Variation in histopathological assessment and association with surgical quality indicators following oesophagectomy.

BACKGROUND: Histopathological outcomes, such as lymph node yield and margin positivity, are used to benchmark and assess surgical centre quality, and are reported annually by the National Oesophago-Gastric Cancer Audit (NOGCA) in England and Wales. The variation in pathological specimen assessment and how this affects these outcomes is not known. METHODS: A survey of practice was circulated to all tertiary oesophagogastric cancer centres across England and Wales. Questions captured demographic data, and information on how specimens were prepared and analysed. National performance data were retrieved from the NOGCA. Survey results were compared for tertiles of lymph node yield, and circumferential and longitudinal margins. RESULTS: Survey responses were received from 32 of 37 units (86 per cent response rate), accounting for 93.1 per cent of the total oesophagectomy volume in England and Wales. Only 5 of 32 units met or exceeded current guidelines on specimen preparation according to the Royal College of Pathologists guidelines. There was wide variation in how centres defined positive (R1) margins, and how margins and lymph nodes were assessed. Centres with the highest nodal yield were more likely to use systematic fat blocking, and to re-examine specimens when the initial load was low. Systematic blocking of lesser curve fat resulted in significantly higher rates of patients with at least 15 lymph nodes examined (91.4 versus 86.5 per cent; P = 0.027). CONCLUSION: Preparation and histopathological assessment of specimens varies significantly across institutions. This challenges the validity of currently used surgical quality metrics for oesophageal and other tumours.

Primary cutaneous peripheral T-cell lymphoma, not otherwise specified: results of a multicentre European Organization for Research and Treatment of Cancer (EORTC) cutaneous lymphoma taskforce study on the clinico-pathological and prognostic features.

BACKGROUND: Cutaneous peripheral T-cell lymphoma, not otherwise specified (PTL NOS) is an aggressive, but poorly characterized neoplasm. OBJECTIVES: The European Organization for Research and Treatment of Cancer cutaneous lymphoma taskforce (EORTC CLTF) investigated 33 biopsies of 30 patients with primary cutaneous PTL NOS to analyse their clinical, histological, immunophenotypic features and outcome. METHODS: Retrospective analysis of clinical data and histopathological features by an expert panel. RESULTS: Cutaneous PTL NOS manifested clinically either with solitary or disseminated rapidly grown ulcerated tumours or disseminated papulo-nodular lesions. Histologically, a mostly diffuse or nodular infiltrate in the dermis and often extending into the subcutis was found. Epidermotropism was rarely present and only mild and focal. Unusual phenotypes were frequent, e.g. CD3+ /CD4- /CD8- and CD3+ /CD4+ /CD8+ . Moreover, 18% of the cases exhibited an aberrant expression of the B-cell marker CD20 by the tumour cells. All solitary tumours were located on the limbs and presented a high expression of GATA-3 but this did not correlate with outcome and therefore could not serve as a prognostic factor. The prognosis was shown to be generally poor with 10 of 30 patients (33%) dying of lymphoma within the follow-up of 36 months (mean value; range 3-144). The survival rates were 61% after 3 years (CI, 43-85%) and 54% after 5 years (CI, 36-81%). Small to medium-sized morphology of tumour cells was associated with a better outcome than medium to large or large tumour cells. Age, gender, clinical stage, CD4/CD8 phenotype and GATA-3 expression were not associated with prognosis. Chemotherapy was the most common treatment modality, but surgical excision and/or radiotherapy may represent an appropriate first-line treatment for solitary lesions. CONCLUSIONS: Cutaneous PTL NOS shows an aggressive course in most patients independent of initial presentation, age and phenotype. Cytomorphology was identified as a prognostic factor. The data indicate a need for more effective treatment modalities in PTL NOS.

Minichromosomal Maintenance Component Complex 5 (MCM5) as a Marker of Barrett's Esophagus-Related Neoplasia: A Feasibility Study.

BACKGROUND: The endoscopic detection of esophageal cancer is suboptimal in both patients referred with dyspeptic symptoms and those enrolled in Barrett's surveillance programs. MCM5 expression in cells collected from gastric fluid may be correlated with the presence of dysplasia or adenocarcinoma. Analysis of this biomarker may improve the detection of cancer. METHODS: Sixty-one patients were enrolled at a single UK referral center. From each patient, 5-10 ml of gastric fluid was aspirated endoscopically. Patients were categorized according to their histology, normal, non-dysplastic Barrett's (NDBE), high-grade dysplastic Barrett's (HGD), and esophageal adenocarcinoma (EAC). All histology was confirmed by Seattle protocol biopsies or endoscopic mucosal resection. Samples were centrifuged, and the cell pellet was lysed. MCM5 expression levels were quantified using a proprietary immunoassay. The mean MCM5 expression was compared between groups by Kruskal-Wallis test. ROC curves were also used to assess diagnostic utility. RESULTS: The mean expression of MCM5 increases as patients progress from a normal esophagus to NDBE, HGD, and EAC (14.4; 49.8; 112.3; and 154.1, respectively). There was a significant difference in the MCM5 expression of patients with a normal esophagus compared to those with EAC (p = 0.04). There was a trend toward higher MCM5 expression in patients with EAC compared to those with NDBE (p = 0.34). MCM5 expression was a fair discriminator (AUC 0.70 [95% CI 0.57-0.83]) between patients without neoplasia (normal and NDBE) and those with early neoplasia (HGD and EAC). CONCLUSION: MCM5 expression in gastric fluid samples can differentiate patients with a histologically normal esophagus compared to those with early adenocarcinoma. Larger, powered studies are needed to assess whether it can be used to differentiate those with HGD from NDBE.

Prognostic markers for colorectal cancer: estimating ploidy and stroma.

Background: We report here the prognostic value of ploidy and digital tumour-stromal morphometric analyses using material from 2624 patients with early stage colorectal cancer (CRC). Patients and methods: DNA content (ploidy) and stroma-tumour fraction were estimated using automated digital imaging systems and DNA was extracted from sections of formalin-fixed paraffin-embedded (FFPE) tissue for analysis of microsatellite instability. Samples were available from 1092 patients recruited to the QUASAR 2 trial and two large observational series (Gloucester, n = 954; Oslo University Hospital, n = 578). Resultant biomarkers were analysed for prognostic impact using 5-year cancer-specific survival (CSS) as the clinical end point. Results: Ploidy and stroma-tumour fraction were significantly prognostic in a multivariate model adjusted for age, adjuvant treatment, and pathological T-stage in stage II patients, and the combination of ploidy and stroma-tumour fraction was found to stratify these patients into three clinically useful groups; 5-year CSS 90% versus 83% versus 73% [hazard ratio (HR) = 1.77 (95% confidence interval (95% CI): 1.13-2.77) and HR = 2.95 (95% CI: 1.73-5.03), P < 0.001]. Conclusion: A novel biomarker, combining estimates of ploidy and stroma-tumour fraction, sampled from FFPE tissue, identifies stage II CRC patients with low, intermediate or high risk of CRC disease specific death, and can reliably stratify clinically relevant patient sub-populations with differential risks of tumour recurrence and may support choice of adjuvant therapy for these individuals.

HPyV6, HPyV7 and TSPyV DNA sequences detection in skin disease patients and healthy subjects.

BACKGROUND: The discovery, from 2007, of eight new human polyomaviruses (HPyVs) has revived interest in the Polyomaviridae family and their association with human diseases and cancer. In particular, HPyV6 and HPyV7 were discovered in skin swabs of healthy donors and TSPyV was discovered in a heart transplant recipient affected by virus-associated Trichodysplasia Spinulosa (TS), a rare skin disease, exclusively found in immunocompromised patients. OBJECTIVE: The presence of HPyV6, HPyV7 and TSPyV DNA in skin biopsies from patients affected by different skin diseases (cancers and inflammatory disorders) has been evaluated to confirm their skin tropism and the possible pathological association. METHODS: DNA extracted was amplified with HPyV6, HPyV7 and TSPyV specific PCR real time on Taqman platform with standard profile. RESULTS: HPyV7 and TSPyV sequences were not found in any skin specimen analysed. HPyV6, on the other hand, was detected in 30% of samples from healthy subjects vs. 14.3% of skin cancer patients and 2.9% of inflammatory disorders. HPyV6 sequences have been detected in primary cutaneous T-cell lymphoma (CTCL) patients (in 18.6% out of Mycosis Fungoides (MF) patients and in 16.7% out of CTCL not MF/SS(Sèzary syndrome) but have not been detected in primary cutaneous B-cell lymphoma (CBCL) patients. CONCLUSION: Our preliminary data suggest that these three novel human polyomaviruses seem not to play a significant role neither in the pathogenesis of cutaneous malignancies nor in that of inflammatory disorders but, according to literature, can inhabit the skin. On the basis of our data regarding the HPyV6 DNA presence with decreasing percentages in healthy subjects, skin cancer and inflammatory disorders patients, it could be an intriguing matter to study if the activated innate immune response in inflammatory disorders can suppress the virus. Further investigations are needed to better understand their relationship with the human host and its innate immune system.

Phenotypical characterization of circulating cell subsets in pyoderma gangrenosum patients: the experience of the Italian immuno-pathology group.

BACKGROUND: No data are available as to the phenotype of circulating lymphocyte subsets in pyoderma gangrenosum (PG). AIM: To analyse the expression of different chemokine receptors associated to T-helper (Th)1 (CCR5), Th2 (CCR4) and Th17 (CCR6), as well as the regulatory T-cell subset (Treg) and dendritic cell polarization in the blood of newly diagnosed untreated PG patients. MATERIALS AND METHODS: Multi-parameter flow cytometry was performed on blood samples from 10 PG patients collected at first diagnosis among centres belonging to the Italian Immuno-pathology Group. Blood samples from 10 age- and sex-matched healthy controls (HC) were used as controls. RESULTS: PG patients are characterized by an over-expression in the blood of the CD4+CCR5+ and CD4+CCR6+ and a down-regulation of CD4+CCR4+ counts with respect to healthy subjects. Moreover, they show increased levels of myeloid derived dendritic cells type1 and reduced levels of the Treg CD4+CD25highFOXP3+ subset. CONCLUSIONS: The pattern of chemokine expression argues in favour of a Th1 (CCR5+) and Th17 (CCR6+) polarization with a down-regulation of Th2 (CCR4+).

Improvement over time in outcomes for patients undergoing endoscopic therapy for Barrett's oesophagus-related neoplasia: 6-year experience from the first 500 patients treated in the UK patient registry.

BACKGROUND: Barrett's oesophagus (BE) is a pre-malignant condition leading to oesophageal adenocarcinoma (OAC). Treatment of neoplasia at an early stage is desirable. Combined endoscopic mucosal resection (EMR) followed by radiofrequency ablation (RFA) is an alternative to surgery for patients with BE-related neoplasia. METHODS: We examined prospective data from the UK registry of patients undergoing RFA/EMR for BE-related neoplasia from 2008 to 2013. Before RFA, visible lesions were removed by EMR. Thereafter, patients had RFA 3-monthly until all BE was ablated or cancer developed (endpoints). End of treatment biopsies were recommended at around 12 months from first RFA treatment or when endpoints were reached. Outcomes for clearance of dysplasia (CR-D) and BE (CR-IM) at end of treatment were assessed over two time periods (2008-2010 and 2011-2013). Durability of successful treatment and progression to OAC were also evaluated. RESULTS: 508 patients have completed treatment. CR-D and CR-IM improved significantly between the former and later time periods, from 77% and 56% to 92% and 83%, respectively (p<0.0001). EMR for visible lesions prior to RFA increased from 48% to 60% (p=0.013). Rescue EMR after RFA decreased from 13% to 2% (p<0.0001). Progression to OAC at 12 months is not significantly different (3.6% vs 2.1%, p=0.51). CONCLUSIONS: Clinical outcomes for BE neoplasia have improved significantly over the past 6 years with improved lesion recognition and aggressive resection of visible lesions before RFA. Despite advances in technique, the rate of cancer progression remains 2-4% at 1 year in these high-risk patients. TRIAL REGISTRATION NUMBER: ISRCTN93069556.

Maternal obesity programs offspring non-alcoholic fatty liver disease through disruption of 24-h rhythms in mice.

BACKGROUND: Maternal obesity increases offspring propensity to metabolic dysfunctions and to non-alcoholic fatty liver disease (NAFLD), which may lead to cirrhosis or liver cancer. The circadian clock is a transcriptional/epigenetic molecular machinery synchronising physiological processes to coordinate energy utilisation within a 24-h light/dark period. Alterations in rhythmicity have profound effects on metabolic pathways, which we sought to investigate in offspring with programmed NAFLD. METHODS: Mice were fed a standard or an obesogenic diet (OD), before and throughout pregnancy, and during lactation. Offspring were weaned onto standard or an OD at 3 weeks postpartum and housed in 12:12 light/dark conditions. Biochemical and histological indicators of NAFLD and fibrosis, analysis of canonical clock genes with methylation status and locomotor activity were investigated at 6 months. RESULTS: We show that maternal obesity interacts with an obesogenic post-weaning diet to promote the development of NAFLD with disruption of canonical metabolic rhythmicity gene expression in the liver. We demonstrate hypermethylation of BMAL-1 (brain and muscle Arnt like-1) and Per2 promoter regions and altered 24-h rhythmicity of hepatic pro-inflammatory and fibrogenic mediators. CONCLUSIONS: These data implicate disordered circadian rhythms in NAFLD and suggest that disruption of this system during critical developmental periods may be responsible for the onset of chronic liver disease in adulthood.

Phase I/II study of verteporfin photodynamic therapy in locally advanced pancreatic cancer.

BACKGROUND: Patients with pancreatic cancer have a poor prognosis apart from the few suitable for surgery. Photodynamic therapy (PDT) produces localised tissue necrosis but previous studies using the photosensitiser meso-tetrahydroxyphenylchlorin (mTHPC) caused prolonged skin photosensitivity. This study assessed a shorter acting photosensitiser, verteporfin. METHODS: Fifteen inoperable patients with locally advanced cancers were sensitised with 0.4 mg kg(-1) verteporfin. After 60-90 min, laser light (690 nm) was delivered via single (13 patients) or multiple (2 patients) fibres positioned percutaneously under computed tomography (CT) guidance, the light dose escalating (initially 5 J, doubling after each three patients) until 12 mm of necrosis was achieved consistently. RESULTS: In all, 12 mm lesions were seen consistently at 40 J, but with considerable variation in necrosis volume (mean volume 3.5 cm(3) at 40 J). Minor, self-limiting extrapancreatic effects were seen in multifibre patients. No adverse interactions were seen in patients given chemotherapy or radiotherapy before or after PDT. After PDT, one patient underwent an R0 Whipple's pancreaticoduodenectomy. CONCLUSIONS: Verteporfin PDT-induced tumour necrosis in locally advanced pancreatic cancer is feasible and safe. It can be delivered with a much shorter drug light interval and with less photosensitivity than with older compounds.

Prognostic impact of genomic instability in colorectal cancer.

BACKGROUND: The prognostic impact of an indication of chromosomal instability (CIN) is evaluated in a consecutive series of 952 colorectal cancer patients treated at Aker University Hospital, Norway, during 1993-2003. Microsatellite instability (MSI) in this case series has recently been reported and made it possible to find the co-occurrence and compare the prognostic significance of CIN and MSI. METHODS: Data sets for overall survival (OS; n=855) and time to recurrence (TTR; n=579) were studied. To reveal CIN we used automated image cytometry (ICM). Non-diploid histograms were taken as indicative of the presence of CIN. PCR-based measures of MSI in this material have already been described. RESULTS: As with MSI, CIN was found to be an independent predictor of early relapse and death among stage II patients (TTR: n=278: HR 2.19 (95% CI: 1.35-3.55), P=0.002). Of the MSI tumours (16%), 71% were found to be DNA diploid, 21% were DNA tetraploid and 8% were DNA aneuploid. Among microsatellite stable tumours, 24% were DNA diploid, 15% were DNA tetraploid and 61% were DNA aneuploid. CONCLUSION: For patients presenting with stage II disease, genomic instability as detected by DNA image cytometry has the potential to provide a useful biomarker for relapse and cancer-related death following surgery with curative intent.

Regulatory T cells in skin lesions and blood of patients with bullous pemphigoid.

BACKGROUND: Although regulatory T cells (Tregs) are affected in several autoimmune skin diseases, only two studies have been performed in patients with bullous pemphigoid (BP) with contrasting results. OBJECTIVE: To characterize Tregs and to determine the serum levels of regulatory cytokines in patients with BP. METHODS: In BP lesional skin, immunohistochemistry and confocal microscopy were performed for CD4(+) , CD25(+) , forkhead/winged helix transcription factor (FOXP3)(+) , transforming growth factor (TGF)-ß(+) and interleukin (IL)-10(+) cells. In addition, the number of CD4(+) CD25(++) FOXP3(+) Tregs in peripheral blood was assessed by flow cytometry, and the levels of TGF-ß and IL-10 were determined in serum samples by enzyme-linked immunosorbent assay before and after steroid therapy. Controls included patients with psoriasis, atopic dermatitis (AD) and healthy donors. RESULTS: The frequency of FOXP3(+) cells was significantly reduced in skin lesions from patients with BP (P < 0.001) compared with psoriasis and AD. Moreover, the number of IL-10(+) cells was lower in BP than in psoriasis (P < 0.001) and AD (P = 0.002), while no differences were observed in the number of TGF-ß(+) cells. CD4(+) CD25(++) FOXP3(+) Treg in the peripheral blood of patients with BP was significantly reduced compared with healthy controls (P < 0.001), and augmented significantly after steroid therapy (P = 0.001). Finally, TGF-ß and IL-10 serum levels were similar in patients with BP compared with healthy controls. However, after therapy, BP patients showed significantly higher IL-10 serum levels than before therapy (P = 0.01). CONCLUSIONS: These data suggest that the depletion of Tregs and of IL-10 in patients with BP may be an important factor in the pathogenesis of the disease.

T staging esophageal tumors with x rays.

With histopathology results typically taking several days, the ability to stage tumors during interventions could provide a step change in various cancer interventions. X-ray technology has advanced significantly in recent years with the introduction of phase-based imaging methods. These have been adapted for use in standard labs rather than specialized facilities such as synchrotrons, and approaches that enable fast 3D scans with conventional x-ray sources have been developed. This opens the possibility to produce 3D images with enhanced soft tissue contrast at a level of detail comparable to histopathology, in times sufficiently short to be compatible with use during surgical interventions. In this paper we discuss the application of one such approach to human esophagi obtained from esophagectomy interventions. We demonstrate that the image quality is sufficiently high to enable tumor T staging based on the x-ray datasets alone. Alongside detection of involved margins with potentially life-saving implications, staging tumors intra-operatively has the potential to change patient pathways, facilitating optimization of therapeutic interventions during the procedure itself. Besides a prospective intra-operative use, the availability of high-quality 3D images of entire esophageal tumors can support histopathological characterization, from enabling "right slice first time" approaches to understanding the histopathology in the full 3D context of the surrounding tumor environment.

Apical MUC1 expression revealed on the foveolar epithelium in H. pylori gastritis.

BACKGROUND: The membrane mucin MUC1 is altered in its pattern of expression in cancer, and also in other pathological situations, including Helicobacter pylori gastritis. Here we investigate the basis for the loss of apical staining of the gastric foveolar epithelium in H. pylori gastritis. METHODS: MUC1 was examined in the gastric antrum from cases of H. pylori gastritis and normal controls. We used tissue sections that were either treated or not treated with periodate to effect deglycosylation, and the monoclonal antibodies LICRLonM8, MUSE-11, CT2 and BC2. RESULTS: We show that the epitopes on the TR domain of MUC1 are partially cryptic due to glycosylation and that MUC1 is present on the apical surface of the gastric foveolar epithelium of gastritis patients. CONCLUSION: This observation suggests that there is no substantial loss of the mucin domain of MUC1 from the apical surface in gastritis, as suggested by others, but rather the H. pylori influences the glycosylation of MUC1. This paper highlights the issue of epitope specificity of monoclonal antibodies directed against disease-associated markers, specifically when they are glycoproteins, as is the case for many cancer markers.

A randomised controlled trial of ALA vs. Photofrin photodynamic therapy for high-grade dysplasia arising in Barrett's oesophagus.

Photofrin photodynamic therapy (PDT) is a licenced treatment for Barrett's oesophagus (BE) with high-grade dysplasia (HGD) but causes strictures and photosensitivity and complete reversal of dysplasia (CR-HGD) by 50 % at 5 years. 5-Aminolaevulinic acid (ALA) is an alternative treatment with non-randomised data suggesting 85 % CR-HGD and a low risk of side effects. We aimed to compare efficacy and side effect profile between the drugs. A single-centre randomised controlled trial was conducted. Presence of HGD was confirmed on three occasions by two specialist GI pathologists. Stratification was by length of BE and extent of dysplasia. Standard protocols for ALA and Photofrin-PDT were followed. Endoscopic follow-up with 2-cm four-quadrant biopsy was at 6 weeks, 4 months, and then annually. All adverse event data were collected. Sixty four patients were randomised, 34 ALA and 30 Photofrin-PDT. Median follow-up is 24 months. On intention-to-treat analysis, CR-HGD was 16/34 (47 %) with ALA-PDT and 12/30 (40 %) with Photofrin-PDT. The overall cancer incidence was 14 % (9/64). On sub-group log-rank analysis, for BE ≤ 6 cm, CR-HGD was significantly higher with ALA-PDT than Photofrin-PDT (χ(2) =5.39, p=0.02). Strictures and skin photosensitivity were significantly more common after treatment with Photofrin-PDT than ALA-PDT (33 vs. 9 % and 43 vs. 6 %, respectively, p<0.05). The rate of buried glands with either drug was significantly higher post-PDT (48 % of patients) than pre-PDT (20 %). ALA-PDT has a better risk profile than Photofrin-PDT. In patients with BE length ≤ 6 cm, preliminary results show ALA-PDT is associated with significantly higher CR-HGD. In longer segments of BE, neither PDT drug is sufficiently efficacious to warrant routine use.

Flow cytometric analyses of circulating regulatory T cells in patients with dermatitis herpetiformis and other immune mediated dermatoses.

Regulatory T cells (Tregs) play a crucial role by maintaining the peripheral tolerance and inhibiting autoimmunity. In recent years, numerous autoimmune and immune-mediated diseases have been shown to present significant number depletion and/or function impairment of this subset. In the present study, we present a brief overview of the results obtained by our group in association with the centers belonging to the Italian Immunopathology Group, as to the expression levels and biological significance of circulating regulatory CD4+CD25+brightFOXP3+ T cells in a variety of immune-mediated skin diseases (such as psoriasis, scleroderma, bullous pemphigoid and GvHD), together with preliminary results achieved in patients with inflammatory bowel disease-related dermatoses. This review shows that this series of different cutaneous diseases characterised by an immune-mediated pathogenesis, share a significant down-regulation of circulating FOXP3+ Treg cells, whilst the treatment and the achievement of clinical response are generally associated with an opposite phenomenon with up-regulation of Treg cells. Future studies are mandatory to identify the effective role of these modifications in the disease pathogenesis as well as its relationship with the clinical response.

Array-comparative genomic hybridization analysis in a cohort of 130 children with Autism Spectrum Disorders: A single Center Italian Study.

Introduction: utism spectrum disorder (ASD) is a heterogeneous clinical condition, and its genetic basis is widely confirmed. The chromosomal microarray analysis (CMA) is a first-line diagnostic test that identifies copy number variants (CNVs). Some of these genomic rearrangements are associated with ASD, but the meaning of most of them is still unknown. Materials and methods: We performed a comparative genome hybridization (array-CGH) analysis in 130 children with confirmed ASD. Genetic results were analyzed and compared to clinical phenotype. Results and discussion.: 61/130 children carry CNVs, 44 presenting variants of unknown significance (u-CNVs), and 17 with susceptibility-CNVs (c-CNVs). Clinical evaluation showed no differences in cognitive abilities, language and EEG abnormalities, ASD symptoms among CNVs group and other patients. Finally, we highlight the role of GPHN, IMMP2L and ZMYND11, as ASD susceptibility genes. Conclusions: Our findings underscore the importance of array-CGH in ASD children since new CNVs and emerging genes appear to be associated with different clinical pictures.

Comparing virtual with conventional microscopy for the consensus diagnosis of Barrett's neoplasia in the AspECT Barrett's chemoprevention trial pathology audit.

AIMS: To compare the diagnostic accuracy of conventional versus virtual microscopy for the diagnosis of Barrett's neoplasia. METHODS AND RESULTS: Sixty-one biopsies from 35 ASPirin Esomeprazole ChemopreventionTrial (AspECT) trial patients were given a Barrett's neoplasia score (1-5) by a panel of five pathologists using conventional microscopy. Thirty-three biopsies positive for neoplasia were digitized and rescored blindly by virtual microscopy. Diagnostic reliability was compared between conventional and virtual microscopy using Fleiss' kappa. There was substantial reliability of diagnostic agreement (κ = 0.712) scoring the 61 biopsies and moderate agreement scoring the subgroup of 33 'positive' biopsies with both conventional microscopy (κ = 0.598) and virtual microscopy (κ = 0.436). Inter-observer diagnostic agreement between two pathologists by virtual microscopy was substantial (κ = 0.76). Comparison of panel consensus neoplasia scores between conventional and virtual microscopy was almost perfect (κ = 0.8769). However, with virtual microscopy there was lowering of the consensus neoplasia score in nine biopsies. CONCLUSIONS: Diagnostic agreement with virtual microscopy compares favourably with conventional microscopy in what is recognized to be a challenging area of diagnostic practice. However, this study highlights possible limitations for this method in the primary diagnostic setting.

Clinical and prognostic reports from 270 patients with multiple primary melanomas: a 34-year single-institution study.

BACKGROUND: Development of more than one primary melanoma in a sole patient is frequent, accounting for 1.2-8.2% of melanoma patients in most recent series. OBJECTIVE AND METHODS: Clinical, histological and epidemiological characteristics of 270 multiple primary melanomas patients were reviewed. RESULTS: Two-hundred and seven patients (76.7%) had two melanomas, whereas in the remaining 63 the number of primary ranged from three to eight; on the whole, 639 multiple primary melanomas were identified. Synchronous melanomas developed more frequently in patients with three or more lesions; median age was significantly lower in the group of patients with more than three melanomas than in the others. Mean Breslow's thickness significantly decreases (P<0.001) from the first (1.77±1.76 mm) to subsequent primaries (0.85±1.25 mm for the second and 0.66±0.48 mm for the third melanoma). Percentage of 'in situ' melanomas was 5.6% as first diagnosis, but increased to 24.8% for the second melanoma; number of nodular melanomas was significantly lower for succeeding diagnosis. AJCC stage at diagnosis showed a statistical prognostic significance, whereas outcome and survival did not depend on the number of primary lesions. Multivariate analysis confirmed the prognostic role of Breslow's thickness, ulceration, gender and patient age, and the better prognosis of patients with multiple melanomas, respect to those with single primary melanoma. CONCLUSIONS: Skin examination and long-term follow-up are mandatory for patients affected by melanoma, with the intent to promptly diagnose not only a disease progression but also possible new primary melanomas.