Analytical validation of HER2DX genomic test for early-stage HER2-positive breast cancer.

BACKGROUND: HER2DX, a multianalyte genomic test, has been clinically validated to predict breast cancer recurrence risk (relapse risk score), the probability of achieving pathological complete response post-neoadjuvant therapy (pCR likelihood score), and individual ERBB2 messenger RNA (mRNA) expression levels in patients with early-stage human epidermal growth factor receptor 2 (HER2)-positive breast cancer. This study delves into the comprehensive analysis of HER2DX's analytical performance. MATERIALS AND METHODS: Precision and reproducibility of HER2DX risk, pCR, and ERBB2 mRNA scores were assessed within and between laboratories using formalin-fixed paraffin-embedded (FFPE) tumor tissues and purified RNA. Robustness was appraised by analyzing the impact of tumor cell content and protocol variations including different instruments, reagent lots, and different RNA extraction kits. Variability was evaluated across intratumor biopsies and genomic platforms [RNA sequencing (RNAseq) versus nCounter], and according to protocol variations. RESULTS: Precision analysis of 10 FFPE tumor samples yielded a maximal standard error of 0.94 across HER2DX scores (1-99 scale). High reproducibility of HER2DX scores across 29 FFPE tumors and 20 RNAs between laboratories was evident (correlation coefficients >0.98). The probability of identifying score differences >5 units was ≤5.2%. No significant variability emerged based on platform instruments, reagent lots, RNA extraction kits, or TagSet thaw/freeze cycles. Moreover, HER2DX displayed robustness at low tumor cell content (10%). Intratumor variability across 212 biopsies (106 tumors) was <4.0%. Concordance between HER2DX scores from 30 RNAs on RNAseq and nCounter platforms exceeded 90.0% (Cohen's κ coefficients >0.80). CONCLUSIONS: The HER2DX assay is highly reproducible and robust for the quantification of recurrence risk, pCR likelihood, and ERBB2 mRNA expression in early-stage HER2-positive breast cancer.

Synchronous primary cutaneous melanomas: a descriptive study of their clinical features, histology, genetic background of the patients and clinical outcomes.

BACKGROUND: Around 0.5% of cutaneous melanoma (CM) patients will present with synchronous melanomas when first seen. Moreover, 26-40% of patients with multiple primary melanomas present with synchronous lesions. OBJECTIVES: To assess the prevalence, clinical and histopathological characteristics, germline mutations and outcome in patients with synchronous melanoma. METHODS: Clinical and histopathological data from 4703 melanoma patients were included. Clinical, histological and genetic mutational status information was analysed. Kaplan-Meier curves were used to investigate survival outcomes. RESULTS: A total of 144 patients (3.06%) presented simultaneously with two or more primary melanomas. During follow-up, 25.7% of patients with synchronous melanoma developed a new primary melanoma compared to 8.6% of patients diagnosed with single melanoma (P < 0.001). Germinal CDKN2A mutations were identified in 10.7% of patients with synchronous melanomas and genetic variants in MC1R in 72%. No significant differences in all survival outcomes between patients with synchronous melanomas and single melanomas were found. CONCLUSION: Synchronous melanomas are more frequent than previously reported and are more frequent in older patients compared to single melanomas. Moreover, these patients have a higher risk of developing a new primary melanoma during follow-up and have higher rates of germline susceptibility variants. Nevertheless, these findings were not associated with worse outcomes.

Cell-free DNA concentration and fragment size fraction correlate with FDG PET/CT-derived parameters in NSCLC patients.

PURPOSE: The aim of our study was to investigate the correlation between cfDNA concentration and fragment size fraction with FDG PET/CT- and CT-derived parameters in untreated NSCLC patient. METHODS: Fifty-three patients diagnosed of locally advanced or metastatic NSCLC who had undergone FDG PET/CT, CT and cfDNA analysis prior to any treatment were included in this retrospective study. CfDNA concentration was measured by fluorometry and fragment size fractions were determined by microchip electrophoresis. [18F]F-FDG PET/CT was performed and standardised uptake values (SUV), metabolic tumour volume (MTV) and total lesion glycolysis (TLG) were calculated for primary, extrapulmonary and total disease. CT scans were evaluated according to RECIST 1.1 criteria. RESULTS: CfDNA concentration showed a positive correlation with extrapulmonary MTV (r2 = 0.36, P = 0.009), and extrapulmonary TLG (r2 = 0.35, P = 0.009) and their whole-body (wb) ratios. Higher concentrations of total cfDNA were found in patients with liver lesions. Short fragments of cfDNA (100-250 bp) showed a positive correlation with extrapulmonary MTV (r2 = 0.49, P = 0.0005) and extrapulmonary TLG (r2 = 0.39, P = 0.006) and their respective wb ratios, and a negative correlation with SUVmean (r2 = -0.31, P = 0.03) and SUVmean/SUVmax ratio (r2 = -0.34, P = 0.02). A higher fraction of short cfDNA fragments was found in patients with liver and pleural lesions. CONCLUSIONS: This study supports the hypothesis that cfDNA concentration and short cfDNA fragment size fraction reflect the tumour burden as well as metabolic activity in advanced NSCLC patients. This suggests their suitability as complementary tests for a more accurate diagnosis of tumour metabolic behaviour and to allow personalised therapies.

Detection of cell-free circulating BRAFV 600E by droplet digital polymerase chain reaction in patients with and without melanoma under dermatological surveillance.

BACKGROUND: The p.V600E mutation in the BRAF protein is the most frequent mutation in cutaneous melanoma and is a recurrent alteration found in common benign naevi. Analysis of the cell-free BRAF c.1799T>A, p.V600E mutation (cfBRAFV 600E ) in plasma has emerged as a biomarker for monitoring prognosis and treatment response in patients with melanoma. OBJECTIVES: To quantify cfBRAFV 600E levels in plasma from patients with melanoma and from patients without melanoma undergoing regular follow-up of their melanocytic lesions, in order to assess the clinical significance of the test. METHODS: We quantified cfBRAFV 600E by droplet digital polymerase chain reaction in plasma from 146 patients without melanoma undergoing continuous dermatological screening, from 26 stage III and seven stage IV patients with BRAF-mutant melanoma, and from 32 patients with melanoma who were free of disease for 3 or more years. RESULTS: Among disease-free patients and individuals without melanoma, 52% presented a high naevus count (> 50) and 49% had clinically atypical naevi. cfBRAFV 600E was detected in 71% of patients with stage IV melanoma and 15% with stage III, and in 1·4% of individuals without melanoma. No cfBRAFV 600E mutation was detected in disease-free patients with melanoma. Individuals without melanoma had lower cfBRAFV 600E levels than patients with melanoma. We established a variant allelic frequency of 0·26% or 5 copies mL-1 of cfBRAFV 600E as the optimal cutoff value for identifying patients with melanoma with > 99% specificity. CONCLUSIONS: This study suggests that naevus-related factors do not influence the detection of cfBRAFV 600E in individuals without melanoma, and supports the clinical diagnostic value of plasma cfBRAFV 600E quantification in patients with melanoma. What's already known about this topic? The analysis of the BRAF c.1799T>A (p.V600E) mutation in cell-free (cf)DNA has emerged as a potential biomarker for monitoring prognosis and treatment response in patients with metastatic BRAFV600E melanoma. The BRAFV600E alteration is a common genetic alteration found in benign proliferations such as melanocytic naevi. No information exists about the impact of the number of common acquired naevi or the presence of clinically atypical naevi in cfBRAFV600E detection in an individual. What does this study add? The cfBRAFV600E mutation is detected in plasma from a reduced number of individuals without melanoma undergoing continuous dermatological follow-up. A high number of naevi or the presence of clinically atypical naevi are factors that do not influence cfBRAFV600E detection in an individual. Both total cfBRAF concentration and cfBRAFV600E frequency are effective biomarkers in patients with advanced melanoma but not in patients at early stages or with micrometastases. What is the translational message? Detection of cfBRAFV600E in an individual is not influenced by naevus-related factors. cfBRAFV600E is a robust and reliable biomarker that can be used in dermatological surveillance programmes.

Fibroblast activation and abnormal extracellular matrix remodelling as common hallmarks in three cancer-prone genodermatoses.

BACKGROUND: Recessive dystrophic epidermolysis bullosa (RDEB), Kindler syndrome (KS) and xeroderma pigmentosum complementation group C (XPC) are three cancer-prone genodermatoses whose causal genetic mutations cannot fully explain, on their own, the array of associated phenotypic manifestations. Recent evidence highlights the role of the stromal microenvironment in the pathology of these disorders. OBJECTIVES: To investigate, by means of comparative gene expression analysis, the role played by dermal fibroblasts in the pathogenesis of RDEB, KS and XPC. METHODS: We conducted RNA-Seq analysis, which included a thorough examination of the differentially expressed genes, a functional enrichment analysis and a description of affected signalling circuits. Transcriptomic data were validated at the protein level in cell cultures, serum samples and skin biopsies. RESULTS: Interdisease comparisons against control fibroblasts revealed a unifying signature of 186 differentially expressed genes and four signalling pathways in the three genodermatoses. Remarkably, some of the uncovered expression changes suggest a synthetic fibroblast phenotype characterized by the aberrant expression of extracellular matrix (ECM) proteins. Western blot and immunofluorescence in situ analyses validated the RNA-Seq data. In addition, enzyme-linked immunosorbent assay revealed increased circulating levels of periostin in patients with RDEB. CONCLUSIONS: Our results suggest that the different causal genetic defects converge into common changes in gene expression, possibly due to injury-sensitive events. These, in turn, trigger a cascade of reactions involving abnormal ECM deposition and underexpression of antioxidant enzymes. The elucidated expression signature provides new potential biomarkers and common therapeutic targets in RDEB, XPC and KS. What's already known about this topic? Recessive dystrophic epidermolysis bullosa (RDEB), Kindler syndrome (KS) and xeroderma pigmentosum complementation group C (XPC) are three genodermatoses with high predisposition to cancer development. Although their causal genetic mutations mainly affect epithelia, the dermal microenvironment likely contributes to the physiopathology of these disorders. What does this study add? We disclose a large overlapping transcription profile between XPC, KS and RDEB fibroblasts that points towards an activated phenotype with high matrix-synthetic capacity. This common signature seems to be independent of the primary causal deficiency, but reflects an underlying derangement of the extracellular matrix via transforming growth factor-ß signalling activation and oxidative state imbalance. What is the translational message? This study broadens the current knowledge about the pathology of these diseases and highlights new targets and biomarkers for effective therapeutic intervention. It is suggested that high levels of circulating periostin could represent a potential biomarker in RDEB.

POT1 germline mutations but not TERT promoter mutations are implicated in melanoma susceptibility in a large cohort of Spanish melanoma families.

BACKGROUND: Germline mutations in telomere-related genes such as POT1 and TERT predispose individuals to familial melanoma. OBJECTIVES: To evaluate the prevalence of germline mutations in POT1 and TERT in a large cohort of Spanish melanoma-prone families (at least two affected first- or second-degree relatives). METHODS: Overall, 228 CDKN2A wild-type melanoma-prone families were included in the study. Screening of POT1 was performed in one affected person from each family and TERT was sequenced in one affected patient from 202 families (26 families were excluded owing to DNA exhaustion/degradation). TERT promoter sequencing was extended to an additional 30 families with CDKN2A mutation and 70 patients with sporadic multiple primary melanoma (MPM) with a family history of other cancers. RESULTS: We identified four families with potentially pathogenic POT1 germline mutations: a missense variant c.233T>C (p.Ile78Thr); a nonsense variant c.1030G>T (p.Glu344*); and two other variants, c.255G>A (r.125_255del) and c.1792G>A (r.1791_1792insAGTA, p.Asp598Serfs*22), which we confirmed disrupted POT1 mRNA splicing. A TERT promoter variant of unknown significance (c.-125C>A) was detected in a patient with MPM, but no germline mutations were detected in TERT promoter in cases of familial melanoma. CONCLUSIONS: Overall, 1·7% of our CDKN2A/CDK4-wild type Spanish melanoma-prone families carry probably damaging mutations in POT1. The frequency of TERT promoter germline mutations in families with melanoma in our population is extremely rare.

Association between dermoscopic and reflectance confocal microscopy features of cutaneous melanoma with BRAF mutational status.

BACKGROUND: Melanomas harbouring common genetic mutations might share certain morphological features detectable with dermoscopy and reflectance confocal microscopy. BRAF mutational status is crucial for the management of metastatic melanoma. OBJECTIVES: To correlate the dermoscopic characteristics of primary cutaneous melanomas with BRAF mutational status. Furthermore, a subset of tumours has also been analysed for the presence of possible confocal features that might be linked with BRAF status. METHODS: Retrospectively acquired dermoscopic and confocal images of patients with melanoma in tertiary referral academic centres: Skin Cancer Unit in Reggio Emilia and at the Melanoma Unit in Barcelona. Kruskal-Wallis test, logistic regressions, univariate and multivariate analyses have been performed to find dermoscopic and confocal features significantly correlated with BRAF mutational status. RESULTS: Dermoscopically, the presence of irregular peripheral streaks and ulceration were positive predictors of BRAF-mutated melanomas with a statistically significance value, while dotted vessels were more represented in wild-type melanomas. None of the evaluated reflectance confocal microscopy features were correlated with genetic profiling. CONCLUSIONS: Ulceration and irregular peripheral streaks represent dermoscopic feature indicative for BRAF-mutated melanoma, while dotted vessels are suggestive for wild-type melanoma.

Genetic susceptibility to cutaneous melanoma in southern Switzerland: role of CDKN2A, MC1R and MITF.

BACKGROUND: Nearly 10% of all cases of cutaneous melanoma (CM) occur in patients with a personal or family history of the disease. OBJECTIVES: To obtain information about genetic predisposition to CM in Ticino, the southern region of Switzerland, a zone with moderate-to-high CM incidence. METHODS: We identified germline mutations in highly CM-associated genes (CDKN2A and CDK4) and low/medium-penetrance variants (MC1R and MITF) in patients with multiple primary CMs or individuals with one or more CM and a positive family history for CM or pancreatic cancer among first- or second-degree relatives. Healthy blood donors (n = 146) were included as a control group. RESULTS: From July 2010 to July 2012, 57 patients (41 pedigrees) were included. Twenty-six were melanoma-prone families (with at least two cases) and 15 had multiple CMs. Pancreatic cancer was found in six families. The CDKN2A mutation p.V126D was identified in seven patients (four families) with a founder effect, whereas CDKN2A A148T was detected in seven cases (five families) and seven healthy donors (odds ratio 2·76, 95% confidence interval 0·83-9·20). At least one MC1R melanoma-associated polymorphism was detected in 32 patients (78%) and 97 healthy donors (66%), with more than one polymorphism in 12 patients (29%) and 25 healthy donors (17%). The MITF variant p.E318K was identified in four patients from three additional pedigrees (7%) and one healthy control (0·7%). CONCLUSIONS: Inclusion criteria for the Ticino population for genetic assessment should follow the rule of two (two affected individuals in a family or a patient with multiple CMs), as we detected a CDKN2A mutation in almost 10% of our pedigrees (four of 41), MITF p.E318K in 7% (three of 41) and a higher number of MC1R variants than in the control population.

Mutational status of naevus-associated melanomas.

BACKGROUND: The origin of melanoma has always been a debated subject, as well as the role of adjacent melanocytic naevi. Epidemiological and histopathological studies point to melanomas arising either de novo or from a naevus. OBJECTIVES: To evaluate the presence of mutations in genes from well-known melanomagenesis pathways in a large series of naevus-associated melanomas. MATERIALS AND METHODS: Sixty-one melanomas found in association with a pre-existing naevus were microdissected, after careful selection of cell subpopulations, and submitted to Sanger sequencing of the BRAF, NRAS, c-KIT, PPP6C, STK19 and RAC1 genes. Each gene was evaluated twice in all samples by sequencing or by sequencing and another confirmation method, allele-specific fluorescent polymerase chain reaction (PCR) and capillary electrophoresis detection or by SNaPshot analysis. Only mutations confirmed via two different molecular methods or twice by sequencing were considered positive. RESULTS: The majority of cases presented concordance of mutational status between melanoma and the associated naevus for all six genes (40 of 60; 66.7%). Nine cases presented concomitant BRAF and NRAS mutations, including one case in which both the melanoma and the adjacent naevus harboured V600E and Q61K double mutations. In two cases, both melanoma and associated naevus located on acral sites were BRAF mutated, including an acral lentiginous melanoma. CONCLUSIONS: To our knowledge this is the largest naevus-associated melanoma series evaluated molecularly. The majority of melanomas and adjacent naevi in our sample share the same mutational profile, corroborating the theory that the adjacent naevus and melanoma are clonally related and that the melanoma originated within a naevus.

Dermoscopic criteria associated with BRAF and NRAS mutation status in primary cutaneous melanoma.

BACKGROUND: The identification of BRAF mutations in melanoma led to the development and implementation of new and effective therapies. Few clinical and histological features have been associated with this mutational status. OBJECTIVES: The main objective of this study was to investigate clinical, histopathological and dermoscopic characteristics of primary melanomas according to BRAF or NRAS mutational status. METHODS: An observational retrospective study including melanoma dermoscopy images assessed for somatic mutations in BRAF and NRAS. RESULTS: Seventy-two patients were included, 30 women (42%) and 42 men (58%), mean age was 59 ± 15.51 years. BRAF-mutated melanomas were more frequently located on the trunk (n = 18, 64% for BRAF-mutated vs. n = 11, 29% for wild-type melanomas, P = 0.013). Histological ulceration was associated with the presence of BRAF mutations [odds ratio (OR) 3.141; 95% confidence interval (CI) 1.289-7.655; P = 0.002]. The Breslow index tended to be thicker in BRAF-mutated compared with wild-type (P = 0.086). BRAF mutations were present in 28 (39%) patients and only four cases were positive for NRAS mutations (6%), BRAF and NRAS mutations being mutually exclusive. The presence of dermoscopic peppering was associated with MAPK mutations (BRAF and NRAS) (OR 1.68; 95% CI 1.089-2.581; P = 0.015). Dermoscopic ulceration was also associated with BRAF mutations excluding acral and facial melanomas (OR 2.64; 95% CI 1.032-6.754). CONCLUSIONS: This study showed a correlation between BRAF and NRAS status and dermoscopic findings of 'peppering' as an expression of regression and melanophages in the dermis, suggesting a morphological consequence of immune behaviour in BRAF-mutated melanomas.

Distribution of MC1R variants among melanoma subtypes: p.R163Q is associated with lentigo maligna melanoma in a Mediterranean population.

BACKGROUND: Cutaneous melanoma tumour is classified into clinicohistopathological subtypes that may be associated with different genetic and host factors. Variation in the MC1R gene is one of the main factors of risk variation in sporadic melanoma. The relationship between MC1R variants and the risk of developing a specific subtype of melanoma has not been previously explored. OBJECTIVES: To analyse whether certain MC1R variants are associated with particular melanoma subtypes with specific clinicohistopathological features. METHODS: An association study was performed between MC1R gene variants and clinicopathological subtypes of primary melanoma derived from 1679 patients. RESULTS: We detected 53 MC1R variants (11 synonymous and 42 nonsynonymous). Recurrent nonsynonymous variants were p.V60L (30·0%), p.V92M (11·7%), p.D294H (9·4%), p.R151C (8·8%), p.R160W (6·2%), p.R163Q (4·2%) p.R142H (3·3%), p.I155T (3·8%), p.V122M (1·5%) and p.D84E (1·0%). Melanoma subtypes showed differences in the total number of MC1R variants (P = 0·028) and the number of red hair colour variants (P = 0·035). Furthermore, an association between p.R163Q and lentigo maligna melanoma was detected under a dominant model of heritance (odds ratio 2·16, 95% confidence interval 1·07-4·37; P = 0·044). No association was found between p.R163Q and Fitzpatrick skin phototype, eye colour or skin colour, indicating that the association was independent of the role of MC1R in pigmentation. No association was observed between MC1R polymorphisms and other melanoma subtypes. CONCLUSIONS: Our findings suggest that certain MC1R variants could increase melanoma risk due to their impact on pathways other than pigmentation, and may therefore be linked to specific melanoma subtypes.

Genetic variations of patients with familial or multiple melanoma in Southern Brazil.

BACKGROUND: Patients with familial melanoma or multiple primary melanoma represent a high-risk population to hereditary melanoma. Mutations in susceptibility genes, such as CDKN2A, CDK4 and MC1R, have been associated with the development of melanoma. OBJECTIVES: The purpose of this study was to determine the genotypic background of patients with familial and/or multiple melanoma in southern Brazil. METHODS: This study analysed 33 cases (5 patients with multiple primary melanoma and 28 patients from families with at least two well documented cases) and 29 controls. Genomic analysis of CDKN2A and CDK4 genes by PCR-SSCP analysis and sequencing and direct sequencing of MC1R were performed in all individuals. RESULTS: No functional mutations in CDKN2A or CDK4 were detected in the 62 individuals. Infrequent variants in polymorphic loci of CDKN2A gene were identified in 15 participants (24.2%) and 24/33 (72.8%) cases and 19/27 (70.4%) controls reported at least one infrequent variant in MC1R (P = 0.372). Furthermore, a non-significant tendency towards an association between melanoma risk and MC1R variants G274A and C451T and a non-significant linear tendency to the number of infrequent high-risk variants in MC1R were observed. CONCLUSIONS: These results suggest that in southern Brazilian population, CDKN2A or CDK4 germinal alterations may have a weaker influence than previously thought and environmental risk factors may play a central role in melanoma susceptibility. However, considering the tendency observed for gene MC1R, low-penetrance genes may be a relevant aetiological factor in southern Brazil with fair skin population and high sunlight exposure.

Benefits of oral Polypodium Leucotomos extract in MM high-risk patients.

BACKGROUND: UV radiation and the presence of melanocytic nevi are the main risk factors of sporadic melanoma (MM). Protection of skin by an oral photoprotective agent would have substantial benefits. OBJECTIVE: We investigated the possible role of an oral Polypodium leucotomos (PL) extract to improve systemic photoprotection in patients at risk of skin cancer analyzing the ability to decrease UV-induced erythema. We also studied the interaction among MC1R polymorphisms and CDKN2A status with the minimal erythematous dose (MED) and their influence in the response after oral PL. METHODS: A total of 61 patients (25 with familial and/or multiple MM, 20 with sporadic MM and 16 with atypical mole syndrome without history of MM) were exposed to varying doses of artificial UVB radiation without and after oral administration of a total dose of 1080 mg of PL. RESULTS: Oral PL treatment significantly increased the MED mean in all group patients (0.123 to 0.161 J/cm(2) , p<0.05). Although not significant, we noticed a stronger effect of PL on the MED of patients with familial MM compared to those with MM (U=273, p=0.06). Among the patients with familial MM, those exhibiting a mutated CDKN2A and/or polymorphisms in MC1R had the bigger differences in response to treatment with PL. LIMITATIONS: Reduced number of patients. No control population. CONCLUSIONS: Administration of PL leads to a significant reduction of sensitivity to UVR (p<0.05) in all patients. Dark-eye patients and patients with higher UVR sensibility (lower basal MED) would be the most benefited from oral PL treatment.

CDKN2A mutations in melanoma families from Uruguay.

BACKGROUND: Familial melanoma, a cluster of several cases within a single family, accounts for approximately 10% of cases of melanoma. Hereditary melanoma is defined as two or more first-degree relatives having melanoma. A member of a melanoma-prone family has a 35-70-fold increased relative risk of developing a melanoma. Genetic susceptibility is linked to the major susceptibility genes CDKN2A and CDK4, and the minor susceptibility gene MC1R. OBJECTIVES: To determine the clinical and genetic characteristics of cutaneous melanoma in melanoma-prone families from Uruguay. METHODS: We studied 13 individuals from six melanoma-prone families living in Uruguay. Phenotype, familial and personal history were recorded. Molecular screening of CDKN2A and CDK4 was done by polymerase chain reaction-single strand conformational polymorphism analysis. The MC1R gene was sequenced. RESULTS: Mutations in CDKN2A were detected in five of six families: c.-34G>T, p.G101W and p.E88X. A novel germline mutation p.E88X, associated with hereditary melanoma in two unrelated families, is described. We hypothesize that a founder effect occurred probably in the Mediterranean region. No mutations in CDK4 were detected. Six different MC1R variants, all previously reported, were present in Uruguayan families. CONCLUSIONS: The overall rate of deleterious CDKN2A mutations in our familial melanoma pedigrees, even though the sample size is small, was considerably higher (83%) than the often quoted range.

Prognostic value of tyrosinase reverse transcriptase PCR analysis in melanoma sentinel lymph nodes: long-term follow-up analysis.

OBJECTIVE: To determine the prognostic value of detecting tyrosinase transcripts in melanoma sentinel lymph nodes (SLNs). METHODS: Reverse transcription (RT) PCR for tyrosinase mRNA was performed on negative SLNs of 76 patients with melanoma. RESULTS: Tyrosinase mRNA was found in 39 patients (51.3%). After a median follow-up period of 51 months, significant differences were found in overall survival (OS) but not in disease-free survival (DFS). The 5-year OS and DFS rates were 97.2% and 80%, respectively, for RT-PCR tyrosinase-negative (TN) patients vs. 78.67% and 66.24% for RT-PCR tyrosinase-positive (TP) patients (P = 0.019 and P = 0.38, respectively). Of four progressing patients in the TN group, three relapsed with subcutaneous, soft-tissue or lymph-node metastases, while seven out of nine progressing patients in the TP group relapsed at visceral sites. CONCLUSIONS: No significant differences in DFS were found by RT-PCR tyrosinase expression analysis at melanoma SLNs. Significant differences in OS could be related to a different pattern of relapse and must be confirmed after a longer follow-up time.

Mutation of the tumour suppressor p33ING1b is rare in melanoma.

BACKGROUND: The p33ING1b gene is involved in the p53-dependent response to DNA damage following exposure to ultraviolet radiation, and has recently been reported to be mutated in 20% of melanoma tumours. OBJECTIVES: We sought to assess the p33ING1b mutation rate in our large panels of fresh melanomas and melanoma cell lines. METHODS: We screened 83 primary melanomas and 55 melanoma cell lines for mutations in p33ING1b by single-strand conformational polymorphism analysis and by direct sequencing. RESULTS: In contrast to previous reports, we found no somatic p33ING1b mutations in our panel of melanomas. We found that some of the discrepancy between our results and previously published studies may be due to inadvertent amplification of the ING1 pseudogene (INGX), and/or contamination of some samples with murine Ing1. CONCLUSIONS: p33ING1b mutations in melanoma are rare. We have highlighted the importance of allele-specific primer design to avoid pseudogene amplification, and also the necessity to confirm the genetic identity and species of origin of individual cell lines. Further studies are needed to clarify the possible role of p33ING1b in melanoma tumorigenesis.