An expanded peripheral T cell population to a cytotoxic T lymphocyte (CTL)-defined, melanocyte-specific antigen in metastatic melanoma patients impacts on generation of peptide-specific CTLs but does not overcome tumor escape from immune surveillance in metastatic lesions.

It is not known if immune response to T cell-defined human histocompatibility leukocyte antigen (HLA) class I-restricted melanoma antigens leads to an expanded peripheral pool of T cells in all patients, affects cytotoxic T lymphocyte (CTL) generation, and correlates with anti-tumor response in metastatic lesions. To this end, a limiting dilution analysis technique was developed that allowed us to evaluate the same frequency of peptide-specific T cells as by staining T cells with HLA-peptide tetrameric complexes. In four out of nine patients, Melan-A/Mart-1(27-35)-specific CTL precursors (CTLp) were >/=1/2,000 peripheral blood lymphocytes and found mostly or only in the CD45RO(+) memory T cell subset. In the remaining five patients, a low (<1/40,000) peptide-specific CTLp frequency was measured, and the precursors were only in the CD45RA(+) naive T cell subset. Evaluation of CTL effector frequency after bulk culture indicated that peptide-specific CTLs could be activated in all patients by using professional antigen-presenting cells as dendritic cells, but CTLp frequency determined the kinetics of generation of specificity and the final number of effectors as evaluated by both limiting dilution analysis and staining with HLA-A*0201-Melan-A/Mart-1 tetrameric complexes. Immunohistochemical analysis of 26 neoplastic lesions from the nine patients indicated absence of tumor regression in most instances, even in patients with an expanded peripheral T cell pool to Melan-A/Mart-1 and whose neoplastic lesions contained a high frequency of tetramer-positive Melan-A/Mart-1-specific T cells. Furthermore, frequent lack of a "brisk" or "nonbrisk" CD3(+)CD8(+) T cell infiltrate or reduced/absent Melan-A/Mart-1 expression in several lesions and lack of HLA class I antigens were found in some instances. Thus, expansion of peripheral immune repertoire to Melan-A/Mart-1 takes place in some metastatic patients and leads to enhanced CTL induction after antigen-presenting cell-mediated selection, but, in most metastatic lesions, it does not overcome tumor escape from immune surveillance.

Multispectral imaging and artificial neural network: mimicking the management decision of the clinician facing pigmented skin lesions.

Various instruments based on acquisition and elaboration of images of pigmented skin lesions have been developed in an attempt to in vivo establish whether a lesion is a melanoma or not. Although encouraging, the response of these instruments, e.g. epiluminescence microscopy, reflectance spectrophotometry and fluorescence imaging, cannot currently replace the well-established diagnostic procedures. However, in place of the approach to instrumentally assess the diagnosis of the lesion, recent studies suggest that instruments should rather reproduce the assessment by an expert clinician of whether a lesion has to be excised or not. The aim of this study was to evaluate the performance of a spectrophotometric system to mimic such a decision. The study involved 1794 consecutively recruited patients with 1966 doubtful cutaneous pigmented lesions excised for histopathological diagnosis and 348 patients with 1940 non-excised lesions because clinically reassuring. Images of all these lesions were acquired in vivo with a multispectral imaging system. The data set was randomly divided into a train (802 reassuring and 1003 excision-needing lesions, including 139 melanomas), a verify (464 reassuring and 439 excision-needing lesions, including 72 melanomas) and a test set (674 reassuring and 524 excision-needing lesions, including 76 melanomas). An artificial neural network (ANN(1)) was set up to perform the classification of the lesions as excision-needing or reassuring, according to the expert clinicians' decision on how to manage each examined lesion. In the independent test set, the system was able to emulate the clinicians with a sensitivity of 88% and a specificity of 80%. Of the 462 correctly classified as excision-needing lesions, 72 (95%) were melanomas. No major variations in receiver operating characteristic curves were found between the test and the train/verify sets. On the same data set, a further artificial neural network (ANN(2)) was then architected to perform classification of the lesions as melanoma or non-melanoma, according to the histological diagnosis. Having set the sensitivity in recognizing melanoma to 95%, ANN(1) resulted to be significantly better in the classification of reassuring lesions than ANN(2). This study suggests that multispectral image analysis and artificial neural networks could be used to support primary care physicians or general practitioners in identifying pigmented skin lesions that require further investigations.

Antiapoptotic role of endogenous nitric oxide in human melanoma cells.

The role of endogenous NO on cell survival was investigated in human melanoma cells and melanocytes. Inducible NO synthase (iNOS) was always expressed in a panel of melanoma cell lines from metastatic lesions and in normal adult melanocytes. iNOS was also detected by immunohistochemistry in melanoma cells from metastases. Release of NO by tumor cells and melanocytes was inhibited by a specific iNOS inhibitor, aminoguanidine (AMG). Inhibition of endogenous NO synthesis did not affect cell cycle progression of melanoma cells but led to cell death by apoptosis, as indicated by Annexin V/propidium iodide and terminal deoxynucleotidyltransferase-mediated dUTP nick end labeling assays. By contrast, iNOS inhibition by AMG did not promote apoptosis in normal adult melanocytes. A mitochondrial pathway was involved in melanoma apop tosis, as indicated by altered mitochondrial membrane potential (delta psi(m)) and down-regulation of Bcl-2 protein level after iNOS inhibition. AMG treatment triggered release of caspase-1, enzymatic activation of caspase-3, and degradation of poly(ADP-ribose) polymerase, one of the main caspase-3 substrates. Melanoma cell apoptosis induced by iNOS inhibition was completely blocked by peptide inhibitors of caspase-1 and caspase-3 (Ac-DEVD-CHO and AC-YVAD-CHO) or by an exogenous NO donor, sodium nitroprusside, or by addition of serum. Finally, comparison of control and AMG-treated melanoma cells by pathway-specific gene array analysis indicated that inhibition of NO synthesis led, before induction of apoptosis, to up-regulation of mRNA levels of genes involved in the apoptosis pathway such as Bax, caspase-1, caspase-3, caspase-6, gadd45beta, mdm2, and TRAIL. Taken together, these results indicate that melanoma cell survival is regulated by endogenous NO resulting from iNOS activity.

Peripheral burst of tumor-specific cytotoxic T lymphocytes and infiltration of metastatic lesions by memory CD8+ T cells in melanoma patients receiving interleukin 12.

Systemic effects on T-cell-mediated antitumor immunity, on expression of T-cell adhesion/homing receptors, and on the promotion of T-cell infiltration of neoplastic tissue may represent key steps for the efficacy of immunological therapies of cancer. In this study, we investigated whether these processes can be promoted by s.c. administration of low-dose (0.5 microg/kg) recombinant human interleukin-12 (rHuIL-12) to metastatic melanoma patients. A striking burst of HLA-restricted CTL precursors (CTLp) directed to autologous tumor was documented in peripheral blood by a high-efficiency limiting dilution analysis technique within a few days after rHuIL-12 injection. A similar burst in peripheral CTLp frequency was observed even when looking at response to a single tumor-derived peptide, as documented by an increase in Melan-A/Mart-1(27-35)-specific CTLp in two HLA-A*0201+ patients by limiting dilution analysis and by staining peripheral blood lymphocytes (PBLs) with HLA-A*0201-melanoma antigen-A/melanoma antigen recognized by T cells (Melan-A/Mart)-1 tetrameric complexes. The CTLp burst was associated, in PBLs, with enhanced expression of T-cell adhesion/homing receptors CD11a/CD18, CD49d, CD44, and with increased proportion of cutaneous lymphocyte antigen (CLA)-positive T cells. This was matched by a marked increase, in serum, of soluble forms of the endothelial cell adhesion molecules E-selectin, vascular cell adhesion molecules (VCAM)-1 and intercellular adhesion molecules (ICAM)-1. Infiltration of neoplastic tissue by CDS+ T cells with a memory and cytolytic phenotype was found by immunohistochemistry in eight of eight posttreatment metastatic lesions but not in five of five pretreatment metastatic lesions from three patients. Increased tumor necrosis and/or fibrosis were also found in several posttherapy lesions of two of three patients in comparison with pretherapy metastases. These results provide the first evidence that rHuIL-12 can boost the frequency of circulating antitumor CTLp in tumor patients, enhances expression of ligand receptor pairs contributing to the lymphocyte function-associated antigen-1/ICAM-1, very late antigen-4/VCAM-1, and CLA/E-selectin adhesion pathways, and promotes infiltration of neoplastic lesions by CD8+ memory T cells in a clinical setting.

Small melanomas: a clinical study on 270 consecutive cases of cutaneous melanoma.

The ABCD (asymmetry, border, colour, dimension) criteria represent a commonly used clinical guide for the diagnosis of early cutaneous melanoma (CM). This guide stipulates that CMs usually are more than 6 mm in diameter. The purpose of this retrospective study was to establish the frequency of occurrence of small (< or =6 mm) melanomas in a clinical context. Our series consisted of 270 consecutive CMs (39 in situ and 231 invasive) in 267 patients. Of these 270 lesions, 47 (17%) were small lesions, ranging from 2 to 6 mm in maximum linear extent, with a median value of 5 mm. Of these small lesions, 14 were in situ and 33 Invasive CMs. The median thickness of the 33 small invasive lesions was 0.31 mm. The clinical features of CMs were sufficiently distinctive to suggest a diagnosis of CM in half of the cases, irrespective of the invasiveness or not of the lesions. Dermatoscopy was performed on 36 of the small lesions and achieved a correct diagnosis in 72% of the cases. The combination of simple visual examination with dermatoscopy allowed a higher rate of recognition (86%) than when the two methods were considered separately. Results of our study show that small CMs represent a considerable clinical subset of all CMs. Clinicians must be aware of this fact in their diagnostic activity.

Clinical and dermatoscopic diagnosis of early amelanotic melanoma.

Amelanotic cutaneous melanoma (ACM) often defies clinical diagnosis because of the lack of pigmentation. In an attempt to find diagnostic clues, we retrospectively studied the clinical features of 15 thin (< 1 mm thick or Clark level I) ACM lesions. The clinical features of early ACMs are identified and illustrated to enable early diagnosis and cure of these lesions. The typical early lesion presents as an asymmetric macula, which may be uniformly pinkish or reddish or, more often, has faint light pigmentation (tan, brown or grey) at the periphery; it has borders that may be well- or ill-defined. In our study, these features suggested the correct clinical diagnosis in only a minority (40%) of cases. Nine cases in this series were also subjected to dermatoscopy. By this technique we identified, as constant feature, the presence of small red dots, evenly distributed or grouped on a whitish or pink-red background. Our results show the importance of dermatoscopy in the evaluation of equivocal pink or reddish lesions. Red dots seen with this technique can be an important sign for the diagnosis of thin ACM. Since this sign does not appear to be pathognomonic, the presence of an associated pigmentary network can be decisive in the differential diagnosis.

Multispectral imaging approach in the diagnosis of cutaneous melanoma: potentiality and limits.

In an attempt to overcome the subjectiveness of clinical observation in the diagnosis of cutaneous melanoma, a computerized method is proposed. Reflectance images of 237 pigmented lesions (67 melanomas and 170 non-melanomas) were analysed using a telespectrophotometric technique. This device consists of a CCD camera with 17 interference filters. Images were acquired at selected wavelengths, from 420 to 1040 nm. Morphological and reflectance related parameters were extracted from the wavelength-dependent images of the lesions. The most significant features in the comparison between benign and malignant lesions were: lesion dimension (P < 10(-8) at 578 nm); mean value (P < 10(-7) at 940 nm) and standard deviation (P < 10(-4) at 904 nm) of lesion reflectance; lesion roundness (P < 10(-5) at 461 nm); and border irregularity (P < 10(-4) at 461 nm). Based on these parameters, a discriminant function between the two populations of lesions (naevi and melanomas) was obtained. By using the results of the analysis of the recruited lesions as 'training data', discriminant functions enabled the assignment of a score, or a 'risk probability', to each studied lesion. By imposing a sensitivity of 80% (a figure that mimics the diagnostic capability of an experienced clinician), entering or not entering the lesion dimension as input data in the discriminant analysis led to a specificity of 51% or 46% respectively. The high number of false-positive cases, which is a consequence of the selection criteria of the lesions, is, at present, the major limitation of the current technique. Nevertheless, our results suggest that an imaging-based computer-assisted device could be capable of discriminating malignant lesions mainly by evaluation of reflectance, especially in the infrared region, and shape properties. The dimension of a lesion should not be essential in the diagnosis of melanoma and, in our opinion, small melanomas should be recognized by a computer system as well as they are on clinical grounds.

Melanotic neuroectodermal tumour of infancy (MNTI). Immunohistochemical and ultrastructural study of a case.

A case of melanotic neuroectodermal tumour of infancy (MNTI) which occurred in the left lower jaw of a 15-months old child was studied by electron microscopy and immunohistochemical methods to verify the neural crest origin of this tumour. Two cell population were observed: pigment melanocyte-like cells and non-pigmented nerve-like cells. Immunohistochemical analysis with neuron-specific enolase (NSE) and S-100 protein visualized two cell subtypes in the non-pigmented cell population. The combined ultrastructural findings and immunohistochemical data confirm the neural crest origin of MNTI.

Melanoma of the external ear.

AIMS AND BACKGROUND: Melanoma of the external ear is a rare disease, and its management is controversial. To address this problem, we reviewed the data concerning the patients observed at our Institution. METHODS: We retrospectively reviewed the clinical records of the 20 patients bearing primary ear melanoma observed over a period of about 20 years at the Istituto Nazionale Tumori of Milan. RESULTS: Initial evaluation of the patients revealed 7 stage I, 12 stage II and 1 stage III. The thickness of the tumors varied from 0.39 to 6.62 mm. Fourteen patients underwent a wedge resection of the skin and cartilage with primary closure, and 6 patients had a partial amputation of the ear. In 8 cases the section was performed at about 1 cm from the border of the tumor, in 6 cases at about 0.5 cm, and in 6 cases at more than 1 cm. The average follow-up was 57 months (range, 1-18 years). Since there was no local recurrence, it could not be related to type and extent of the local resection performed. In contrast, the development of metastases was related to tumor thickness. CONCLUSIONS: A conservative excision with margins of 1 cm can be a safe procedure for invasive ear melanoma, irrespective of tumor thickness. Like melanomas of other sites, the prognosis is linked to the thickness of the tumor.

Image analysis in the RGB and HS colour planes for a computer-assisted diagnosis of cutaneous pigmented lesions.

AIMS AND BACKGROUND: A study was carried out to evaluate the effectiveness of image analysis performed by the two color representation models when a computer-assisted diagnosis of melanoma is involved. METHODS: Color images of 40 skin pigmented lesions, which included 12 melanomas, were acquired by a standard color RGB video camera and stored in a PC for off-line processing. Image analysis was performed in the red green and blue color representation model and using hue and saturation color components. To describe shape and color characteristics of each lesion, including area, roundness and color variegation, 16 parameters were derived from red, green, blue, hue and saturation color planes and tested as possible variables useful to differentiate melanomas from benign nevi. RESULTS: The test gave a result of significance for six of the 16 derived image descriptors. The general trend of our data was in agreement with clinical observations according to which melanoma is usually darker, more variegated and less round than a benign nevus, whereas lesion dimension of melanomas and benign lesions was not significantly different. CONCLUSIONS: Our preliminary results suggested that image analysis performed on hue and saturation-derived and red green and blue-derived data could better discriminate melanoma from nevi than separately using the two color representation models.

Sentinel node biopsy and selective dissection for melanoma nodal metastases.

BACKGROUND: Early detection of nodal metastases still represents an important goal in the management of melanoma patients. A sentinel node is defined as the first colored node in the regional lymphatic basin following injection of blue dye in the site of the primary melanoma. Sentinel node biopsy may represent a feasible technique for early identification of occult disease. A therapeutic dissection is then performed only in patients with proven nodal disease, thus introducing the concept of selective dissection. METHODS: At the National Cancer Institute of Milan from February 1994 to October 1996, 74 patients with a melanoma of the trunk or limbs and without clinically detectable node metastases were submitted to sentinel node biopsy and eventual selective dissection. RESULTS: The sentinel node was identified in 67 patients (90%). Nodal metastases were detected in 11 patients (16%); 5 of these were identified by an intraoperative frozen section examination. In all but one case, only the sentinel node was affected at radical dissection. Incidence of positive sentinel nodes was correlated with depth of infiltration of the primary lesion. Mapped nodal basin failures were observed in 3 patients with negative sentinel node biopsy. All patients but one, presenting distant metastases, are alive at this writing and free of disease with a follow-up ranging from 2 to 34 months. CONCLUSIONS: Our study adds to accumulating evidence supporting the efficacy of sentinel node biopsy in detecting occult localizations and the potential of the technique to better select the group of patients that may benefit from nodal dissection.

Association between cutaneous melanoma and neurofibromatosis type 1: analysis of three clinical cases and review of the literature.

The authors report a rare association between cutaneous melanoma and Von Recklinghausen's disease (NF-1) and analyze the possible meaning of this occurrence. Various types of tumors have been associated with NF-1, in particular those of neuroectodermal origin, such as malignant peripheral nerve sheath tumors (MPNST) and phaeochromocytoma. The development of malignant melanoma in NF-1 patients is rare. Data from the literature is enable to demonstrate an increased incidence of cutaneous melanoma in patients with neurofibromatosis but the association of these two disorders seems reasonable in theory, as both are abnormalities of a neural crest origin. The cases described may represent not only a clinical report of two rarely associated disorders, but may also confirm the biological mechanisms responsible for these infrequent diseases.

[Impact of sentinel lymph node biopsy on the treatment of melanoma]. / Impatto della biopsia del linfonodo sentinella sul trattamento del melanoma.

In the National Cancer Institute and S Pio X Hospital series we registered 981 patients with primary cutaneous melanoma and no evidence of clinically detectable regional node metastases underwent sentinel node (SN) dissection to microscopically define the tumor status of the regional lymph nodes. In 62.2% of cases, only one SN was detected; 26.4% of patients had two SNs and 11.4% had three or more SNs. A positive SNB was demonstrated in 18.1%. Analysis of survival indicated that the tumor status of the nodes was the most important prognostic factor. Breslow's thickness had a significant impact on survival in tumors of 4 mm or thicker, and ulceration dropped to a borderline significant P-value. To assess the tumor burden in positive SNB, all slides (148 SN pos) were reviewed. Twenty per cent of these patients had evidence of metastasis in other nodes. Of the remaining 80% with a single tumor-involved SN, 62% had a single metastatic deposit. Preliminary data from this study indicate that several subgroups may be identified among patients with 1 positive node, but adequate analysis of survival requires a larger number of patients and a multicentric study.

Surgical treatment of gastric metastases from cutaneous melanoma: experience of the National Cancer Institute of Milan.

AIM: To evaluate the role of a surgical approach in patients affected with gastric metastases from cutaneous melanoma. METHODS: A retrospective review of our local melanoma database of 2100 patients identified 31 cases with gastric metastatic deposits. Nine of them were considered candidates for surgical resection. RESULTS: Median overall survival of the 9 patients who underwent surgery was 14.2 months. Six (67%) underwent a local radical resection of disease, and 3 (33%) had a simple exploratory laparotomy. The median survival was 21.6 months (range, 4-32 months) for the subset receiving radical surgery and 3.6 months (range, 2-6 months) for the patients who had no resection. Median follow-up was 14.2 months. No specific correlation of serologic LDH levels and final outcome, as documented elsewhere, was observed. A marked decreased or substantial remission of symptoms with an improvement in quality of life was observed in all radically resected patients. CONCLUSIONS: Patients with gastric metastases from melanoma may benefit from surgery if all macroscopic disease can be removed. In addition, gastric resection in patients with symptomatic melanoma spread to the stomach provides important symptomatic palliation.

In situ and very thin melanomas (< 0.75 mm) are similarly and commonly recognizable.

AIMS AND BACKGROUND: Recent data have suggested that cutaneous melanoma in situ can be clinically recognized in most cases by its features, which resemble those of early invasive melanoma. The aim of the study was to verify whether the diagnostic sensitivity of melanoma in situ is actually equivalent to that of early invasive melanoma. METHODS: We retrospectively reviewed the clinical diagnosis of 274 consecutive cutaneous melanoma < 0.75 mm thick. The series consisted of 84 in situ and 190 invasive lesions. RESULTS: The clinical diagnosis of melanoma was performed in 62% (52/84) of cases of in situ melanoma and in 68% (130/190) of the cases of invasive melanoma. The differences were not statistically significant. CONCLUSIONS: Our results show that cutaneous melanoma can be clinically diagnosed at a very early stage. In situ and very thin melanomas (< 0.75 mm) are similarly recognizable.

[The autologous splenic reimplant in the rat]. / Reimpianto splenico autologo nel ratto.

In order to evaluate the clinical utility of autologous splenic transplantation in the omental pouch, a pneumococcal challenge was performed in 3 groups of rats, after demonstration of vitality of the intraperitoneal inoculum: Group A: splenectomized rats; Group B: reimplanted rats; Group C: sham operation. No statistically significant difference was found between the first two groups regarding resistance against infection (p less than 0.982), while normal rats proved more resistant (p less than 0.031). Between group A and B significant differences (p less than 0.001) exists only for a more precocious mortality in the first group. The poor clinical utility of the technique is demonstrated.

Lymphocyte subsets in bone marrow lymphoid nodules and malignant lymphoma nodular involvement.

The lymphocyte subsets in 67 cases of bone marrow benign or reactive lymphoid nodules (LN) and 23 cases of nodular involvement by B malignant lymphoma (B-ML) have been immunohistochemically characterized on paraffin embedded trephine biopsies utilizing a panel of 10 monoclonal antibodies. LCA was positive in 90% LN and B-ML lymphocytes; LN2, MB2 stained more than 50% LN and B-ML lymphocytes; MT2 stained more than 50% LN and less than 50% B-ML lymphocytes; UCHL1 stained the 20% of LN lymphocytes; LN1 stained only B cells of the rare germinal centers; MT1 only myeloid cells, L26 only plasma cells. DF-T1 and Leu 22 failed to stain LN or B-ML lymphocytes. While anti-T lymphocyte antibodies reacted inconstantly with lymphoid cells in decalcified and embedded specimens, LCA and some anti-B lymphocyte antibodies gave constantly reproducible results for bone marrow LN and B-ML. They permitted an easy recognition and exact evaluation of the size of LN, the identification of scattered B cells, the detection of the residual or minimal involvement by B-ML and the exact burden of the invasion, but could not allow a convincing differential diagnosis between LN and small cell B-ML nodular involvement.

The ABCD system of melanoma detection: a spectrophotometric analysis of the Asymmetry, Border, Color, and Dimension.

BACKGROUND: The ABCD (Asymmetry, Border, Color, and Dimension) criteria represent a commonly used clinical guide for the diagnosis of early melanoma. The authors revised these criteria in the light of objective measurements of the features of pigmented skin lesions obtained by telespectrophotometric analysis (TS) in the visible and near-infrared wavelengths. METHODS: This study involves a consecutive series of 186 patients with 195 cutaneous pigmented lesions (53 melanomas and 142 nonmelanoma lesions). Each lesion was subjected to TS in vivo, before surgery. For this purpose, the authors used four spectrophotometric parameters that could be closely related to the four criteria of the ABCD guide, namely, roundness (an estimate of how a lesion contour resembles a circle), smoothness (an indicator of the regularity of a lesion border), mean reflectance (the ability of a lesion to diffuse or reflect the incident light), and size (the greatest dimension of a lesion). RESULTS: When melanomas and nonmelanoma lesions were compared by univariate analysis, all four spectrophotometric parameters considered proved to be significantly different (P=0.05). Multivariate logistic analysis showed that mean reflectance in the infrared (P < 0.01) and size (P=0.03) were parameters independently associated with melanoma. Melanoma showed lower reflectance and greater size than benign lesions. CONCLUSIONS: Information provided by TS substantially validates the importance of the ABCD clinical guide and suggests that color is the most important parameter in discriminating melanoma from nevi. In particular, melanoma appears darker than other pigmented lesions.