Lymph node mapping and sentinel lymph node biopsy are currently used to stage patients with cutaneous malignant melanoma. Immunohistochemical stains contribute to the detection of micrometastases; however, molecular biology techniques are associated with better diagnostic sensitivity. Sixty sentinel lymph nodes were included in this study. The primary lesions were malignant melanoma stage I or II, with a follow-up of longer than 2 years. Sentinel lymph nodes were studied with hematoxylin-eosin, immunohistochemistry for S-100 and HMB-45, and molecular biology techniques (reverse transcription (RT)-PCR) for the detection of tyrosinase messenger RNA. In 15 of 60 cases (25%), tyrosinase was detected by RT-PCR; three of these cases were also positive by immunohistochemistry. The population was divided into three groups: (i) hematoxylin-eosin-/immunohistochemistry+/molecular biology techniques+ (3 cases); (ii) hematoxylin-eosin-/immunohistochemistry-/molecular biology techniques+ (12 cases); (iii) hematoxylin-eosin-/immunohistochemistry-/molecular biology techniques- (45 cases). Correlation of the groups with overall survival showed the following: (i) 2 of 3 patients died (67%); (ii) 5 of 12 died (42%), and (iii) all 45 patients are alive, with no lymphadenectomy and a median follow-up of 84 months. The inclusion of molecular biology techniques appears to be of great value for the detection of sentinel lymph node micrometastases in patients with cutaneous malignant melanoma. In our series, those patients who showed negativity with all the three methods had a null recurrence rate. Therefore, this triple negativity could be a positive prognostic factor for overall survival. Our findings suggest the possibility of molecular oncological staging, which would allow the selection of patients with submicroscopic metastases for a complete treatment.
[Show abstract][Hide abstract] ABSTRACT: The main cause of death in melanoma patients is widespread metastases. Staging of melanoma is based on the primary tumor thickness, ulceration, lymph node and distant metastases. Metastases develop in regional lymph nodes, as satellite or in-transit lesions, or in distant organs. Lymph flow and chemotaxis is responsible for the homing of melanoma cells to different sites. Standard pathologic evaluation of sentinel lymph nodes fails to find occult melanoma in a significant proportion of cases. Detection of small numbers of malignant melanoma cells in these and other sites, such as adjacent to the primary site, bone marrow or the systemic circulation, may be enhanced by immunohistochemistry, reverse transcription PCR, evaluation of lymphatic vessel invasion and proteomics. In the organs to which melanoma cells metastasize, extravasation of melanoma cells is regulated by adhesion molecules, matrix metalloproteases, chemokines and growth factors. Melanoma cells may travel along external vessel lattices. After settling in the metastatic sites, melanoma cells develop mechanisms that protect them against the attack of the immune system. It is thought that one of the reasons why melanoma cells are especially resistant to killing is the fact that melanocytes (cells from which melanoma cells derive) are resistant to such noxious factors as ultraviolet light and reactive oxygen species. Targeted melanoma therapies are, so far, largely unsuccessful, and new ones, such as adjuvant inhibition of melanogenesis, are under development.
Expert Review of Dermatology 11/2008; 3(5):569-585. DOI:10.1586/17469818.104.22.1689
[Show abstract][Hide abstract] ABSTRACT: Molecular techniques have provided us with a wealth of information about biological events in healthy individual, and improved tremendously our understanding about the pathogenesis of a huge variety of cutaneous diseases. Those methods have originally been invented to support basic scientific investigations on a molecular level and are translated increasingly into sophisticated diagnostic tools changing the classic paradigm of diagnostic pathology; among them are immunohistochemistry (IHC), polymerase chain reaction (PCR), G-banding, loss of heterozygosity, fluorescence in situ hybridization (FISH), chromogen in situ hybridization (CISH), comparative genomic hybridization on chromosomes and microarray technology. Some of them such as IHC and PCR have already been standardized to a level that allows its utility in daily routine diagnostics for several dermatological diseases. For others like array-based technologies, their optimal indications await to be fully determined. These ancillary methods have the great potential to contribute important new information to challenging cases, and will help to improve diagnostic accuracy particularly in cases in which conventional histopathology is ambiguous. Thus, they will broaden our armamentarium for diagnostic pathology.
Herein, some key techniques will be reviewed and their applicability towards the diagnosis of dermatological diseases critically discussed.
[Show abstract][Hide abstract] ABSTRACT: Sentinel lymph node (SLN) biopsy has become integral in the staging of patients with melanoma, and entails detailed histologic examination with immunohistochemistry. Reverse transcriptase-polymerase chain reaction (RT-PCR) for tyrosinase transcripts has been used to increase sensitivity but requires a dedicated piece of tissue that does not undergo histologic examination. We developed a nested RT-PCR assay for tyrosinase applicable on paraffin-embedded tissue and applied this to a series of SLNs from pediatric patients with melanoma. Thirty-six SLNs from 4 females and 4 males were included in the study. Eight lymph nodes with reactive changes were included as controls. SLNs were examined histologically and immunohistochemically for S100, tyrosinase, and MART1. Seven patients had between 1 and 4 morphologically-positive SLNs and one patient had negative SLNs (HISTO+; 12/36, 33%). Three lymph nodes were excluded from molecular analysis owing to inadequate RNA, and 29 of the remaining 33 nodes were positive (MOL+; 88%). All patients had at least 1 SLN positive by RT-PCR. Twelve were HISTO+/MOL+; 17 were HISTO-/MOL+; and 4 were HISTO-/MOL-. All control lymph nodes were negative for tyrosinase transcripts. The application of RT-PCR for tyrosinase to paraffin-embedded tissue significantly increased the number of positive SLNs and upstaged one patient from negative to positive. The prognostic implications of such findings require further investigation, especially in the pediatric age group. Nonetheless, this technique provides a useful tool to determine the clinical significance of RT-PCR positivity in melanoma SLNs.
Applied immunohistochemistry & molecular morphology: AIMM / official publication of the Society for Applied Immunohistochemistry 07/2010; 18(4):365-70. DOI:10.1097/PAI.0b013e3181ce1e61 · 2.01 Impact Factor
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