Sentinel node in melanoma patients: triple negativity with routine techniques and PCR as positive prognostic factor for survival.
ABSTRACT 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.
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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. · 1.63 Impact Factor
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ABSTRACT: Despite their relevance in clinical medicine, the extension and activity of the bone marrow (BM) cannot be directly evaluated in vivo. We propose a new method to estimate these variables by combining structural and functional maps provided by CT and PET. BM extension and glucose uptake were estimated in 102 patients undergoing whole-body PET/CT because of a history of nonmetastatic melanoma. Image analysis assumed that the BM is surrounded by compact bone. An iterative optimization scheme was applied to each CT slice to identify the external border of the bone. To identify compact bone, the algorithm measured the average Hounsfield coefficient within a two-pixel ring located just inside the bone contour. All intraosseous pixels with an attenuation coefficient lower than this cut-off were flagged as 1, while the remaining pixels were set at 0. Binary masks created from all CT slices were thus applied to the PET data to determine the metabolic activity of the intraosseous volume (IBV). Estimated whole-body IBV was 1,632 ± 587 cm(3) and was higher in men than in women (2,004 ± 498 cm(3) vs. 1,203 ± 354 cm(3), P < 0.001). Overall, it was strictly correlated with ideal body weight (r = 0.81, P = 0.001) but only loosely with measured body weight (r = 0.43, P = 0.01). The average FDG standardized uptake value (SUV) in the thoracic and lumbar vertebrae was 2.01 ± 0.36, Accordingly, intraosseous voxels with SUV ≥ 1.11 (mean spine SUV - 2.5 × SD) were considered as active "red" BM and those with SUV <1.11 as "yellow" BM. Estimated red BM volume was 541 ± 195 ml, with a higher prevalence in the axial than in the appendicular skeleton (87 ± 8 % vs. 10 ± 8 %, P < 0.001). Again, red BM volume was higher in men than in women (7.8 ± 2.2 vs. 6.7 ± 2.1 ml/kg body weight, P < 0.05), but in women it occupied a greater fraction of the IBV (32 ± 7 % vs. 36 ± 10 %, P < 0.05). Patient age modestly predicted red BM SUV, while it was robustly and inversely correlated with red BM volume. Our computational analysis of PET/CT images provides a first estimation of the extension and metabolism of the BM in a population of adult patients without haematooncological disorders. This information might represent a new window to explore pathophysiology the BM and the response of BM diseases to chemotherapy.European Journal of Nuclear Medicine 05/2012; 39(8):1326-38. · 4.53 Impact Factor
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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.Experimental Dermatology 11/2008; 18(1):12-23. · 3.58 Impact Factor