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.

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.

[Endocrine differentiation in basocellular carcinoma]. / Differenziazione endocrina nei carcinomi basocellulari.

Three (propositus) cases of basal cell carcinoma (BCC) showing endocrine differentiation at the immunohistochemical level were studied using reverse transcriptase-polymerase chain reaction (RT-PCR) to determine the presence of mRNA of chromogranin A. Moreover, 20 (consecutive) cases of BCC were studied with immunohistochemistry alone using chromogranin A, synaptophysin, S100 protein, cytokeratin 20, and neuron-specific enolase antibodies (NSE). The three propositus cases of BCC showed positive results when RT-PCR for mRNA of chromogranin A was performed. Eleven out of 20 consecutive cases of BCC were focally positive for chromogranin A antibody. These results confirm the presence of endocrine differentiation in BCC, demonstrated both with immunohistochemistry and with RT-PCR.

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.

[Rhabdomyolysis caused by improper intraoperative positioning. Clinical and medico-legal aspects]. / Rabdomiolisi da malposizionamento intraoperatorio. Aspetti clinici e medicolegali.

The etiology, diagnosis, pathology and treatment of rhabdomyolysis due to intraoperative malpositioning and the medico-legal implications of physicians involved in the surgical treatment and anesthesia of the patient are described. According to the Italian law, the anesthesiologist is the only physician of the surgery-anesthesia team responsible for the patient's positioning. The anesthesiologist must assume primary responsibility for protecting the patient from iatrogenic injuries due to improper positioning, and/or inadequate preventive measures.

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.

Chromogranin A gene expression in non-small cell lung carcinomas.

Evidence for the existence of neuroendocrine (NE) differentiation in non-small cell lung carcinomas (NSCLCs) is at present based on histochemical, ultrastructural, and immunohistochemical data. The aim of this study was to investigate the extent of NE differentiation in NSCLCs as revealed by mRNA analysis. Different techniques including immunohistochemistry (IHC), northern blot analysis (NBA), and reverse transcriptase-polymerase chain reaction (RT-PCR) were employed in parallel to reveal the panendocrine marker chromogranin A (CgA). The data were related to pathological, immunocytochemical (PGP 9.5, synaptophysin, Leu-7 and neuron-specific enolase), and prognostic indicators. Forty surgically resected cases of NSCLC (24 squamous cell carcinomas, 12 ordinary type adenocarcinomas, 3 bronchiolo-alveolar carcinomas, and 1 anaplastic large cell carcinoma), in which fresh frozen material was available for mRNA analysis, were collected. CgA immunoreactivity was present in five cases (12.5 per cent), generally confined to a minority of the neoplastic cell population. By RT-PCR, CgA mRNA was found in 20 cases (50 per cent), including the five tumours positive by IHC. A statistically significant correlation was found between the two techniques. By NBA, no CgA mRNA expression was detected. Leu-7 immunoreactivity was present in 15 per cent of cases, NSE in 52.5 per cent, synaptophysin in 10 per cent, and PGP 9.5 in 82.5 per cent. In NSCLC, no correlations were found between CgA production, as detected by IHC or RT-PCR methods, and the histological type, stage, grade and proliferative activity of tumours, or the disease-free interval. It is concluded that CgA gene expression can be revealed in NSCLC at both mRNA and protein levels and that RT-PCR is a valuable tool for identifying NE differentiated NSCLCs. Our data suggest that NE differentiation does not represent an independent prognostic factor in surgically resected NSCLCs.

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.

[The diagnostic reliability of percutaneous biopsy specimens obtained under instrumental guidance using 4 different types of needles]. / Attendibilità diagnostica dei prelievi bioptici percutanei eseguiti con guida strumentale utilizzando quattro diversi tipi di aghi.

To assess if the effectiveness of biopsy sampling, whose usefulness is widely recognized, can be influenced by different cutting mechanisms, we compared four different types of needles (A, B, C, D) in 76 patients, each needle being of the same length (15 cm) and calibre (18 G) but with different tip shapes. Forty biopsy samples were obtained with each type of needle for a total of 160 samples in 76 patients; 61 samples were acquired under CT guidance and 99 under US guidance. The results were subdivided in 6 categories based on biopsy result: PI (diagnosis histotype in malignant lesions), PN (diagnosis of malignancy in neoplastic lesions), P (correct diagnosis in benign lesions), S (suspicion of neoplastic lesion), E (misdiagnosis), NI (insufficient material). The results, respectively, for each needle type are: 32.5%, 30%, 2.5%, 7.5%, 12.5% and 15% with needle A; 35%, 30%, 7.5%, 0%, 15% and 12.5% with needle B; 27.5%, 17.5%, 10%, 7.5%, 22.5% and 15% with needle C; 30%, 15%, 7.5%, 15%, 17.5% and 15% with needle D. The sensitivity and specificity for each needle type were, respectively, 84.4% and 85.29% for needle A; 86.66% and 88.57% for needle B; 70% and 73.52% for needle C; 77.41% and 79.41% for needle D. No statistically significant difference was found in the effectiveness of the four needles (chi 2); on the contrary, lesions's size can affect sampling quality.

Intranodular reactive endothelial hyperplasia in adenomatous goitre.

Endothelial hyperplasia has rarely been recognized in the thyroid. Ischaemic events or hemorrhage, sometimes as a result of fine needle aspiration procedures, have been regarded as possible causes. To clarify the morphological pattern and the clinical significance of this lesion, we studied a series of adenomatous goitres and selected 11 cases showing prominent endothelial hyperplasia in an individual nodule. Grossly, the parenchyma of the affected nodule was substituted by greyish, friable tissue surrounded by a fibrous capsule and by a thin rim of residual thyroid parenchyma. Microscopically, fibrinous and haemorrhagic material was crossed by intercommunicating vascular channels or papillary structures. These were lined by plump endothelial cells. In only one case had pre-operative fine needle aspiration biopsy been performed: smears yielded a haemorrhagic background, fibrin and numerous elongated cells; colloid and follicular cells were virtually absent. In both surgical and cytological specimens a differential diagnosis with a vascular neoplasm, a rare occurrence in the thyroid, was taken into consideration. We conclude that intranodular reactive endothelial hyperplasia is a relatively common occurrence (1.6%) in adenomatous goitre and is of diagnostic interest in both surgical and cytological specimens, since it can mimic vascular tumours and may lead to unnecessary treatment if misdiagnosed.

The Reed-Sternberg cells of Hodgkin disease are clonal.

Relatively little progress has been made in understanding the nature of the Reed-Sternberg (RS) cell and its morphologic variants in Hodgkin disease (HD). This is primarily due to the fact that RS cells represent a minute subpopulation within HD lesions. To investigate the clonal origin of RS cells and variants, we studied 27 HD lesions obtained from 11 patients. Using an image analyzer (CAS 200) we were able to demonstrate that CD30-positive RS cells are clonal elements with unique and individualized DNA profiles and that the DNA content of any given patient RS cell population is constant over time and in different pathologic sites. Using 1, 9, 11, and X alpha satellite chromosome probes and interphase cytogenetics, we also demonstrated that RS cells obtained from different tissue samples of the same patient have a unique and often abnormal chromosomal pattern. To definitively prove the hypothesis that CD30-positive RS cells are clonal elements, we investigated the presence of point mutations within p53 gene exons 5 through 9 and found that only a single patient possessed a nonsense p53 somatic point mutation (Arg to His). This same mutation could be identified in all of his available biopsies. Altogether, these findings demonstrate that RS cells and variants in HD are clonal and represent the neoplastic elements of this entity.

Molecular characterization of CD30+ anaplastic large-cell lymphoma: high frequency of c-myc proto-oncogene activation.

Anaplastic large-cell lymphoma (ALCL) represents a morphologically distinct type of non-Hodgkin's lymphoma (NHL) characterized phenotypically by the expression of the CD30 antigen, a new member of the nerve growth factor gene family. The lymphoid origin of ALCL has been documented using immunohistochemical and molecular genetic analyses. However, very little is known so far regarding the precise pathogenetic mechanisms involved in its development and progression. Therefore, we investigated bcl-2, p53, and retinoblastoma gene (Rb) expression immunohistochemically; the occurrence of bcl-2, c-myc, and Rb gene rearrangements using Southern blotting; and the presence of ras and p53 gene somatic mutations by single-strand conformation polymorphism assay in a panel of 18 well-characterized ALCLs. In addition, the presence of Epstein-Barr (EBV) and human T-cell lymphotropic virus type I (HTLV-I) genomes were investigated using polymerase chain reaction. We identified abnormal c-myc gene products in 6 of 18 cases (33%) of ALCL. On the other hand, the bcl-2 and Rb genes were not rearranged and K-, N-, and H-ras gene somatic mutations were not found. Significant levels of p53 protein expression were found in more than 60% of ALCLs, but only a single ALCL carried a p53 gene mutation (exon 5). Only 3 ALCL cases, all occurring in human immunodeficiency virus-infected patients, were positive for EBV genomes. On the other hand, contrary to previous findings, no HTLV-I products could be identified. Despite the fact that the c-myc proto-oncogene appears to be frequently altered in ALCL, no pathognomonic abnormality could be identified and therefore additional studies and new strategies should be designed to identify the pathogenetic mechanisms involved in the development of ALCL.

Immunophenotypic properties and estrogen dependency of budding cell structures in the developing mouse mammary gland.

The initial phase of growth of the parenchymal component of the mouse mammary gland is ductal clongation, which is mainly accomplished by proliferating cells in a specialized structure termed end bud. End buds are composed of multiple layers of epithelial cells (so called body cells) which are capped by a single layer of morphologically unique cells termed cap cells. We sought to examine the interrelationship between cap cells and other epithelial cell subclasses using a variety of antibodies to different keratin proteins and also antibodies to vimentin, actin and collagen IV. An extensive immunohistochemical characterization of the epithelial components of the developing and differentiating mammary gland demonstrated that cap cells were devoid of any immunohistochemically-detectable keratins but were positive for collagen IV. In contrast, the majority of cells in the end bud along with the luminal epithelial and myoepithelial cells were keratin positive. The body cells of the end bud were the only cells which were positive for antibody to keratin 6, a keratin which previously has been reported to be expressed in proliferating mammary epithelial cells. In addition, estrogen receptor was localized only to epithelial cells of ducts, alvcoli and body cells of end buds, but not to cap cells or myoepithelial cells. We interpret these results to suggest that cap cells are not totpotent stem cells but rather cells specialized in paving the way for ductal elongation as well as serving as precursors to myoepithelial cells.

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.

Cytological analysis of benign breast disease.

The different histological lesions of benign breast disease (BBD) are the result of interactions and regional prevalence of epithelial, myoepithelial, apocrine, and "null" (undifferentiated) cell types. We conducted an immunocytochemical analysis on 14 cases of BBD. Specific markers were employed to identify the different cell types; proliferation in these cells was revealed either by bromo-deoxyuridine uptake or PCNA ("cyclin") localization. The results indicate that apocrine cells do not undergo proliferation, representing therefore a terminally differentiated cell. The question related to the preneoplastic potential of BBD and on which cell type might possibly represent the precursor of in situ cancerous lesions remains unanswered. However, our data tend to exclude that such a putative proliferating precursor might be represented by apocrine or myoepithelial cells or even by the epithelial cells featuring epitheliosis.

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.