Sentinel Lymph Node Biopsy in Thick-Melanoma Patients (N=350): What is Its Prognostic Role?

BACKGROUND: Sentinel lymph node biopsy (SLNB) is currently recommended for patients with intermediate-thickness melanomas (T2-T3). Historically, T4 melanoma patients have not been considered good candidates for SLNB because of the high risk of distant progression. However, some authors suggest that T4 melanoma patients could be considered as a heterogeneous group that could benefit from SLNB. METHODS: We retrospectively analyzed 350 patients with thick (>4 mm) melanomas between 1999 and 2011. Patients were stratified into three groups depending on the results of SLNB: (1) 94 SLNB-negative; (2) 84 SLNB-positive; and (3) 172 SLNB not performed (observation group). The associations of clinical-pathologic features with the result of SLNB, disease-free interval (DFI), and disease-specific survival (DSS) were analyzed. RESULTS: Multivariate analyses confirmed a better prognosis for SLN-negative patients compared with patients in the observation group (DSS hazard ratio [HR] 0.62, p = 0.03; DFI HR 0.47, p < 0.001). The observation group was shown to have the same prognosis as the positive-sentinel lymph node group, when adjusted for principal confounders in the model. CONCLUSIONS: We confirmed that thick-melanoma patients are a heterogeneous group with different prognosis. In our experience, SLNB allowed for an appropriate stratification of patients in different survival groups. On the basis of our results, we strongly recommend the routine execution of SLNB in cases of primary melanoma thicker than 4 mm.

Relevance of multiple basin drainage and primary histologic regression in prognosis of trunk melanoma patients with negative sentinel lymph nodes.

BACKGROUND: Lymphatic drainage to multiple basins (MLBD) is frequently observed in patients with primary melanoma located in the trunk. Conflicting data regarding the prognostic impact of MLBD are reported. OBJECTIVE AND METHODS: We reviewed our case series of 352 patients with trunk melanoma to evaluate the pattern of basin drainage and to analyse whether different basin drainages may have different significance in negative sentinel lymph node (SLN) patients. The presence of single/multiple basin drainage, the status of SLN, the presence of melanoma regression, Breslow thickness, ulceration and type of melanoma were recorded for each patients and correlated to Disease Free Survival (DFS) and Overall Survival (OS). RESULTS: MLBD occurred in 77 patients (21.9%) and single basin lymphatic drainage (SLBD) occurred in 275 patients (79.1%). The presence of metastases in SLN was not significantly different in patients with MLBD compared to those with SLBD (26% vs. 19.6%). No differences in OS and DFS were found in SLBD/MLBD independently from SLN status. However DFS was higher in patients with MLBD and negative SLN (P = 0.0001), in addition, in patients with negative SLN and SLBD disease recurrence was 19% while was only 7% in patients with negative SLN obtained from MLBD (P = 0.03). Multivariate analysis showed that Breslow thickness <2 mm, MLBD pattern and regression of melanoma were favourable variables for DFS of patients with negative SLN. CONCLUSIONS: An accurate study of the drainage basin and of all the SLNs obtained from MLBD is recommended because of the impact in prognosis of melanoma of the trunk.

Tissue engineering for cutaneous wounds: selecting the proper time and space for growth factors, cells and the extracellular matrix.

Currently, autografts are the best treatment to any substantial cutaneous injury, and their success is well known as a burn therapy. However, autografts have been less successful in the treatment of chronic ulcers, and are usually a last-resort therapy because of infection at the injured site, high surgical expense, additional morbidity and engraftment failure. In addition, patients with burns covering more than 50% of their skin have limited donor sites for autograft harvest. Therefore, there is a great need for a cost-effective, user-friendly, tissue-engineered construct (TEC) that can provide successful treatments to both acute and chronic wounds in a wider repertoire of patients, including diabetics and the elderly. One approach to the challenge is to create a substitute for skin in vitro that can integrate into the engraftment site in vivo. An alternative is to engineer a biocompatible, resorbable matrix that can recruit the proper, native tissue cells to the injured site and induce them to heal the wound without scarring. This chapter reviews the 3 essential components of cutaneous wound healing, that is, cells, extracellular matrix molecules and bioactive molecules, that must be considered for designing TECs to potentially enhance the healing process. In nature, a 'dynamic reciprocity' exists amongst cells and extracellular matrix that is mediated by bioactive molecules at the site of injury. Thus, it is important to examine the interplay of all 3 components when engineering a TEC. This chapter also includes examples of commercially available products to highlight how researchers have already begun to find success in tissue engineering.

Satisfaction with dental appearance in 8-9 years-old children. Validation of COAS questionnaire for Italian-speaking children and evaluation of social and geographical context.

AIM: This study was designed to adapt the original English-language COAS (Children's Orthodontic Attitude Survey) for third- grade schoolchildren to the Italian cultural environment and to investigate its properties in typical populations, as well as to evaluate children self-perception of their dental appearance. STUDY DESIGN: The COAS questionnaire for third-grade schoolchildren was translated and culturally adapted for Italian-speaking children. The Italian version of the questionnaire was tested on 169 (73 females, 96 males) children. Test-retest reliability was assessed on 34 children one week after the first administration. We also analysed correlations between social status and questionnaire findings. METHODS: All children filled in the questionnaire and then they were clinically examined by three residents. The clinical parameters were correlated with the questionnaire findings to evaluate children's satisfaction with their dental appearance. RESULTS: Ninety-five per cent of children thought it was important to have straight teeth and 87 per cent considered that crooked teeth were ugly. Comparison with clinical parameters showed a statistically significant correlation between crowding and overjet and some answers. Urban children have a better opinion on braces: they would like to have braces and they think they need braces statistically more than rural subjects. CONCLUSION: The Italian version of the modified-COAS questionnaire had a very good reliability. Social status and geographical context play a very important role in children's satisfaction with dental appearance. Children with different social context demonstrate they have very different approaches towards their dental aspect and braces.

Immediate tangential excision accelerates wound closure but does not reduce scarring of mid-dermal porcine burns.

Current evidence supports the use of excision to remove eschar from deep dermal and full-thickness burns. However, the role of excision of mid-dermal burns remains unclear. This study aimed to develop a porcine model that could produce reproducible middermal thermal burns that undergo tangential excision; and investigate the effects of immediate tangential excision (30 minutes postburn) on healing and scarring. An aluminum bar preheated in hot water (70°C) was applied for 20 or 30 s to produce a total of sixteen mid-dermal burns per pig on each of six pigs. Thirty minutes after burn creation, half of the burns were tangentially excised. Four partial- thickness wounds per pig were created as controls. Depth of burn injury (1 and 24 h), reepithelialization (7 and 10 d) and scar depth (28 d) were assessed microscopically. Total scar surface area was grossly evaluated on day 28. Exposure of porcine skin to a preheated aluminum bar at 70 °C for 20 or 30 sec resulted in reproducible mid-dermal burns, where immediate excision enhanced complete wound closure as judged by complete re-epithelialization, but did not reduce initial depth of injury, scar contraction and scar depth. Immediate surgical intervention is sufficient to enhance wound closure, but not to mitigate mid-dermal burn scar formation. This work provides a suitable animal model to evaluate novel therapies that may be used to inhibit burn progression, accelerate wound closure and decrease scarring, especially those therapies unable to penetrate burn eschar.

Les données actuelles des connaissances sont en faveur de l'excision des brûlures des 2ème degré profond et 3ème degré. L'intérêt de l'excision des brûlures intermédiaires reste mal précisé. Cette étude se penche sur un modèle porcin destiné à la réalisation de brûlures intermédiaires reproductibles et à l'évaluation de l'effet l'excision ultra précoce (30 mn après la brûlure) sur l'épidermisation et la cicatrisation de ces brûlures. Six porcs ont subi chacun un total de 16 brûlures intermédiaires infligées au moyen d'une barre d'aluminium chauffée à 70°C et appliquée pendant 20 à 30 s. La moitié des zones brûlées étaient excisées à la trentième minute. Quatre brûlures superficielles servaient de contrôle. La profondeur de la brûlure (à h1 et h24), la réépithélialisation (à J7 et J10) et l'épaisseur de la cicatrice (à J28), étaient étudiées microscopiquement. La surface cicatricielle totale était évaluée à J28. L'exposition pendant 20 à 30s de la peau d'un porc à de l'aluminium préalablement chauffé à 70°C entraîne une brûlure intermédiaire reproductible. L'excision immédiate en favorise la guérison lorsqu'elle est jugée sur la réépithélialisation mais n'en réduit ni la profondeur, ni la rétraction cicatricielle, pas plus que l'épaisseur de la cicatrice. L'excision immédiate favorise la fermeture de la plaie mais pas son évolution vers des séquelles. Ce travail permet de décrire un modèle animal fiable dans le but d'évaluer de nouvelles thérapeutiques destinées à limiter le progression des lésions, accélérer la fermeture et diminuer la survenues de séquelles, en particulier celles incapables de pénétrer dans une lésion constituée.

Oxytocin enhances myoepithelial cell differentiation and proliferation in the mouse mammary gland.

Using immunocytochemistry and electron microscopy, we demonstrate that oxytocin (OT) exerts a trophic effect on its target myoepithelial cells in the mammary gland. In vitro, in organotypic cultures of mouse mammary gland, we examined proliferation and differentiation of the different cell types induced by OT added to the medium. In vivo, we studied the effect of OT on the structure and cell composition of developing glands. Uptake of 5-bromo-2'-deoxyuridine was used as proliferation marker, while antibodies to smooth muscle alpha-actin (specific for myoepithelial cells) and keratin (MoAb AE1; selective for epithelial cells) were used to identify differentiated cell types. By electron microscopy, we studied structural modifications induced by OT on the extreme projections of the developing gland (sc end buds). The results indicate that OT induces myoepithelial cell differentiation and proliferation, enhancing the effect of mammotrophic hormones in nonlactating mouse mammary gland. A less marked effect was observed in luminal epithelial cells. No significant effect of OT alone was detected in cultured glands from unprimed animals.

Immunocytochemical identification of proliferating cell types in mouse mammary gland.

To study cell proliferation in different cell types and segments of the mammary gland, we devised a dual staining procedure, combining nuclear labeling by 5-bromo-2'-deoxy-uridine (BrdU) uptake (revealed by a dark-brown precipitate) and an alternative (red or blue) cytoplasmic labeling by antibodies specific for the differentiation proteins of epithelial, myoepithelial, and secretory cell types. The following markers, revealed by APAAP or beta-galactosidase procedure, were selected: alpha-smooth muscle actin for the myoepithelial cells, keratin (detected by AE1 monoclonal) for the luminal epithelial cells, alpha-lactalbumin and beta-casein for the secretory cells. To follow the full process of organogenesis, the study was conducted in mouse mammary glands from virgin, primed, and lactating animals and from glands cultured in vitro under specific hormone stimulation. Cell proliferation was localized mainly in focal areas (end buds), and mostly corresponded to "null" undifferentiated cells. Estrogen and progestin stimulation induced a relative increase of proliferating differentiated cells of either epithelial or myoepithelial type, localized in ducts and alveolar structures. Prolactin stimulation induced proliferation in secretory cells.

Specific demonstration of myoepithelial cells by anti-alpha smooth muscle actin antibody.

The myoepithelial cells of the sweat, mammary, tracheobronchial, and salivary glands are specifically identified by monoclonal antibody alpha-SM-1, which recognizes alpha smooth muscle actin and not the other actin isoforms. Basal or "reserve" cells in the stratified epithelia and excretory ducts of the salivary glands are negative, as well as all other epithelial cells in various organs. The reaction can be performed in routinely fixed and embedded tissues and is of practical interest in diagnostic histopathology. In immunoelectron cytochemistry, alpha-SM-1 antibody binds to the microfilament bundles in myoepithelial cells of the breast, but does not stain luminal cells and occasional basally located epithelial cells. These basal cells are morphologically and immunocytochemically distinct from the myoepithelial cells, and their nature and significance remain to be clarified.

Neuroendocrine differentiation in Ewing's sarcomas and primitive neuroectodermal tumors revealed by reverse transcriptase-polymerase chain reaction of chromogranin mRNA.

Ewing's sarcomas (ESs), primitive neuroectodermal tumors (PNETs), and neuroblastomas (NBs) are closely related neoplasms supposedly derived from the neural crest and belonging to the family of the small blue round cell tumors of infancy and childhood. We investigated the expression of the neuroendocrine and neuroectodermal markers chromogranin A (CgA) and secretogranin II (SgII) in ESs, PNETs, and NBs, both in primitive tumors (five, nine, and four cases, respectively) and in established cell lines (three ES and two PNET cell lines). Different technical approaches, namely immunohistochemistry, Northern blot analysis, and reverse transcriptase-polymerase chain reaction (RT-PCR) were used in parallel. Chromogranin A and secretogranin II production was constantly detectable in NBs by all procedures. CgA mRNA was detectable in most ESs and PNETs only by RT-PCR, whereas SgII mRNA was detectable in some ESs and PNETs by Northern blot analysis and in all tumors by RT-PCR. CgA and SgII proteins were never detectable by immunohistochemistry in ESs and PNETs. We conclude that neuroendocrine differentiation is shared by all three tumor entities, being more overt in NBs and rudimentary in ESs and PNETs; traces of chromogranin mRNA are detectable only by a highly sensitive RT-PCR procedure.

Correlative immunohistochemical and reverse transcriptase polymerase chain reaction analysis of somatostatin receptor type 2 in neuroendocrine tumors of the lung.

Somatostatin receptors type 2 (sst2) have been frequently detected in neuroendocrine tumors and bind somatostatin analogues, such as octreotide, with high affinity. Receptor autoradiography, specific mRNA detection and, more recently, antisst2 polyclonal antibodies are currently employed to reveal sst2. The aim of the present study was to investigate by three different techniques the presence of sst2 in a series of 26 neuroendocrine tumors of the lung in which fresh frozen tissue and paraffin sections were available. It was possible, therefore, to compare, in individual cases, RNA analysis studied by reverse transcriptase polymerase chain reaction (RT-PCR), in situ hybridization (ISH), and immunohistochemistry. A series of 20 nonneuroendocrine lung carcinoma samples served as controls. RT-PCR was positive for sst2 in 22 of 26 samples, including 15 of 15 typical carcinoids, 5 of 6 atypical carcinoids, and 2 of 5 small-cell carcinomas. The sst2 mRNA signal obtained by RT-PCR was strong in the majority (87%) of typical carcinoids and of variable intensity in atypical carcinoids and small-cell carcinomas. A weakly positive signal was observed in 5 of 20 control samples. In immunohistochemistry, two different antibodies (anti-sst2) were employed, including a monoclonal antibody, generated in the Department of Pathology, University of Turin. In the majority of samples a good correlation between sst2 mRNA (as detected by RT-PCR) and sst2 protein expression (as detected by immunohistochemistry) was observed. However, one atypical carcinoid and one small-cell carcinoma had focal immunostaining but no RT-PCR signal. ISH performed in selected samples paralleled the results obtained with the other techniques. A low sst2 expression was associated with high grade neuroendocrine tumors and with aggressive behavior. It is concluded that 1) neuroendocrine tumors of the lung express sst2, and there is a correlation between the mRNA amount and the degree of differentiation; 2) immunohistochemistry and ISH are reliable tools to demonstrate sst2 in these tumors; and 3) sst2 identification in tissue sections may provide information on the diagnostic or therapeutic usefulness of somatostatin analogues in individual patients with neuroendocrine tumors.

Models for the action of barley alpha-amylase isozymes on linear substrates.

The formation of maltodextrins, G1 to G12, during the hydrolysis of amylose by alpha-amylases 1 and 2 from barley malt was followed by HPLC. Similar, but not identical, distributions of products were obtained with the two alpha-amylase components. Maltose, G6, and G7 were major products, but G7 was degraded as hydrolysis proceeded. alpha-Amylase 1 produced more G1 and G3 than did alpha-amylase 2 at all stages of hydrolysis. Products formed during the hydrolysis of G9, G10, G11, and G12 by the two alpha-amylases were also determined. A different spectrum of products was observed with each substrate and small differences were observed in the action pattern of the two alpha-amylases, e.g., G3 and G7 were the major products formed during the hydrolysis of G10 by alpha-amylase 1, whereas G2 and G8 were the major products formed by alpha-amylase 2 on the same substrate. These results were used to develop a model of the active site of barley malt alpha-amylases. This site contains ten contiguous subsites with the catalytic site situated between subsites 7 and 8. The model can be used to predict hydrolysis patterns of amylose and maltodextrins by cereal alpha-amylases.

Neuroendocrine differentiation in carcinomas of the breast: a study of 51 cases.

A group of human breast carcinomas shows morphologic and histochemical evidence of neuroendocrine (NE) differentiation. This study presents a structural, immunologic, and electron microscopic analysis of 51 cases in order to establish positive criteria for identification of these tumors, their incidence, variants, and biological behavior. Argyrophilia (by the Grimelius procedure), presence of chromogranin A and/or B, and of synaptophysin are the most reliable histochemical features, correlating with the ultrastructural demonstration of dense-core secretory granules and of clear vesicles of the synaptic type. Structural features alone may be suggestive, but do not prove NE differentiation, which has to be established by additional techniques. Seven histologic types were identified, but those herein described as types A, B, and C, which show cohesive, mucoid, and mixed patterns, respectively, comprise the vast majority of the tumors. Rare NE carcinomas of the breast show structural similarities to Merkel cell and oat cell carcinomas, and behave as highly aggressive tumors. Type B (mucinous) tumors proved to be relatively indolent, while the most frequent types of endocrine tumors (types A and C) have an intermediate grade of malignancy. The analysis of a consecutive series of 100 cases of breast cancer indicates that about 8% of breast carcinomas display some degree of NE differentiation.

Pancreatic oncocytic endocrine tumors. Cytologic features of two cases.

BACKGROUND: Oncocytoma designates a usually benign tumor consisting of oncocytes (cells rich in mitochondria). Rarely do endocrine pancreatic tumors show oncocytic transformation, and consequently their liver metastases may resemble a hepatocellular carcinoma. CASES: Case 1, a 36-year-old male, presented with an 8-cm pancreatic mass with multiple liver metastases. Fine needle aspiration (FNA) biopsy was performed on the liver. The cytologic features were highly cellular material; numerous isolated cells and irregular, loose cellular aggregates; rare mitoses; round or polygonal cell shape; rosette formation; and large, granular, eosinophilic cytoplasm (suggestive of poorly differentiated hepatocellular carcinoma). Case 2, a 57-year-old female with hypoglycemia, had a 13-cm pancreatic mass. FNA material showed the same cytologic features as case 1. In situ hybridization to detect albumin mRNA was negative in both cases, while immunocytologic reactions for glandular epithelial cytokeratin and chromogranin A were positive. Case 2 was also positive for insulin. CONCLUSION: Oncocytic transformation in endocrine tumors of the pancreas is a rare occurrence and must be kept in mind in the diagnostic workup of FNA material from tumors of the hepatopancreatic region.

Analysis of deaths due to injury in Milano: preliminary results.

We reviewed all trauma deaths occurring in the urban area of Milan during one year. Autopsy reports were cross-referenced with pre- and in-hospital records and the Injury Severity Score was calculated by a senior surgeon. Causes of deaths were defined as central nervous system injury (CNS), hemorrhage (HEM), combined central nervous system injury and hemorrhage (CNS + HEM), and burns (BURN). Places of death were considered the scene (DOS), during transportation (DOA), the emergency room (DER), and hospital. Two multidisciplinary commissions reviewed patient reports and deaths were judged non-preventable, possibly preventable or frankly preventable, using the unanimous decision rule. The TRISS method was used to calculate the probability of survival for in-hospital deaths. Overall trauma deaths were 255 with 78.04% blunt and 16.08% penetrating traumas. Burns accounted for 5.88%. CNS and CNS + HEM caused 171 (67.05%) deaths. DOS were 91, DOA 48, DER 34, and in-hospital deaths 33. Victims found dead (49 individuals) were excluded from further analysis. The commissions classified 56.31% of deaths as non-preventable, 32.03% as possibly preventable and 11.65% as frankly preventable. The Injury Severity Score decreased from DOS to in-hospital deaths (p < 0.05). The preventability rate was higher for in-hospital deaths (p < 0.05). The results of this study suggest that the development of a tiered trauma system in Milan is mandatory.

Clinico-pathologic features of primary melanoma and sentinel lymph node predictive for non-sentinel lymph node involvement and overall survival in melanoma patients: a single centre observational cohort study.

OBJECTIVE: Completion Lymph Node Dissection (CLND) is the current standard of practice for patients with a positive Sentinel Lymph Node Biopsy (SLNB). Significant morbidity is associated to CLND, so we tried to evaluate which prognostic variables could predict NSLN invasion in SLN-positive patients and their impact on the overall survival (OS). METHODS: A retrospective chart review of 603 patients that had undergone SLNB for melanoma between 2000 and 2009 at our department was done. 100 SLN were positive at the histopathological analysis of SLN. Demographic variables, primary melanoma, SLN pathologic features and results of CLND were analysed. Multivariate logistic regression and OS analyses were carried out to test the prognostic relevance of clinico-pathologic variables on CLND results and disease course. RESULTS: Breslow thickness, ulceration and micro/macrometastatic pattern of SLN invasion carried a significantly independent higher likelihood of NSLN involvement; Starz classification did not maintain a statistical significance in multivariate analysis. Only one patient (4.3%) without adverse prognostic factors showed NSLN involvement, which was found in 33.3% of patients with one and 55.9% with two or more adverse parameters (p = 0.0001). OS analyses confirmed the prognostic significance of these factors. CONCLUSION: Waiting for the results of Multicenter Selective Lymphadenectomy Trial II, our study suggests a clinically useful and easily applicable means of identifying patients with an unfavourable disease course. The presence of one or more adverse factors identifies patients in whom CLND is mandatory to include thereafter in a more strict follow-up program. Moreover, the finding of no adverse prognostic indicators associated to the presence of significant co-morbidities and/or elderly age, could be useful in identifying patients not to treat by CLND.