Detection Of Tyrosinase mRNA Research Articles

Tumor-Related Methylated Cell-Free DNA and Circulating Tumor Cells in Melanoma.

Solid tumor release into the circulation cell-free DNA (cfDNA) and circulating tumor cells (CTCs) which represent promising biomarkers for cancer diagnosis. Circulating tumor DNA may be studied in plasma from cancer patients by detecting tumor specific alterations, such as genetic or epigenetic modifications. Ras association domain family 1 isoform A (RASSF1A) is a tumor suppressor gene silenced by promoter hypermethylation in a variety of human cancers including melanoma. The aim of the present study was to assess the diagnostic performance of a tumor-related methylated cfDNA marker in melanoma patients and to compare this parameter with the presence of CTCs. RASSF1A promoter methylation was quantified in cfDNA by qPCR in a consecutive series of 84 melanoma patients and 68 healthy controls. In a subset of 68 cases, the presence of CTCs was assessed by a filtration method (Isolation by Size of Epithelial Tumor Cells, ISET) as well as by an indirect method based on the detection of tyrosinase mRNA by RT-qPCR. The distribution of RASSF1A methylated cfDNA was investigated in cases and controls and the predictive capability of this parameter was assessed by means of the area under the ROC curve (AUC). The percentage of cases with methylated RASSF1A promoter in cfDNA was significantly higher in each class of melanoma patients (in situ, invasive and metastatic) than in healthy subjects (Pearson chi-squared test, p < 0.001). The concentration of RASSF1A methylated cfDNA in the subjects with a detectable quantity of methylated alleles was significantly higher in melanoma patients than in controls. The biomarker showed a good predictive capability (in terms of AUC) in discriminating between melanoma patients and healthy controls. This epigenetic marker associated to cfDNA did not show a significant correlation with the presence of CTCs, but, when the two parameters are jointly considered, we obtain a higher sensitivity of the detection of positive cases in invasive and metastatic melanomas. Our data suggest that cell-free tumor DNA and CTCs represent two complementary aspects of the liquid biopsy which may improve the diagnosis and the clinical management of melanoma patients.

Circulating tyrosinase mRNA detection in a preclinical xenograft mouse melanoma model and in advanced melanoma patients

22204 Background: Although it is still under debate, molecular detection of circulating Tyrosinase is believed to be a good prognostic marker for melanoma progression. Methods: We have designed high-affinity primers to amplify the human Tyrosinase mRNA sequence to test the value of molecular detection of circulating melanoma cells by reverse transcription-PCR. The optimization process included in vitro settings and in vivo studies in a xenograft mouse model. Results: We were able to detect Tyrosinase expression with as few as 40 pg and 1.5 pg of total RNA from SKmel human melanoma cells, using PCR and nested PCR, respectively. Human Tyrosinase expression was found in the blood of nude mice bearing s.c. SKmel tumors, and expression bands were stronger after manipulation of the tumor mass. In this melanoma model, Tyrosinase expression declined in blood 6 hours after a direct intravenous injection of SKmel cells. A preliminary study in human blood samples demonstrated a baseline positive Tyrosinase determination in 64% (16/25) of advanced melanoma patients using the RT-PCR nested assay. Baseline Tyrosinase expression was significantly associated with disease progression after 12 months, and sequential determination during follow up of the remaining disease-free patients showed a progressive increase of negative results. Conclusions: We have developed sequential in vitro and in vivo preclinical validations to improve the optimization of the detection system. Our data indicate that molecular detection of circulating Tyrosinase mRNA in advanced melanoma patients could be considered a prognostic parameter. No significant financial relationships to disclose.

Detection of tyrosinase mRNA in the sentinel lymph nodes of melanoma patients is not a predictor of short-term disease recurrence

Detection of tyrosinase mRNA in the sentinel lymph nodes of melanoma patients is not a predictor of short-term disease recurrence

Detection of tyrosinase mRNA in the sentinel lymph nodes of melanoma patients is not a predictor of short-term disease recurrence

AbstractSentinel lymph node evaluation has enabled identification of patients with cutaneous melanoma who might benefit from elective regional lymph node dissection. Sentinel nodes are currently assessed by histologic and reverse transcription polymerase chain reaction (RT–PCR) evaluation for melanocyte-specific markers. The clinical significance of positive findings by RT–PCR in the absence of histologic evidence of metastasis (HISNEG/PCRPOS) remains unclear. Examination of 264 lymph nodes from 139 patients revealed histopathologic positivity in 34 patients (24.5%), in which 26 also demonstrated simultaneous RT–PCR positivity (HISPOS/PCRPOS). Of 35 HISNEG/PCRPOS patients (25.2%), five also had nodal capsular nevi. In total, capsular nevi were detected in 13 patients (9.4%). A total of 70 patients (50.4%) had negative sentinel nodes by both histopathology and RT–PCR (HISNEG/PCRNEG). Over a median follow-up of 25 months, local and/or systemic recurrence developed in 31 patients (22.3%). Recurrence rates were similar among patients with histopathologic evidence of sentinel lymph node metastasis, irrespective of RT–PCR status (HISPOS/PCRPOS 62%; HISPOS/PCRNEG 75%). In contrast, only 10% of HISNEG/PCRNEG patients developed recurrence, significantly less than those in either HISPOS group (P<0.0001). Recurrence in the HISNEG/PCRPOS/CNNEG group (7.7%) was comparable to that in HISNEG/PCRNEG patients and significantly lower than that in either HISPOS group (P<0.0001). The only independent prognostic factors identified by multivariate analysis were the Breslow thickness of the primary tumour and histopathologic positivity of sentinel nodes. Our findings support previous observations that histopathologic evidence of metastatic melanoma in sentinel lymph nodes is an independent predictor of disease recurrence. In contrast, detection of tyrosinase mRNA by RT–PCR alone does not appear to increase the likelihood of short-term disease recurrence.

Comparison between three molecular methods for detection of blood melanoma tyrosinase mRNA. Correlation with melanoma stages and S100B, LDH, NSE biochemical markers

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.

Molecular Staging in Stage II and III Melanoma Patients and Its Effect on Long-Term Survival

To assess the prognostic value of serial reverse transcriptase polymerase chain reaction (RT-PCR) -based measurements of tyrosinase mRNA in peripheral blood of stage II and III melanoma patients. During routine follow-up of American Joint Committee on Cancer stage II and III melanoma patients, serial testing for tyrosinase transcripts in peripheral blood was performed by RT-PCR. The PCR results were compared with the clinical data collected during the follow-up. Over a period of 3 years, 111 patients (78 stage II and 33 stage III patients) were enrolled, and tyrosinase determinations were carried out. The 6-year disease-specific survival probability was 97% for patients always showing negative RT-PCR results and 67% for patients who tested positive at least once. In a Cox proportional hazards model, the prognostic value of sex, age, site of primary tumor, histologic subtype, stage, Breslow's tumor thickness, Clark level, and the time-dependent variable PCR result was assessed. Patients with a positive RT-PCR test had a distinctly higher risk of dying from melanoma, with a hazard ratio of 12.6 (95% CI, 3.4 to 46.3; P < .001). Our study shows a strong association between PCR and disease-specific survival time. Detection of tyrosinase mRNA in peripheral blood may be of similar importance for the clinical course of melanoma as the detection of micrometastatic disease in the sentinel lymph node. Whether a combination of these two factors leads to a better definition of the prognosis of melanoma patients is under investigation in current studies.

Vitiligo: analysis of grafting versus curettage alone, using melanocyte morphology and reverse transcriptase polymerase chain reaction for tyrosinase mRNA

Recent studies have indicated that vitiligo areas contain inactive or dormant melanocytes. Melanin synthesis is related to tyrosinase presence and indicative of active metabolic state. The aim of this study was to compare repigmentation, epidermal melanocyte distribution and tyrosinase mRNA detection through reverse transcriptase polymerase chain reaction, in tissue samples of vitiligo, before and after curettage, with or without subsequent autologous skin graft using a new method. Prospective, in the Department of Dermatology, Faculdade de Medicina do ABC, Santo André. Two vitiligo areas were curetted. One subsequently received grafted normal sacral autologous skin, whereas the other had no further treatment. The curetted areas were examined after 30 days, to evaluate the degree of repigmentation. The melanocyte percentages and tyrosinase mRNA presence in normal skin and vitiligo areas, before and after curettage and grafting, were compared. Complete repigmentation was seen in all grafted areas, whereas non-grafted curetted vitiligo presented partial repigmentation. The melanocyte percentage in grafted areas was greater than in non-treated vitiligo skin (p = 0.01) and skin with curettage alone (p = 0.015). Tyrosinase mRNA was negative in 93.75% of non-treated vitiligo areas. After treatment (curettage alone or curettage and grafting), all lesions became positive for tyrosinase mRNA. Metabolically inactive or dormant melanocytes are probably present within vitiligo areas, and may be activated by exogenous or endogenous stimuli.

Prognostic significance of detecting micrometastases by tyrosinase RT/PCR in sentinel lymph node biopsies: lessons from 322 consecutive melanoma patients

This prospective study was performed to determine the prognostic value of tyrosinase mRNA detection in sentinel lymph nodes (SLN) of melanoma patients. About 847 SLNs from 322 consecutive patients were assessed by histopathology and immunohistochemistry as well as tyrosinase-reverse transcriptase-polymerase chain reaction (RT/PCR) for the presence of micrometastases. The results were correlated with the prognostic parameters employing a multivariate analysis after a median follow-up of 37 months. Histopathological analysis revealed metastases in 34/322 patients (10.6%). Among the 288 patients with histopathologically negative SLN, tyrosinase-mRNA was detected in 39 patients. A relapse of the tumour occurred in 44.1% of the patients with histopathologically positive SLN, in 25.6% with histopathologically negative, but tyrosinase-RT/PCR-positive SLN, and 8.0% with “double-negative” SLN. A multivariate analysis identified tumour thickness, the histopathological SLN status, and the ulceration of the primary tumour as independent prognostic factors. Thus, by assessing tyrosinase mRNA in the SLN of melanoma patients, we identified a subgroup with histopathologically negative, but Tyr-RT-PCR-positive SLN who have a high risk of disease relapse.

Tyrosinase RT-PCR as a Supplement to Histology for Detecting Melanoma and Nevus Cells in Paraffin Sections of Sentinel Lymph Nodes

The detection of tyrosinase mRNA in sentinel lymph nodes (SLNs) by reverse transcription polymerase chain reaction (RT-PCR) is a sensitive indicator for the presence of melanoma or nevus cells, but it does not enable a distinction between both. We have established an efficient method for extraction and reverse transcription of tyrosinase mRNA from paraffin sections that permits the close correlation of the RT-PCR results with (immuno)histologic findings in adjacent sections. One hundred fifty-three SLNs and 6 non-SLN specimens originating from 92 melanoma and 4 nonmelanoma patients were studied to test the reliability of this approach. The predictive value of positive RT-PCR results was 0.98 for the presence of melanoma or nevus cells; the corresponding negative predictive value was 0.83. Furthermore, the detection rate of tyrosinase mRNA significantly correlated with tumor burden. Among the 33 melanoma-positive SLNs without nevus cells, positive RT-PCR results were obtained in all specimens with extended peripheral (S2) or deeply invasive (S3) micrometastases but in only 46% of the cases with few localized melanoma cells in the subcapsular zone (S1). Routine (immuno)histologic evaluation alone had missed microclusters of melanoma cells in one SLN and small nevus cell aggregates in six other SLNs. They were detected only during microscopic reexamination caused by a positive RT-PCR result. We conclude that histology and immunohistochemistry remain the indispensable gold standard for the identification of melanoma and nevus cells in SLNs. Additional molecular analyses using adjacent paraffin sections may further improve the diagnostic accuracy by sensitizing and guiding the microscopist’s attention.

The detection of tyrosinase mRNA in peripheral blood samples is unlikely to aid in the management of patients with localised malignant melanoma

A number of authors have reported the detection of tyrosinase mRNA in the peripheral blood of patients with malignant melanoma using the reverse transcription polymerase chain reaction (RT-PCR). The precise value of this assay as a prognostic tool, however, remains in doubt. This is particularly so with relation to localised disease, where relatively little data has been accumulated. In this study we analysed the peripheral blood of 50 consecutive patients with primary malignant melanoma referred to a plastic surgical centre with the facility of a pigmented lesion clinic. Samples were analysed from an additional 35 patients with advanced melanoma disease and 35 patients with benign pigmented cutaneous lesions. We were able to identify tyrosinase transcripts in the peripheral blood of only two of 50 patients with localised disease. Of those with more advanced disease, a positive finding was found in three with regional disease and four patients with metastatic spread. Stage of disease was found to correlate significantly with PCR status. No correlation was identified with other prognostic markers or with outcome over a three-year period. This data would support the conclusion that the detection of tyrosinase mRNA in peripheral blood is likely to be of little value as an aid in the management of patients with early malignant melanoma.

Tyrosinase-mRNA in verschiedenen Kompartimenten als Tumormarker des malignen Melanoms

Der valide, prospektive Nachweis einer Tumorzellaussaat zum Zeitpunkt der Primärexzision ist bisher bei Patienten mit malignem Melanom nicht möglich. Tyrosinase ist ein Enzym der Melanogenese, dessen Expression durch RT-PCR bestimmt werden kann. Untersuchungen im Blut erbrachten jedoch selbst bei Patienten mit manifesten Fernmetastasen in neueren Publikationen nur bei einem Teil der Patienten positive Werte. Es stellte sich die Frage, ob durch Bestimmung in anderen Kompartimenten zum Zeitpunkt des Eintritts ins klinische Stadium der Fernmetastasierung vor Beginn einer systemischen Therapie die Prävalenz positiver Werte gesteigert werden kann. Durch die zusätzliche Bestimmung von Tyrosinase-mRNA im Knochenmark ließ sich die, durch Bestimmung im Blut erzielte Prävalenz jedoch nur unwesentlich steigern. Auch im Hinblick auf die mittlere Überlebenszeit unterschieden sich die positiven Patienten mit metastasiertem Melanom nicht von den Patienten mit metastasiertem Melanom, bei denen kein Nachweis in Blut oder Knochenmark möglich war. In dieser Konstellation kommt der Detektion von Tyrosinase-mRNA durch RT-PCR keine Rolle als Tumormarker zu.

Prognosis of metastatic melanoma: no correlation of tyrosinase mRNA in bone marrow and survival time.

Recent publications suggest that tyrosinase mRNA in blood as well as in bone marrow is detectable only in a subgroup of patients with metastatic melanoma. This would imply that tyrosinase mRNA is of limited value as a tumor marker. We addressed the question of whether patients with metastatic melanoma and RT-PCR-detectable tyrosinase mRNA in blood or bone marrow have a different prognosis than tyrosinase mRNA-negative patients. Twenty melanoma patients with widespread clinical metastases were enrolled; the survival time after first diagnosis of visceral metastases was correlated to tyrosinase mRNA presence in blood and bone marrow samples. The time of survival of eight patients with metastatic melanoma and detectable tyrosinase mRNA in either blood or bone marrow was not different from the prognosis of 12 patients without detectable tyrosinase mRNA in either blood or bone marrow. Detection of tyrosinase mRNA in blood or bone marrow samples of melanoma patients with advanced disease seems to have no substantial relevance for survival time and outcome of disease. In this constellation, detection of tyrosinase mRNA by RT-PCR is not a valid tumor marker. Nevertheless, tyrosinase positivity in bone marrow in earlier tumor stages might indicate increased risk for the development of distant metastases. This should be addressed in further studies.

Facts and pitfalls in the detection of tyrosinase mRNA in the blood of melanoma patients by RT-PCR.

Reverse transcription (RT) of tyrosinase mRNA and specific cDNA amplification to facilitate the early detection of circulating tumor cells in melanoma patients have been reported. The significance and practical value of these procedures for the diagnosis of tumor dissemination in melanoma patients is, however, still unclear. We analyzed peripheral blood samples of melanoma patients of different clinical stages for the presence of tyrosinase mRNA by reverse transcriptase polymerase chain reaction (RT-PCR). In addition to a nested RT-PCR-based system, we evaluated the new PCR enzyme-linked immunosorbent assay tyrosinase system for sensitivity and specificity in detecting circulating melanoma cells. Our results showed a high sensitivity and specificity for this system in detecting one melanoma cell in 1 ml of whole blood. Using different methods of detection, no tyrosinase mRNA was detectable in blood samples of patients with primary melanoma and regional lymph node metastases. In a small number of patients with visceral metastases (10-30%), we found tyrosinase mRNA-positive results. Analyses of different blood samples taken at 2-h intervals indicate that tumor cells persist only transiently in the peripheral blood. Successful establishment of melanoma cell growth from tyrosinase mRNA-positive samples indicates that viable tumor cells exist in melanoma patients' peripheral blood. Our results indicate a low amount of tyrosinase-specific transcripts in a small subset of stage IV patients and suggest that the analysis of tyrosinase mRNA in peripheral blood samples is not helpful as a prognostic marker or monitoring tool in melanoma patients.

The detection of tyrosinase mRNA in the peripheral blood of stage I melanoma patients is not of clinical relevance in predicting metastasis risk and survival

The presence of tyrosinase mRNA in the peripheral blood cells of melanoma patients has been recently studied as a possible marker of haematogenous dissemination. However, considerable variations in the rates of detection have been noted. We determined the presence of tyrosinase mRNA-positive circulating cells using reverse transcriptase-polymerase chain reaction (RT-PCR) in 35 patients with stage I melanoma, two patients with stage II melanoma and two patients with stage III melanoma. Among the patients with stage 1, 13 were tested before and after surgery (< 1 h). Twenty healthy subjects served as negative controls. Out of the melanoma patients, the tyrosinase gene was expressed in three of the 52 samples tested. Tyrosinase mRNA was present in the circulating cells of only one patient with stage I melanoma after intra-congenital naevi resection. However, two other stage I patients developed rapidly lethal metastasis within the following 6 months, despite the lack of detectable tyrosinase mRNA. None of stage II patients were positive for the tyrosinase transcripts, while both patients with stage III melanoma showed enzyme expression. Our results confirm those of previous studies, showing that a small proportion of stage I melanoma patients have tyrosinase-positive circulating cells. Moreover, the lack of tyrosinase mRNA detection in the blood does not necessarily exclude metastatic progression. Therefore, this study indicates that the detection of tyrosinase mRNA-positive circulating cells by RT-PCR is not a predictive biomarker of a metastasis risk in patients with stage I melanoma.

The detection of tyrosinase mRNA in the peripheral blood of stage I melanoma patients is not of clinical relevance in predicting metastasis risk and survival

The detection of tyrosinase mRNA in the peripheral blood of stage I melanoma patients is not of clinical relevance in predicting metastasis risk and survival

No Correlation of Tyrosinase mRNA in Bone Marrow with Prognosis of Metastatic Melanoma

Background: Recent publications suggest that tyrosinase mRNA in blood as well as in bone marrow is detectable only in a subgroup of patients with metastatic melanoma. Objective: We addressed the question, whether patients with metastatic melanoma and with RT-PCR-detectable tyrosinase mRNA in blood or bone marrow have a different prognosis compared to tyrosinase mRNA-negative patients. Methods: 20 melanoma patients with widespread clinical metastases were enrolled and the survival time after first diagnosis of visceral metastases was correlated to tyrosinase mRNA presence in blood and bone marrow samples. Results: The time of survival of 8 patients with metastatic melanoma and detectable tyrosinase mRNA in either blood or bone marrow was not different from the prognosis of 12 patients without detectable tyrosinase mRNA in either blood or bone marrow. Conclusion: Although based on a limited number of patients our results suggest that detection of tyrosinase mRNA in blood or bone marrow samples of melanoma patients with advanced disease seems to have no substantial relevance for survival time and outcome of disease. For this purpose, detection of tyrosinase mRNA by RT-PCR is not a valid tumor marker. Nevertheless, tyrosinase positivity in bone marrow in earlier tumor stages might indicate increased risk for the development of distant metastases. This should be addressed in further studies.

Detection of tyrosinase mRNA by RT-PCR in the peripheral blood of patients with advanced metastatic melanoma.

Detection of melanoma cells in the peripheral blood has been facilitated by the reverse transcriptase-polymerase chain reaction (RT-PCR), but their presence is of uncertain importance in the evolution of the disease. We studied the detection of melanoma cells using RT-PCR in the peripheral blood of 21 patients, four with regional lymph node metastases (American Joint Committee on Cancer [AJCC] stage III) and 17 with disseminated disease (AJCC stage IV). RNA was extracted from 10 ml of heparinized blood following density gradient centrifugation and converted into cDNA for PCR analysis. Assay sensitivity of 10 cells in 10(7) mononuclear cells and granulocytes obtained from 10 ml of peripheral blood was achieved using the G361 and C32 melanoma cell lines. Tyrosinase mRNA was not detected in control samples from healthy volunteers or patients with non-malignant disease. Six patients (one stage III, five stage IV) tested positive for tyrosinase mRNA (28.6%); with one exception, all patients were receiving chemotherapy at the time of sampling. Of the six positive results, three were from patients who initially tested negative but were subsequently positive after a 3-4 week interval. The low detection rates of melanoma cells in the peripheral blood of patients with widely disseminated disease is consistent with recent reports and correlates poorly with the clinical stage of melanoma. This may be partly explained by the clinically observed intermittent and random evolution of melanoma metastases.

P29 Detection of tyrosinase mRNA by RT-PCR in peripheral blood of patients with melanoma

P29 Detection of tyrosinase mRNA by RT-PCR in peripheral blood of patients with melanoma

Detection of tyrosinase mRNA by reverse transcription-polymerase chain reaction in melanoma sentinel nodes.

Sentinel lymph node (SLN) biopsy is an alternative to elective dissection or observation for management of lymph node basins in patients with cutaneous melanomas. The detection of tyrosinase mRNA in melanoma SLN specimens by reverse transcription-polymerase chain reaction (RT-PCR) has been reported to be a more sensitive method to detect subclinical metastases, compared with histological analysis. The aims of this study were to (1) define the yield of RT-PCR in assessing SLNs, compared with histological analysis, (2) identify the incidence of false-positive results in SLNs, and (3) report the rate of actin PCR negativity (i.e., samples with degraded RNA) in SLNs. Twenty-eight patients with 1.2-9.6-mm cutaneous melanomas underwent SLN biopsy (between October 1996 and March 1997). One half of each SLN was analyzed by nested RT-PCR for tyrosinase mRNA. The other half of the SLN was examined by routine microscopy. Twenty-one lymph nodes from patients without melanoma were evaluated as controls. Two of the 28 patients with melanoma were excluded because of RNA degradation, as indicated by actin negativity. Six of the remaining 26 patients exhibited melanoma metastases in routine histological examinations. All histologically positive lymph nodes were RT-PCR-positive. Thirteen of the 20 (65%) histologically negative cases were RT-PCR-positive. Of 21 control lymph nodes, 3 were actin-negative and were not assessable for tyrosinase mRNA. Two of the remaining 18 (11%) negative-control nodes were RT-PCR-positive. Among patients undergoing SLN biopsy, tyrosinase mRNA was detectable in 73% of SLNs from patients at risk for regional nodal metastases, including all of those with histologically positive SLNs. There is a definable false-positive rate for tyrosinase mRNA detection in the lymph nodes of patients who do not have melanoma. Actin verification of RNA integrity is necessary to ensure the accuracy of this test in detecting tyrosinase mRNA. Ongoing follow-up monitoring will define the prognostic value of this assay.

Detection of tyrosinase mRNA in paraffin-embedded sections of melanoma, using RT in situ PCR

Departments of Pathology and Laboratory Medicine and of Surgery, Jonsson Comprehensive Cancer Center, University of California Los Angeles, Los Angeles, USA