Final trial report of sentinel-node biopsy versus nodal observation in melanoma.

BACKGROUND: Sentinel-node biopsy, a minimally invasive procedure for regional melanoma staging, was evaluated in a phase 3 trial. METHODS: We evaluated outcomes in 2001 patients with primary cutaneous melanomas randomly assigned to undergo wide excision and nodal observation, with lymphadenectomy for nodal relapse (observation group), or wide excision and sentinel-node biopsy, with immediate lymphadenectomy for nodal metastases detected on biopsy (biopsy group). Results No significant treatment-related difference in the 10-year melanoma-specific survival rate was seen in the overall study population (20.8% with and 79.2% without nodal metastases). Mean (± SE) 10-year disease-free survival rates were significantly improved in the biopsy group, as compared with the observation group, among patients with intermediate-thickness melanomas, defined as 1.20 to 3.50 mm (71.3 ± 1.8% vs. 64.7 ± 2.3%; hazard ratio for recurrence or metastasis, 0.76; P=0.01), and those with thick melanomas, defined as >3.50 mm (50.7 ± 4.0% vs. 40.5 ± 4.7%; hazard ratio, 0.70; P=0.03). Among patients with intermediate-thickness melanomas, the 10-year melanoma-specific survival rate was 62.1 ± 4.8% among those with metastasis versus 85.1 ± 1.5% for those without metastasis (hazard ratio for death from melanoma, 3.09; P<0.001); among patients with thick melanomas, the respective rates were 48.0 ± 7.0% and 64.6 ± 4.9% (hazard ratio, 1.75; P=0.03). Biopsy-based management improved the 10-year rate of distant disease-free survival (hazard ratio for distant metastasis, 0.62; P=0.02) and the 10-year rate of melanoma-specific survival (hazard ratio for death from melanoma, 0.56; P=0.006) for patients with intermediate-thickness melanomas and nodal metastases. Accelerated-failure-time latent-subgroup analysis was performed to account for the fact that nodal status was initially known only in the biopsy group, and a significant treatment benefit persisted. CONCLUSIONS: Biopsy-based staging of intermediate-thickness or thick primary melanomas provides important prognostic information and identifies patients with nodal metastases who may benefit from immediate complete lymphadenectomy. Biopsy-based management prolongs disease-free survival for all patients and prolongs distant disease-free survival and melanoma-specific survival for patients with nodal metastases from intermediate-thickness melanomas. (Funded by the National Cancer Institute, National Institutes of Health, and the Australia and New Zealand Melanoma Trials Group; ClinicalTrials.gov number, NCT00275496.).

IL-10 expression by primary tumor cells correlates with melanoma progression from radial to vertical growth phase and development of metastatic competence.

Downregulation of the immune system facilitates tumor progression at different stages of cutaneous melanoma. Sentinel nodes, the first lymph nodes on lymphatics draining directly from a primary melanoma, are immune downregulated by tumor-generated immunosuppressive cytokines, including interleukin-10 (IL-10). To better understand the kinetics of sentinel node suppression, we investigated IL-10 expression by melanoma cells and tumor-associated macrophages and lymphocytes at different stages of primary melanoma evolution. We used reverse-transcriptase in situ PCR to identify the cellular sources of IL-10 mRNA in 39 melanomas. IL-10 mRNA was identified in tumor cells of 2 of 6 melanomas in situ (33%), of 17 of 21 invasive melanomas (81%) and of 11 of 12 metastatic melanomas (92%). Higher IL-10 expression correlates with tumor progression, with differences between melanoma in situ, invasive melanoma and metastatic melanoma. In primary melanomas, the IL-10 mRNA content of tumor cells correlates with Clark's level. There was significantly more IL-10 mRNA in vertical growth-phase melanoma cells than in radial growth-phase cells. In a logistic regression model, moderate-to-high IL-10 mRNA expression by tumor cells was significantly associated with vertical growth-phase melanoma. IL-10 mRNA was detected in melanoma-associated macrophages and lymphocytes. In invasive melanomas, IL-10 mRNA reactivity of macrophages decreased as Clark's level increased. Alterations of immunity by IL-10 derived from melanoma cells and melanoma-associated macrophages and lymphocytes potentially facilitate evolution of the primary melanoma and render regional lymph nodes susceptible to metastases.

Treatment of locoregional metastases of malignant melanomas with radiotherapy and intralesional beta-interferon injection.

Intralesional injection of beta-interferon can lead to remission of metastatic melanomas. Simultaneous radiotherapy may augment the effect of these injections. The effect of intralesional beta-interferon injections combined with radiotherapy was examined in patients with metastatic malignant melanoma. A total of 20 patients with inoperable, incapacitating and disfiguring malignant melanoma metastases were treated with simultaneous external beam radiotherapy (electrons and/or photons) and intralesional injection of beta-interferon. The total radiation dosage ranged from 40-50 Gy, fractionated as 1.8 Gy five times per week. beta-Interferon (Fiblaferon) 3-5 million units per injection was administered three times weekly until symptoms disappeared or until termination of therapy. One patient was treated with beta-interferon alone; the area harbouring the tumour had been previously irradiated. Five patients treated in this way showed partial remission after combined therapy and 12 showed complete remission, with permanent regression of metastases. The latter group included five patients with lengthy survival times, including one patient who has been free of symptoms for 7 years following treatment. Injected metastases showed complete regression in the patient who was treated exclusively with beta-interferon post-irradiation. In conclusion, combined treatment with radiotherapy and intratumoral injection of beta-interferon controlled local tumour growth in inoperable metastatic malignant melanomas.

RT in situ PCR detection of MART-1 and TRP-2 mRNA in formalin-fixed, paraffin-embedded tissues of melanoma and nevi.

Melanoma antigen recognized by T cells 1 (MART-1) and tyrosinase-related protein-2 (TRP-2) are two useful markers for immunohistochemical detection of melanocytic tumors. However, these markers may be passively acquired (phagocytosed) rather than actively synthesized. Reverse transcriptase in situ polymerase chain reaction (RT in situ PCR) can amplify even small amounts of specific mRNA in cells and therefore confirm the cellular source of a marker. We developed a one-step RT in situ PCR procedure in which Thermus thermophilus DNA polymerase synthesizes and amplifies cDNA from mRNA in a single reaction mixture. To examine its practicability and feasibility with formalin-fixed, paraffin-embedded (FFPE) tissue, we compared the results of one-step RT in situ PCR with those of immunohistochemistry (IHC). MART-1 mRNA was identified in the cytoplasm of lesional cells from 23/26 primary melanomas (92%), 9/9 metastatic melanomas (100%) and 5/6 nevi (83%). MART-1 epitope was detected by IHC in 23/24 primary melanomas (96%), 9/9 metastatic melanomas (100%) and 5/6 nevi (83%). TRP-2 mRNA was identified in the cytoplasm of lesional cells from 17/26 primary melanomas (65%), 6/9 metastatic melanomas (67%) and 4/6 nevi (67%). TRP-2 epitope was detected by IHC in 20/24 primary melanomas (83%), 9/9 metastatic melanomas (100%) and 4/6 nevi (67%). Both techniques detected MART-1 and TRP-2 in FFPE melanoma cell lines. Neither marker was detected in squamous cell carcinomas or basal cell carcinomas by RT in situ PCR or IHC. We conclude that the RT in situ PCR technique can be successfully applied to FFPE tissue to determine the cellular sources of gene expression observed by conventional PCR approaches.

Proteomics analysis of hypothalamic response to energy restriction in dairy cows.

The hypothalamus is the central regulatory unit that balances a number of body functions including metabolic rate, hunger, and satiety signals. Hypothalamic neurons monitor and respond to alterations of circulating nutrients and hormones that reflect the peripheral energy status. These extracellular signals are integrated within the cell at the ATP:AMP ratio and at the level of ROS, triggering gene expression associated with glucose and lipid metabolism. In order to identify new molecular factors potentially associated with the control of energy homeostasis, metabolic adaptation, and regulation of feed intake, hypothalami from ad libitum fed and energy restricted cows were characterized using 2-DE and MALDI-TOF-MS. Among 189 different protein spots identified, nine proteins were found to be differentially expressed between groups. Beside the 5-aminoimidazole-4-carboxamide ribonucleotide formyltransferase/IMP cyclohydrolase, stress-induced phosphoprotein-1, heat shock protein 70 kDa-protein-5, dihydropyrimidinase-related protein-2, [Cu-Zn]-superoxide dismutase, ubiquitin carboxy-terminal hydrolase-L1, and inorganic pyrophosphatase were found to be up-regulated, whereas glyceraldehyde 3-phosphate dehydrogenase and aconitase-2 were down-regulated in the restricted group. In conclusion, differentially expressed proteins are related to energy and nucleotide metabolism and cellular stress under conditions of dietary energy deficiency. These proteins may be new candidate molecules that are potentially involved in signaling for maintaining energy homeostasis.

High-grade trichoblastic carcinoma arising in trichoblastoma: a rare adnexal neoplasm often showing metastatic spread.

It has been debated whether malignant transformation of trichoblastoma occurs. The concept was recently forwarded that basal cell carcinoma is as a malignant neoplasm of follicular germinative cells and should be named trichoblastic carcinoma to show its relationship to trichoblastoma. Almost all basal cell carcinomas are low-grade malignant neoplasms and develop metastases only very rarely, and if so, only after very long duration and untreated growth. Only rare basal cell carcinomas arise in trichoblastomas. Up to now there have only been two reports of high-grade trichoblastic carcinoma arising in trichoblastoma, showing systemic metastatic spread and death. We add two further cases of trichoblastic carcinoma with anaplastic nuclei, arising in trichoblastoma. One of the tumors arose in a small nodular trichoblastoma on the right forearm of an 84-year-old male patient. The other one was a trichoblastic carcinoma at the base of a trichoepithelioma on the right thigh of an 87-year-old woman with Brooke-Spiegler syndrome. Our cases emphasize that high-grade trichoblastic carcinoma develops via malignant transformation of trichoblastoma, and is very rare.

Detection of multiple melanoma-associated markers in melanoma cell lines by RT in situ PCR.

New surgical oncology techniques, such as lymphatic mapping and sentinel node biopsy, require precise identification of the presence of even very small numbers of tumor cells. The gold standard for such analysis remains microscopic assessment of tissue sections, stained conventionally or by immunohistochemistry for appropriate tumor markers. This approach is limited by sampling constraints and requires a high degree of expertise from the microscopist. Recent studies have demonstrated a subgroup of patients whose sentinel nodes are negative on microscopy, but whose nodes yield an enhanced signal for melanoma markers when evaluated by RT-PCR. These enhanced signals reflect a mixture of signal sources, including small numbers of melanoma cells and cells other than melanoma cells that express the relevant markers(s). Because the preparative techniques for RT-PCR destroy the structural integrity of the tissues and disrupt individual cells, the exact cellular source of enhanced signal from a tissue cannot be demonstrated by conventional RT-PCR. RT in situ PCR, in which the RT-PCR technique is applied on a tissue section, does identify the cells that are the source of signal. We have attempted to optimize this interesting approach and have applied it to the detection of relevant melanoma markers in tissue culture lines.

sE-selectin for stratifying outcome in rheumatoid arthritis.

OBJECTIVES: To determine the usefulness of sE-selectin as a marker for early diagnosis and stratification of rheumatoid arthritis. METHODS: We investigated several markers of disease activity, including circulating adhesion molecules and other standard laboratory tests, in a 2-3 year followup analysis of patients with rheumatoid arthritis. RESULTS: The mean +/- SD levels of sE-selectin (91.68 +/- 31.8 ng/ml versus 49.83 +/- 14.76 ng/ml) and rheumatoid factor (375.7 +/- 394.4 U versus 44.66 +/- 37.63 U) were strongly elevated in severe (n = 15) versus mild (n = 7) courses of disease. Statistical calculation of mean and standard deviation revealed that sE-selectin represents a highly significant marker for the presence of persistent and aggressive disease over time, regardless of therapeutic intervention and observation time points (P = 0.0004). Notably, regression analysis identified constant values for all parameters analyzed and, therefore, a stable course of the disease could be predicted from the beginning. CONCLUSION: sE-selectin appears to be a powerful marker to predict the severity of rheumatoid arthritis.