BACKGROUND:Oral cancer is a major public health problem worldwide, with a poor survival. Our aim was to evaluate several protein markers in oral squamous cell carcinomas (OSCC) and analyse their prognostic value on patient's survival.METHODS:We analysed the expression of EGFR, p53, p27, p16, cyclin D1, cyclin A2, COX-2, Ki-67, Bcl-2, VEGFR-1 and VEGFR-2, by immunohistochemistry on 67 primary OSCC. Cancer-specific survival (CSS) analysis was evaluated by the Cox regression model.RESULTS:Markers showed variable expression between 27.9% and 95.2%. In univariate analysis for CSS, we found that four of the tested markers, namely high expression of p53 (P = .001), EGFR (P = .003), cyclin A2 (P = .005) and low expression of p16 (P = .019), along with clinical stage (P < .001), tumour size (P < .001), presence of nodal metastasis (P < .001) and perineural permeation (P = .039) were related to decreased survival. On the basis of these results, we constructed an immunohistochemical score hinging on the possibility that any tumour could express none of these four markers (score 0), one or two markers (score 1) and three or more markers (score 2). In multivariable analysis, this immunohistochemical score revealed an independent prognostic value on cancer-specific survival (P = .001; HR: 3.7: 95%CI 1.7-7.9). Moreover, we confirmed that in early-stage tumours (stage I or II) this score maintained its independent prognostic value (P = .025; HR: 7.9, 95%CI 1.3-49.1) on CSS.CONCLUSION:The expression of the markers p53, p16, EGFR and cyclin A in OSCC, combined to give an immunohistochemical score, may identify high-risk subgroups for decreased survival and to further guide therapeutic decisions.

Aim The aim of our study was develop and validate an algorithm system based on morphological features for finding the differences between recurrent hepatitis C virus ( HCV ) and acute cellular rejection ( ACR ) in liver biopsies of HCV ‐transplanted patients. Methods Two hundred and eighty‐eight liver biopsies were analyzed from 121 patients transplanted for HCV . A diagnostic consensus was reached between clinicians and pathologists in 214 biopsies for the diagnosis of recurrent HCV or ACR . A random sample of 114 liver biopsies (derivation cohort) was taken to generate the diagnostic tree and was subsequently evaluated using the validation cohort in 100 liver biopsies by recursive partitioning analysis of morphological variables and time since transplantation. Results The presence of endotheliitis together with a time of less than 6 weeks since LT definitely excluded recurrent HCV . After obtaining the regression tree, diagnostic accuracy was 96% and 93% in the derivation and validation cohort, respectively. Both cases surpassed the pathologist's original diagnosis, which had a diagnostic accuracy of 91% ( P < 0.05, for both comparisons). Conclusion A recursive partitioning analysis of the morphological features in liver biopsies from HCV ‐transplanted patients may be useful for easily distinguishing between recurrent HCV and ACR .

AIMS the assessment of the human epidermic growth factor receptor 2 (HER2) is currently performed in most laboratories using two techniques: Fluorescence in situ hybridisation (FISH) and immunohistochemistry (IHC), and novel methodology is being investigated continuously in the assessment of HER2, such as SISH, CISH, DNA chips, ELISA or real time PCR to make assessment easier, faster or more accurate. RNA-ISH (RNA in Situ Hybridisation) is a new technique designed to detect mRNA expression levels, conducted by light microscope without the need for counting or grading systems in a total processing time of 4 hours. This study aims to determine if RNA-ISH is a viable and effective technique and a possible alternative to the currently used techniques by analysing and comparing genetic amplification (FISH) and protein levels (IHC) with mRNA over-expression (RNA-ISH) in 141 cases of breast cancer. RESULTS This study demonstrated a 96.5% concordance between over-expression of HER2 as determined by RNA-ISH and gene amplification as determined by FISH. The relationship between RNA-ISH-evaluated and IHC-evaluated over-expression was equally well reflected with a 95.2% concordance. Importantly, a considerable reduction in processing and evaluation time was achieved of only 4 hours. CONCLUSIONS We conclude that the probe developed for RNA-ISH represents a viable, effective possible alternative to FISH and IHC for analysing HER2 status in primary breast tumours.

OBJECTIVE:Our aim was to evaluate the expression of several molecules that regulate growth, the cell cycle and signalling pathways including EGFR, p53, p16 and p27 in oral squamous cell carcinomas (OSCC). We examined their utility as prognostic markers by relating to clinicopathological characteristics and the clinical outcome.PATIENTS AND METHODS:Using tissue microarray technology, we analysed 67 primary OSCC and examined immunohistochemical expression of EGFR, p53, p16 and p27. Multivariate analysis was conducted to examine their role in survival.RESULTS:Many of the markers were highly expressed in these cancers. Membranous EGFR expression in 95.2%, both membrane and cytoplasm expression in 35%, p53 expression in 61.6%, p27 expression in 89.5% and p16 expression in 27.9% of cases. In the multivariate analysis, independent prognostic influence of a lower overall survival was determined only for advanced tumour stage (P < 0.001), p53 overexpression (P = 0.004), EGFR cytoplasm and membrane co-expression location (P = 0.002) and p16 reduced expression (P = 0.002). When considering a subgroup of early stage tumours, p53 overexpression (P = 0.028) and combined membranous and cytoplasm EGFR co-expression (P = 0.039) were indicators of a lower overall survival. For disease-free survival, in addition to these three factors, the histological grade (P = 0.011) showed independent prognostic values.CONCLUSION:The independent value of EGFR subcellular location (cytoplasm and membrane) and p53 overexpression in overall survival even in early stages of OSCC suggests that these markers may serve as reliable biological markers to identify high-risk subgroups and to guide therapy.

Pancreatic cancer is a malignant neoplasm with an extremely poor prognosis. The mechanisms of aggressive growth and metastasis are currently not well understood. Expression of epidermal growth factor receptor (EGFR) has been suggested to be associated with the malignant transformation of pancreatic cancer. In this study, we examined the EGFR status of 52 pancreatic tumors by PCR-sequencing (exons 19 and 21), immunohistochemistry and FISH probes. We subsequently investigated the relationship between EGFR status and clinicopathological factors. Somatic alterations in EGFR (R841R, T571T and R831C) were observed only in ductal adenocarcinoma (3/34). In 4 (8%) of the 52 tumors analyzed EGFR was overexpressed, 6 (12%) of the tumors showed moderate expression while 19 (32%) were weakly stained. EGFR overexpression (3+ score) was frequently found in endocrine tumors (29%) followed of ampullary tumors (13%; p < 0.01). No significant correlation was observed between the presence of a somatic EGFR mutation and clinicopathological variables. Fluorescence in situ hybridization (FISH) analysis did not demonstrate amplification in any tumors. Only three somatic mutations in the EGFR gene were detected in pancreatic ductal adenocarcinoma and no association was observed with the clinical variables. Our results suggest that EGFR mutations are rare in pancreatic tumors and not associated with clinical prognosis, and treatment response.

Endoscopic ultrasound (EUS)-guided fine needle aspiration (FNA) is an efficacious technique for sampling pancreatic solid lesions. However, cytology possesses limitations to its final yield and accuracy, which may be overcome if histological specimens are provided to the pathologist. Aim of the study was to evaluate the accuracy of the newly developed 19-gauge and 22-gauge Procore needles, compare to the standard FNA needles. 83 consecutive patients (mean age 65.5±10.6 years, range 42-86, 47 male), who underwent EUS for the evaluation of solid pancreatic masses were prospectively included in the study. EUS were performed using a convex array echoendoscope (Pentax EG-3870UTK), connected to an ultrasound equipment (Hitachi). Biopsies were performed with the new 19-gauge and 22-gauge Procore needles (Cook Medical Inc, Limerick Ireland). FNA were performed with the standard 22-gauge and 25-gauge (Cook Medical Inc, Limerick Ireland). With Procore needles, sample obtained was recovered into cytolit and processed for histological analysis, after one pass. With standard cytology needles, samples were recoverd into slides and fixed for cytological evaluation, following the standard methodology. Results were compared to the gold standard of surgical histopathology, or global clinical and radiological assessment and follow-up in non-operated cases. Results are shown as mean or percentage (95%CI), and compared by chi-square test. Diagnostic accuracy was also evaluated. Size of solid pancreatic masses was 41.5±19.8 mm (mean±SD). 50 tumors were located in the head of the pancreas, 29 in the body, and 4 in the tail. 38 punctures (45.8%) were performed with Procore needles (21 with 19-gauge and 17 with 22-gauge), and 45 (54.2%) with standard cytology needles (25 with 25-gauge and 20 with 22-gauge). In 36 cases (85.96%) diagnosis proved to be correct with Procore needles (94.7%) (16/17 with 22-gauge, and 20/21 with 19-gauge), while with the standard needles diagnosis proved to be correct in 36 cases (80.0%) (15/20 with 22-gauge, and 21/25 with 25-gauge) (p=0.046). Performing a EUS-guided biopsy with the new Procore needles is more accurate than with the standard cytology needles.

Despite improvement in the treatment of advanced classical Hodgkin lymphoma, approximately 30% of patients relapse or die as result of the disease. Current predictive systems, determined by clinical and analytical parameters, fail to identify these high-risk patients accurately. We took a multistep approach to design a quantitative reverse-transcription polymerase chain reaction assay to be applied to routine formalin-fixed paraffin-embedded samples, integrating genes expressed by the tumor cells and their microenvironment. The significance of 30 genes chosen on the basis of previously published data was evaluated in 282 samples (divided into estimation and validation sets) to build a molecular risk score to predict failure. Adequate reverse-transcription polymerase chain reaction profiles were obtained from 262 of 282 cases (92.9%). Best predictor genes were integrated into an 11-gene model, including 4 functional pathways (cell cycle, apoptosis, macrophage activation, and interferon regulatory factor 4) able to identify low- and high-risk patients with different rates of 5-year failure-free survival: 74% versus 44.1% in the estimation set (P < .001) and 67.5% versus 45.0% in the validation set (P = .022). This model can be combined with stage IV into a final predictive model able to identify a group of patients with very bad outcome (5-year failure-free survival probability, 25.2%).

OBJECTIVEOur aim was to validate the use of tissue microarrays (TMA) in oral squamous cell carcinomas (OSCC) to analyse epidermal growth factor receptor (EGFR) and Ki-67 expression. We also analysed the relationship that the expression of these markers may have with clinical, pathological and survival variables.PATIENTS AND METHODSThe study sample comprised 39 unselected patients diagnosed and treated for OSCC. We analysed Ki-67 and EGFR expression by immunohistochemistry on formalin-fixed, paraffin-embedded surgical specimens. Whole sections (WS) were compared with double 1.5 mm core-tissue microarrays.RESULTSHigh EGFR expression was observed both on TMA (in 98% of the cases) and WS (in 100% of the cases) with substantial agreement kappa value (0.720). EGFR expression was not significantly associated with clinical, pathological and survival variables on TMA and WS. Ki-67 analysis showed a Spearman correlation of 0.741 with a Ki-67 mean labelling index of 45% in TMA and 56.8% in WS. We found a significant relationship between gender and Ki-67 labelling index on WS (P = 0.022) and TMA (P = 0.002). Clinical stage was the only parameter in multivariate analysis that had a significant predictive value.CONCLUSIONWe demonstrate that dual 1.5 mm core TMA is a valid, rapid, economical and tissue-saving way to study OSCC biopsies and that it presents strong correlation with the WS. EGFR overexpression in OSCC suggests that these tumours may be a candidate for therapy investigation directed to EGFR.

To the Editor.—We read the outstanding article by Miller and Tazelaar1 recently published in the Archives of Pathology & Laboratory Medicine about the main features of 5 cardiovascular pseudoneoplasms (inflammatory myofibroblastic tumor, hamartoma of mature cardiac myocytes, mesothelial monocytic cardiac excrescences, calcified amorphous tumor, and lipomatous hypertrophy of the atrial septum). Although lacking malignant potential, these lesions can be associated with morbility and occasional mortality, and their recognition is important for appropriate patient management.We describe the main features of the cystic tumor of the atrioventricular node (CTAVN), another pseudoneoplastic lesion that may cause sudden death. First described in 1911,2 its controversial histogenesis resulted in several names3,4 such as mesothelioma, lymphangioendothelioma, endodermal inclusion, hamartoma, and so forth. We showed that CTAVN and solid cell nests of the thyroid gland are identical structures and indicated that CTAVN is an ultimobranchial heterotopia secondary to an alteration in cardiac neural crest cell development.3 Its association with several congenital abnormalities3,4 and possible familial occurrence5 suggest a genetic defect in cardiac neural crest cell development.3Grossly, the heart can appear normal or show small cysts in the triangle of Koch.6 Cystic tumor of the atrioventricular node runs from the ostium of the coronary sinus in the right atrium to the membranous septum, along the top of the tricuspid valve septal leaflet. Histologically, it is composed of squamoid solid cell nests and cystic structures with a minor proportion of neuroendocrine cells3–10 (Figure, A through E). Squamoid cells (main cells) are polygonal to elongated with centrally located oval nuclei and eosinophilic cytoplasm. The neuroendocrine cells show clear cytoplasm and compact nuclei. Cysts usually contain mucosubstances and are lined by mucinous, ciliated, transitional, and/or squamous cells. Sebaceous and goblet cells are described. Mitotic figures are usually absent. The main cells are immunoreactive for cytokeratins (cytokeratin 7 and clones CAM 5.2, AE1/AE3, and 34βE12), epithelial membrane antigen, carcinoembryonic antigen, carbohydrate antigen 19.9, p63, bcl-2, and galectin-3 with no reaction for vimentin, cytokeratin 20, CD31, factor VIII–related antigen, thrombomodulin, Wilms tumor 1, or calretinin. The neuroendocrine cells are positive for cytokeratin 7, CAM 5.2, and AE1/AE3, epithelial membrane antigen, carcinoembryonic antigen, thyroid transcription factor 1, calcitonin, serotonin, chromogranin, and synaptophysin. Ultrastructurally,3–5 the main cells are characterized by cytoplasmic tonofilaments and desmosome-like structures, while the neuroendocrine cells showed electron-dense secretory granules. An angiomatous variant of CTAVN has also been reported.11One patient8 was reported to have developed symptoms during her pregnancy and the tumors cells were positive for estrogen and progesterone receptors suggesting that the CTAVN might have been progressive during the pregnancy stage. Another case12 treated with long-acting gonadotropin-releasing hormone decreased in size; however, with a combination of estrogen and progestin, the tumor increased in size and progressed even after the discontinuation of all therapy. These findings suggest that there might be a correlation between the progression of CTAVN and the levels of circulating hormones.8 However, we were unable to find positivity for estrogen and progesterone receptors in this tumor type.3 Clinically, CTAVN can cause heart blockage and sudden death as a result of ventricular tachycardia or ventricular fibrillation.5,7,9 The mean age of presentation is 38 years (range, birth to 78 years), with a female to male ratio of 3∶1.9 Most of these tumors were diagnosed postmortem3–9; some of them as an incidental finding, because they were clinically silent. Antemortem diagnosis and successful excision of CTAVN was reported in 7 cases,6,8 and in 1 of them the tumor was an unexpected finding in an explanted heart.10 Cystic tumor of the atrioventricular node is an important differential diagnosis in young patients with syncopal attacks and varying degrees of heart blockage.10 It is also important to note that in patients with CTAVN, pacemaker implantation does not prevent death because these patients are at risk of lethal arrhythmias unconnected to the heart blockage.6 Complete surgical resection is essential, even if a subsequent pacemaker implant is required.6

Fluorescent in situ hybridization (FISH) is a very useful tool for diagnostic and prognostic purposes in pathology. However, many laboratories still experience troubles when applying FISH to paraffin material. To overcome these difficulties, different pretreatments which include enzymatic digestion have been described. Usually, previous to digestion, a heating step is performed. The aim of this study was to compare the efficiency of the heating step with different buffers and different heating methods. We conclude that the main factor in the heating pretreatment is the temperature control, irrespective of the buffer used. Best results are obtained with any buffer by heating the slides to 99°C for 15 min followed by 10 min at room temperature.

Differential diagnosis between pancreatic cancer (PC) and inflammatory masses (IM) in chronic pancreatitis (CP) remains a challenge. Analysis of tissue stiffness by quantitative EUS-elastography may be of help in this setting. Aim of the study was to evaluate the accuracy of quantitative EUS-elastography for the differential diagnosis between PC and IM in CP.

BACKGROUND & AIMS: Qualitative endoscopic ultrasound (EUS) elastography is an accurate but subjective tool for the differential diagnosis of solid pancreatic masses. Second-generation EUS elastography allows quantitative analysis of tissue stiffness. We evaluated the accuracy of quantitative, second-generation EUS elastography in the differential diagnosis of solid pancreatic masses. METHODS: The study included 86 consecutive patients who underwent EUS for the evaluation of solid pancreatic masses. EUS elastography was performed with the linear Pentax EUS and the Hitachi EUB900. Representative areas from the mass (A) and soft reference areas (B) were analyzed. The result of the elastographic evaluation was defined by the quotient B/A (strain ratio). Final diagnosis was based on histology of surgical specimens and cytology of EUS‐fine-needle aspiration samples. The diagnostic accuracy of EUS elastography in detecting malignancy was calculated using receiver operating curve analysis. RESULTS: The mean size of the pancreatic masses was 31.4 12.3 mm. The final diagnoses were pancreatic adenocarcinoma (n 49), inflammatory mass (n 27), malignant neuroendocrine tumor (n 6), metastatic oat-cell lung cancer (n 2), pancreatic lymphoma (n 1), and pancreatic solid pseudopapillary tumor (n 1). The strain ratio was significantly higher among patients with pancreatic malignant tumors compared with those with inflammatory masses. The sensitivity and specificity of strain ratio for detecting pancreatic malignancies were 100% and 92.9%, respectively (area under the receiver operating curve, 0.983). CONCLUSIONS: Quantitative, second-generation EUS elastography is useful for differential diagnosis of solid pancreatic masses. It allows for a quantitative and objective evaluation of tissue stiffness, which indicates the malignant or benign nature of the pancreatic lesion.

Laser Confocal Endomicroscopy (CEM) enables real time in vivo microscopic images during an ongoing endoscopy. Although CEM has been evaluated in different clinical settings, correlation studies with specific histological parameters are lacking. UC requires endoscopic surveillance due to a high risk of colon dysplasia and cancer. Aim of the study was to evaluate the accuracy of CEM for the analysis of specific histopathological parameters in patients with longstanding UC, and its correlation with conventional histopathology as reference method.

Intestinal spirochetosis (IS) was described in 1967 associated to chronic diarrhea. However, the clinical significance of this finding remains controversial, and it is not clear if they are commensals or pathogenic organisms. In many cases, the diagnosis has been made based on the histopathology, demonstrating the presence of the microorganisms attached to the luminal surface of the colonic mucosa. In developed countries, the prevalence of IS in colonic biopsies ranges from 2% to 7% in patients with digestive disorders. In all, 2 different spirochete species, Brachyspira aalborgi and Brachyspira (formerly Serpulina) pilosicoli, have been identified as the pathogenic agents of IS in humans. A 68-year-old man presented with persistent diarrhea for the past 6 months. Clinical investigation included stool examination, which revealed the presence of isolated Blastocystis hominis, Pentatri chomona hominis, Entamoeba dispar, and abundant spirochetes. Total colonoscopy was performed showing no lesions. Several mucosal biopsies were taken and histopathological examination revealed a thin, band-like growth of spirochetes adherent to the colonic luminal surface, giving a bluish hue to the luminal surface (Figure 1). Staining with Warthin– Starry (Figure 2) and acridine orange (Figure 3) helped to highlight the organisms. No significant changes in the underlying mucosa were noted. Treatment with metronidazole (750 mg/8 h) was given for 10 days, achieving the complete resolution of the symptoms. Nevertheless,

INTRODUCTION:Rheumatoid arthritis is an autoimmune disease in which joint inflammation leads to progressive cartilage and bone erosion. Matrix metalloproteinases (MMPs) implicated in homeostasis of the extracellular matrix play a central role in cartilage degradation. However, the role of specific MMPs in arthritis pathogenesis is largely unknown. The aim of the present study was to investigate the role of Mmp-8 (collagenase-2) in an arthritis model.METHODS:Arthritis was induced in Mmp8-deficient and wildtype mice by K/BxN serum transfer. Arthritis severity was measured by a clinical index and ankle sections were scored for synovial inflammation, cartilage damage and bone erosion. cDNA microarray analysis, real-time PCR and western blot were performed to identify differential changes in gene expression between mice lacking Mmp8 and controls.RESULTS:Mmp8 deficiency increased the severity of arthritis, although the incidence of disease was similar in control and deficient mice. Increased clinical score was associated with exacerbated synovial inflammation and bone erosion. We also found that the absence of Mmp8 led to increased expression of IL-1β, pentraxin-3 (PTX3) and prokineticin receptor 2 (PROKR2) in arthritic mice joints.CONCLUSIONS:Lack of Mmp-8 is accompanied by exacerbated synovial inflammation and bone erosion in the K/BxN serum-transfer arthritis model, indicating that this Mmp has a protective role in arthritis.