[The sentinel lymph node in melanoma: utilization of molecular biology (RT-PCR) to detect occult metastases]. / II linfonodo sentinella nel melanoma: utilizzo della biologia molecolare (RT-PCR) nella ricerca delle metastasi occulte.

Sentinel lymph node (SLN) analysis allows the detection of occult metastases in patients with melanoma. The use of serial sections and immunohistochemical investigations (ICH) increases the chance of identifying metastases. Nevertheless, detection of mRNA of the tyrosinase gene through reverse transcription-polymerase chain reaction (RT-PCR) is the most sensitive tool for detection of occult melanoma cells in SLN. From September 1999 to August 2001, in the Anatomic Pathology Unit of M. Bufalini Hospital of Cesena, 489 SLNs from 332 patients with primary melanoma in clinical stages I and II (according to AJCC) were examined. There were 66 (13.5%) SLNs and 58 (17.4%) cases with metastasis revealed by histology and ICH. A single case with metastatic SLN was found in patients with melanomas < or = 1 mm in thickness. The percentage of cases with metastases in SLN correlated with thickness of the primary melanoma (p < 0.0001). RT-PCR for tyrosinase was carried out in 448 SLNs from of 308 cases. Overall, the RT-PCR results were positive in 149 (48.4%) patients and in 169 SLNs (37.9%). RT-PCR results showed a strong positive correlation with tumor thickness of primary melanoma (p < 0.0001) and with the clinical stage (p < 0.0001). Of the RT-PCR-positive cases, 18 showed intracapsular aggregates of nevus cells. Besides the percentage of positive cases, once those with nevus aggregates were excluded, overall, the RT-PCR revealed the presence of tyrosinase mRNA in 34.5% of patients with negative histology and ICH. Ongoing monitoring is necessary to define the real prognostic implication of the presumed presence of occult melanoma deposits disclosed by RT-PCR in SLNs.

Sentinel lymph node biopsy in patients with primary cutaneous melanoma: study of 455 cases.

BACKGROUND: The role of elective lymph node dissection in the treatment of patients with primary melanoma is a debated topic in surgical oncology. However, recent data assure a survival improvement with this technique only for patients harbouring nodal metastases. The emergence of a new procedure of lymphatic mapping permits the identification of the sentinel lymph node (SLN), the first draining node from the site of cutaneous melanoma, which has demonstrated to be predictive of staging of the entire regional lymphatic basin and useful in selecting for lymph node dissection only those patients who have early micrometastases. OBJECTIVES: To verify in a large series of cases whether a combination of preoperative lymphoscintigraphy and intraoperative mapping with both vital blue dye and a hand-held gamma probe would permit an increase of the rate of successful SLN localization up to 100%; to check the utility of a wider application of SLN biopsy in patients with thin melanomas owing to a favourable risk-benefit ratio; to determine the predictive value of SLN biopsy by performing regional lymphadenectomy in patients who have pathological evidence of metastases in the SLN; to observe whether the use of SLN technique and selective lymphadenectomy might improve the clinical evolution of patients and favour low rates of recurrence. METHODS: In 425 AJCC stage I or II melanoma patients, preoperative lymphoscintigraphy by intracutaneous injection of Tc99m-labelled albumin nanocolloids around the tumour or the tumour's excision scar was combined with the intraoperative use of a hand-held gamma probe and patent blue V mapping technique, in order to identify and harvest the SLN. In five cases the blue dye was voluntarily not used because of previous allergic reactions. In other 25 preliminary cases the procedure was performed using the blue dye alone (10 cases) or combined with a preoperative lymphoscintigraphy (15 cases). A wide excision of the primary site was then undertaken in all cases. SLNs were sent to the pathologist for serial sectioning and permanent preparations with histological and immunohistochemical examination. Patients with pathological evidence of metastatic disease in SLN returned for regional lymphadenectomy. RESULTS: The combined use of lymphoscintigraphy, blue dye and gamma probe allowed us to identify one or more SLNs in all cases except for two (99.5% rate of success). In 70 melanomas less than 0.76 mm thick, SLNs were negative for metastases, whereas in 380 patients with thicker tumours micrometastases were demonstrated in 75 cases (19.7%). In patients with SLN metastases who underwent regional lymph node dissection, no other metastases were found three times out of four. After a median follow-up period of 18 months the rate of recurrence of the disease in 335 patients with SLN free of metastasis was low (5.4%) with a very low regional nodal recurrence (1.2%). Moreover, the worsening of the disease did not exceed 18.5% of cases with metastasis in SLN. CONCLUSIONS: Our data confirm in a large series of cases that the SLN biopsy is extremely selective and useful to find early micrometastases and to identify patients needing regional lymphadenectomy and adjuvant immunotherapy. Patients with intermediate thickness melanoma (0.76-4.0 mm) should be informed on the availability of such a revolutionary procedure, which represents a new opportunity in primary melanoma surgery.

Sentinel node biopsy and selective lymph node dissection in cutaneous melanoma patients.

Sentinel node biopsy allows an accurate selection of melanoma patients to be submitted to therapeutic dissection. From February 1994 to August 1998, at the National Cancer Institute, S. Pio X Hospital in Milan and Bufalini Hospital in Cesena, 580 sentinel node biopsies were performed in 540 stage I melanoma patients (242 males; 298 females; median age 47). Primary melanoma was located in the trunk in 201 patients, in lower limbs in 242 cases, in upper limbs in 80 cases and in head and neck in 17 patients. Injection of blue dye for sentinel node identification was performed in all cases; 372 patients were submitted to preoperative lymphoscintigraphy and in 272 cases an intraoperatory probe for a radioguided biopsy was utilized. Sentinel node identification rate was 91%. Sentinel node positivity rate was 15%. Frozen sections were examined in 199 cases. Distribution of positive cases according to primary thickness is the following: <1 mm: 1%; 1-1.99 mm: 5%; 2-2.99 mm: 18% and > or =3 mm: 27%. Sentinel node appeared to be the only metastatic node in 77% of patients submitted to dissection. The adoption of preoperative lymphoscintigraphy and the intraoperative use of the gamma probe contributed substantially in S.N. identification. No complications caused by the procedure were reported. Eight patients had a regional node relapse after a negative sentinel node biopsy and were submitted to therapeutic distant dissection. Currently 513 patients are alive with no evidence of disease. Present data confirm the feasibility and safety of sentinel node technique for selection of patients to be submitted to radical node dissection and to eventual adjuvant treatments.

[Anatomo-pathologic study of sentinel lymph nodes in melanoma. Analysis of 200 cases]. / Studio anatomo-patologico del linfonodo sentinella nei melanomi. Analisi di 200 casi.

Pathologic evaluation of sentinel lymph node represents a new technique for managing high-risk primary melanoma. We examined the sentinel lymph node biopsies of 200 patients affected by primary melanomas of trunk, limbs, head and neck, who had been operated at "M. Bufalini" Hospital between April 1996 and July 1998. The lymphatic mapping has been performed through the preoperative intradermal injection of vital blue dye and technetium-labelled albumin. 319 sentinel lymph nodes were harvested and the 11.3% (15% of patients) were positive for melanoma metastases. No metastases were found in melanomas < or = 1 mm. The percentage of positive sentinel lymph nodes in patients with melanomas > 1 mm in thickness was 16.3% (22% of patients). In 5 cases (2.5%) nodal nevi were found, 1 of which was associated with micrometastasis. All 30 patients with positive sentinel lymph nodes underwent regional lymph node dissection and 555 lymph nodes were harvested. Melanoma metastases were found in only 7 patients, in 31 lymph nodes. The procedure of SLN detection and biopsy is a feasible surgical approach to melanoma patients. It is extremely useful in finding early metastases and in effective pathologic staging. As a consequence of the very low incidence of metastases in the sentinel lymph nodes of patients with thin melanomas, we suggest the sentinel lymph node mapping should be offered to patients with primary melanomas at least 1 mm in depth.

Effects of heavy ions on nucleic acids: measurement of the damage.

In this short survey the main, available information on the molecular mechanisms of action of heavy ions on DNA is critically reviewed. Formation of single- and double-stranded DNA breaks in cells exposed to heavy particles is well established. On the other hand, base damage and, in a more general way, clustered lesions, whose formation should be increased upon exposure to heavy ions, have not yet been isolated and characterized. Efforts should be made to identify this important class of DNA damage in both isolated and cellular DNA. Sensitive and specific assays involving chemical and biochemical approaches have to be developed for such a purpose.

Ascorbic acid-2-0-beta-glucuronide, a new metabolite of vitamin C identified in human urine and uremic plasma.

A new metabolite of ascorbic acid has been isolated by a multi-step chromatographic procedure both from normal human urine and uremic plasma. Nuclear Magnetic Resonance studies, and chemical and enzymic analyses indicated that the compound is a conjugated structure consisting of equimolar ascorbic and beta-D-glucuronic acids. We determined the pKa value of the ascorbic acid moiety of the compound on the basis of variations of ultraviolet absorbances as a function of pH. Results showed that glucuronic acid is coupled to the 2-position of ascorbic acid.

Simple chromatographic systems permitting both DNA purification and separation of 2'-deoxyribonucleoside 3'-monophosphates as substrates for 32P-postlabelling studies.

The 32P-postlabelling method has recently been applied to the measurement of oxidative DNA damage. The assay requires the isolation of 2'-deoxyribonucleoside 3'-monophosphates subsequent to the extraction of DNA followed by its enzymatic digestion. As an alternative to the use of toxic and oxidizing solvents such as phenol, a simple purification method is proposed, based mainly on size-exclusion chromatography carried out either with ready-made columns (NAP-10, SEC-2000) or, more conveniently, with stainless-steel laboratory-packed columns (Fractogel HW 65 F). This method was applied to the purification of the DNA extracted from seeds of Lactuca sativa. After enzymatic digestion of DNA, the 2'-deoxyribonucleoside 3'-monophosphates may be further separated in less than 30 min by high-performance liquid chromatography on a Hypersil octadecylsilylsilica gel column in the ion-suppression mode by using either ammonium formate (0.05 M, pH 6.5) or sodium succinate (0.02 M, pH 6.0). The use of these eluent systems is compatible with straightforward 32P-labelling of the 2'-deoxyribonucleoside 3'-monophosphates without any concentration and desalting steps.

Measurement of oxidative base damage to DNA by using HPLC-32P-postlabelling and GC/MS-selective ion monitoring assays.

A 32P-postlabelling assay has been developed for singling out specific oxidized base lesions. Emphasis was placed on the quantitative aspect and the accuracy of the assay, which require the use of calibration curves and microreactions, respectively. The method was successfully applied to the detection and the measurement of adenine N1-oxide and 5-hydroxymethyluracil in cells exposed to agents inducing oxidative stress including H2O2 and UV-A radiation. The sensitivity of the assay allows the detection of one lesion in 10(6) normal bases in 1 microgram of DNA. The GC/MS method when coupled to the selective ion monitoring (SIM) technique is about twenty times less sensitive, even for suitable substrates such as 5-hydroxymethyluracil and 5-hydroxyuracil, than the 32P-postlabelling assay. However, the former assay is much easier to apply, even though a derivatization step is necessary, and provides unambiguous structural information on the compound to be measured. Accurate quantitative measurements can be obtained when stable, isotopically labelled standards are available.

Chemical and biochemical postlabeling methods for singling out specific oxidative DNA lesions.

A survey of the main available chemical and biochemical postlabeling assays for measuring oxidative DNA damage is reported. Two main approaches, radio and fluorescent postlabeling, have been used in order to reach a high level of sensitivity of detection. This is required for the measurement of DNA damage within cells and tissues upon exposure to agents of oxidative stress. Most of the methods are based on liquid chromatographic separation of defined DNA modifications following either acidic hydrolysis or enzymic digestion of DNA. In a subsequent step, the isolated base or sugar damages are either radiolabeled or made fluorescent by chemical or enzymatic reactions. Emphasis is placed on the recently developed high performance liquid chromatographic 32P-postlabeling assay, which allows the specific and sensitive measurement of various base damages including adenine N-1 oxide and 5-hydroxymethyluracil at the level of one modification per 10(7) normal bases in a sample size of 1 microgram of DNA. Examples of application of radioactive postlabeling to the measurement of DNA base damage following exposure of human cells to oxidizing agents including hydrogen peroxide and UVA radiation are provided.

Ionic and radical oxidations of DNA by hydrogen peroxide.

Hydrogen peroxide mediated oxidation of 2'-deoxyadenosine and isolated DNA was investigated. Reaction of hydrogen peroxide with 2'-deoxyadenosine under non radical conditions led to the formation of a predominant decomposition product. This was identified as 2'-deoxyadenosine N-1-oxide on the basis of detailed 1H and 13C NMR analysis and further confirmed by photolysis experiments. Quantitative determination of both radical and ionic DNA type damage was based on the use of a 32P-postlabeling method (5-hydroxymethyluracil and adenine N-1-oxide) and of an HPLC-EC assay (8-hydroxyguanine). Adenine N-1-oxide was shown to be the predominant ionic DNA base damage under non radical conditions. The presence of Fe(II)-DTPA complex in the reaction medium led to a reduction in the amount of adenine N-1-oxide by a factor of 4 whereas radical DNA type damages including 8-hydroxyguanine and 5-hydroxymethyluracil was increased by a factor of 2-3.

32P-postlabeling measurement of adenine N-1-oxide in cellular DNA exposed to hydrogen peroxide.

A 32P-postlabeling assay has been developed for monitoring the formation within DNA of adenine N-1-oxide, the specific H2O2-mediated oxidation product under nonradical conditions. This has required the chemical synthesis of both 2'-deoxyadenosine N-1-oxide 3'-monophosphate and 2'-deoxyadenosine N-1-oxide 5'-monophosphate, the substrate and the product of polynucleotide kinase mediated phosphorylation. Isolation of the substrate from the other nucleotides was found to be necessary in order to improve the rate of phosphorylation and to prevent self-radiolysis processes. [32P]-2'-deoxyadenosine N-1-oxide 5'-monophosphate was obtained after successive ion exchange and reverse-phase HPLC and was characterized by a microreaction. The sensitivity of the assay, which is close to 1 modified adenine N-1-oxide/10(6) bases, allowed the determination of this lesion within the DNA of cells exposed to nonlethal levels of H2O2.

Identification of N1-glycolylbiuret in the gamma radiolysis of aerated aqueous solution of cytosine. Influence of the pH.

N1 glycolylbiuret has been identified as a radiation product of cytosine in aerated aqueous solution at pH 4.5. When varying the pH of the solution before irradiation from acidic values towards neutral ones, G value of N1 glycolylbiuret reached a maximum at pH 4.5.