ABSTRACT
The ST18 -497-65050â¯Tâ¯>â¯C polymorphisms (rs17315309) exhibit a very strong association in the pathogenesis of Pemphigus Vulgaris (PV) and could represent a new potential molecular target for the treatment of disease. The present study aimed to establish a low-cost, sensitive and reliable assay using high-resolution melting curve analysis (HRMA) on magnetic induction rotor-based platform, the Magnetic Induction Cycler (MIC) (Bio molecular Systems). HRMA assay was able to identify easily and unambiguously the c.-497-65050â¯Tâ¯>â¯C genotypes evaluating melting curve shape and melting temperature (Tm). The results of HRMA were validated by direct DNA sequencing. The HRMA is rapid, sensitive, low-cost and high-throughput assay to screen the rs17315309 variant and could be used in clinical diagnostic laboratories.
Subject(s)
Genetic Predisposition to Disease/genetics , Nucleic Acid Denaturation , Pemphigus/genetics , Polymorphism, Single Nucleotide , Repressor Proteins/genetics , Genotype , Humans , Molecular Diagnostic Techniques , Pemphigus/diagnosis , Reproducibility of Results , Risk Factors , Sensitivity and Specificity , Sequence Analysis, DNA/methodsABSTRACT
OBJECTIVE: The aim of this study was to evaluate if our molecular algorithm, based on tumor circulating transcripts, may predict relapse risk in cutaneous malignant melanoma (CMM). RESULTS: The multi-marker panel was able to differentiate patients with CMM from HC with high diagnostic sensitivity and specificity, especially for MITF-m and TGFB2 (91-100%) whose levels decreased during follow-up of recurrence-free patients, and remained stable in the case of relapse. PAX3d higher than 2.76 copies/µL emerged as a promising biomarker [specificity = 75-93% and negative predictive value = 75-98%] to stratify subjects at high risk of CMM recurrence independently of age, gender and AJCC staging [OD = 9.5(3.2-28.0), p < 0.001]. The survival analysis confirmed PAX3d performance in relapse prediction with significant differences in recurrence risk 12 months after the basal time-point (p = 0.008). MATERIALS AND METHODS: Peripheral blood was collected from 111 CMM patients and from 87 healthy controls (HC) randomly selected. Each specimen was examined by qRT-PCR analysis for the expression of 3 tumor-related transcripts (PAX3d, MITF-m and TGFB2) at diagnosis, and at the following 6 and 12 months during clinical monitoring. CONCLUSIONS: We demonstrated the usefulness of our molecular algorithm to indirectly detect circulating melanoma cells in blood, along with PAX3d capability to assess patients' progression and relapse prediction.
ABSTRACT
BACKGROUND: IGF-I-(CA) repeats have been previously analysed in few types of cancer and the results, although discordant in different studies, showed possible associations between cancer and IGF-I(CA)(19) repeats. Aim of this pilot study was to detect a possible association between some of the IGF-I(CA) repeats and the presence of malignant melanoma and its Breslow index. METHODS: Two hundred patients affected with cutaneous malignant melanoma and 100 control healthy subjects were analysed for IGF-I(CA) repeats by fragment analysis sequencing and, partially, confirmed by direct sequencing. RESULTS: A significant association of IGF-I(CA)(19) repeats was observed with melanoma higher Breslow indices (P<0.001), while no association between melanoma patients and the different genotypes of IGF-I(CA) was found. The above mentioned association was confirmed after Bonferroni's correction for multiple comparisons and also by logistic regression analysis adjusted for age, sex and BMI variables. A slight, significant difference (P=0.03) was observed for serum IGF-I values in IGF-I(CA)(19)-positive or IGF-I(CA)(19)-negative subjects. DISCUSSION: The association observed for IGF-I(CA)(19) and malignant melanoma is in keeping with similar results obtained in prostate or breast cancers, suggesting that this type of repeat may be directly or indirectly important in controlling cancer induction and its severity.
Subject(s)
Insulin-Like Growth Factor I/genetics , Melanoma/genetics , Adult , Aged , Base Sequence , DNA Primers , Female , Humans , Male , Melanoma/pathology , Middle Aged , Pilot Projects , Polymorphism, GeneticABSTRACT
BACKGROUND: The molecular monitoring of circulating tumor cells by reverse transcriptase-PCR (RT-PCR) for patients with melanoma, is still under debate. It may be affected by: a) pre-analytical variability, b) frequency of melanoma-associated gene transcripts and c) the reliability of the methods employed. Few commercial methods are available for the detection of tyrosinase mRNA in blood. OBJECTIVE: Comparison between two RT-PCR-nested methods with a third one based on real-time methodology, for detection and quantitation of tyrosinase transcripts, respectively. METHODS: Sixty-two melanoma patients with different AJCC stages and 20 healthy subjects were enrolled. All blood samples were extracted in duplicate with two different methods. Two nested-PCR methods (one commercial and one in house) plus a real time commercial kit were employed. RESULTS: The two nested PCR methods employed were overimposable, specific and sensitive, at least in the stage III, where there was a concordance between sentinel lymph nodes detection and blood tyrosinase positivity. The different extraction methods did not affect the quality of results, while the commercial real-time kit cannot be used. CONCLUSION: Tyrosinase mRNA detection may be therefore employed to monitor the melanoma patients over time in function of response to therapy.