatch repair proteins, was performed for patients with MSI-positive tumors. Sporadic MSI-positive tumors were identified by screening for BRAF mutation and MLH1 promoter methylation. RESULTS: Ninety patients (42.1%) met the revised guidelines. Twenty-one patients (9.8%) had MSI-positive tumors. Germline testing identified eight mutations (3.7%) (MSH2 N = 5, MLH1 N = 2, MSH6 N =1). The revised guidelines failed to identify 2 of the 8 probands (aged 67 and 81 yr, both with no family history). In contrast, the molecular strategy identified all patients requiring testing for germline mutation. The percentages of patients selected for germline testing by the revised guidelines and the molecular strategy were 4.2% and 5.1%, respectively. CONCLUSIONS: The revised Bethesda guidelines did not identify all HNPCC cases in our series. The molecular approach identified all HNPCC patients with MSI-positive tumors, increasing the workload for germline testing only slightly.

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.

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.

[Pleomorphic carcinoma of the large intestine]. / Localisation colique inhabituelle d'un carcinome à cellules pléomorphes.

Pleomorphic carcinoma, firstly described in the lung, is exceptional in the digestive tract. This tumor is often associated with fever and peripheral blood leukocytosis. The histological feature is characterized by giant pleomorphic often multinucleated cells with dense neutrophil infiltration and frequent phagocytosis of neutrophils by the tumor cells. Expression of epithelial markers confirm the diagnosis. The prognosis is very poor. We hereby report one case of pleomorphic carcinoma unusually situated in the large intestine, emphasizing difficulties encountered in establishing diagnosis.

Detection of BRAF p.V600E Mutations in Melanoma by Immunohistochemistry Has a Good Interobserver Reproducibility.

CONTEXT: Assessment of BRAF p.V600E mutational status has become necessary for treatment of patients with metastatic melanoma. Detection of p.V600E mutation by immunohistochemistry was recently reported in several tumor types. OBJECTIVE: To evaluate the interobserver reproducibility of BRAF p.V600E detection by immunohistochemistry in melanoma. DESIGN: Immunohistochemistry with VE1 antibody was performed on metastatic melanomas of 67 patients. Staining interpretation was performed on digital image virtual slides of tissue microarrays. The p.V600E status was determined by 7 pathologists from 3 European laboratories, blinded for other interpretations and for molecular biology results. RESULTS: Melanomas had p.V600E (n = 30), p.V600K (n = 4), p.K601E (n = 1), p.600-601delinsE (n = 1), or no p.V600 mutations (n = 31). Staining of p.V600E within mutated cells was cytoplasmic and diffuse, and for each case the staining on the 3 tissue microarray cores was similar. In 53 cases (79.1%) the 7 pathologists had perfect concordance. Agreement of interobserver reproducibility was almost perfect (κ = 0.81 [0.77-0.85]). Only 2 false-positive responses (0.9%) were obtained. The specificities reported were 100% for 5 pathologists (two of whom previously trained for p.V600E interpretation), and 97% for 2 untrained pathologists. CONCLUSIONS: Detection of BRAF p.V600E mutation by immunohistochemistry in melanomas has an excellent interobserver reproducibility. Our results suggest that immunohistochemistry could be used as a first step for detection of BRAF p.V600E mutation, to identify patients with melanoma as candidates for BRAF inhibitors.

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.

Expression of endothelin receptor subtypes in bronchial tumors.

The importance of endothelin-1 (ET-1) in cell growth, migration and stimulation of angiogenesis suggests that ET-1 may play a role in tumor progression. The expression of the ET-1 receptors ETA (ET(A)R) and ETB (ET(B)R) was analyzed by immunohistochemistry in fragments of human lung carcinomas. Samples were obtained from 11 patients with adenocarcinoma (ADK), 12 with squamous cell carcinoma (SCC) and 8 patients with small cell carcinoma (SCLC). Morphologically normal airway areas adjacent to the tumors served as controls. ADK and SCC samples had ET(A)R and ET(B)R levels similar to normal tissues; however, the ET(A)R/ET(B)R ratio was higher in ADK than in SCC. We also observed the presence of endothelin receptors in SCLC, although the ET(A)R levels and the ratio ET(A)R/ET(B)R were lower than in normal tissue and in other carcinomas. In conclusion, both ET(A)R and ET(B)R are present in lung carcinomas but at different levels, according to the histological type of tumor.

Foreign body granulomas after all injectable dermal fillers: part 1. Possible causes.

SUMMARY: Genuine granuloma formation following implantation of injectable dermal fillers is a rare complication, with incidences ranging from one in 100 patients (1 percent) to one in 5000 (0.02 percent). Foreign body granulomas occur several months to years after injection at all implantation sites at the same time. Without treatment, they may grow to the size of beans, remain virtually unchanged for some years, and then resolve spontaneously. Three clinical and histologic types of foreign body granulomas can be distinguished:Permanent implants are not characterized by a higher rate of foreign body granuloma per se than temporary implants; however, their clinical appearance is more pronounced and their persistence longer if not treated adequately.

Improved detection of urothelial carcinomas with fluorescence immunocytochemistry (uCyt+ assay) and urinary cytology: results of a French Prospective Multicenter Study.

The aim of the study was to assess the sensitivity and specificity of fluorescence immunocytochemistry (uCyt+ assay) as combined with urinary cytology for detection of primary and recurrent urothelial carcinomas. We analyzed 694 urinary samples from 236 new symptomatic patients and 458 patients followed after transurethral resection (TUR) for bladder tumor. Lesions suspicious for cancer at cystoscopy were sampled by biopsies or TUR. Sensitivity and specificity of tests were calculated using cystoscopy and histopathology, whether or not combined as gold standards. In new symptomatic patients, sensitivity of uCyt+ was 40%, 88.2%, and 76.7%, whereas that of urinary cytology was 30%, 70.6%, and 83.3%, respectively, in G1, G2, and G3 tumors. In follow-up cases, sensitivity of uCyt+ was 61.9%, 66.7%, and 76.9%, whereas that of urinary cytology was 38.1%, 58.3%, and 64.1%, respectively, in G1, G2, and G3 tumors. The combination of uCyt+ and urinary cytology significantly increased mean sensitivity in newly diagnosed cases (86.4% versus 71.2% with urinary cytology only, p < 0.05), as well as in patients followed after TUR (79.3% versus 55.2%, p < 0.001). Specificity of uCyt+ and urinary cytology was identical in new patients (83.3%) and was 81.9% and 86.2%, respectively, in patients followed after TUR. In patients with negative cystoscopy, positive uCyt+ tests had a strong predictive value for tumor recurrence at 1 year (47.0% versus 11.9% in patients with negative assay, p < 0.01). We conclude that combining uCyt+ with urinary cytology improves the detection of urothelial carcinomas as well in patients with symptoms suggesting bladder cancer as in those followed after treatment.