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Background The thoracic lymphadenectomy during an esophagectomy for esophageal cancer includes resection of the thoracic duct (TD) compartment containing the thoracic duct lymph nodes (TDLN). However, the role of TD compartment resection is still a topic of debate since metastatic TDLNs have only been demonstrated in squamous cell carcinomas in Eastern esophageal cancer patients. Therefore, the aim of this study was to assess the presence and metastatic involvement of TDLNs in a Western population, in which adenocarcinoma is the predominant type of esophageal cancer. Methods From July 2017 to May 2020 all consecutive patients undergoing an open or robot-assisted transthoracic esophagectomy with concurrent lymphadenectomy and resection of the TD compartment in the University Medical Center Utrecht, The Netherlands and the Citta della Salute e della Scienza University Hospital in Turin, Italy were included. The TD compartment was resected en bloc and was separated in the operation room by the operating surgeon after which it was macro- and microscopically assessed for (metastatic) TDLNs by the pathologist. Results A total of 117 patients with an adenocarcinoma (73%) or squamous cell carcinoma (27%) of the esophagus were included. In 61 (52%) patients TDLNs were found, containing metastasis in 9 (15%) patients. No major complications related to TD compartment resection were observed. Conclusions This is the first study to demonstrate the presence of metastatic TDLNs in adenocarcinomas of the esophagus. This result provides a valid argument to routinely extend the thoracic lymphadenectomy with resection of the TD compartment during an esophagectomy for esophageal cancer.

Background Gallbladder hemangioma is an exceptionally rare entity, with only ten cases reported in literature hitherto. The here described case is the first report of a gallbladder hemangioma coexisting with gallstones. Case presentation A 76-year-old male was hospitalized following repeated episodes of epigastric pain. Patient’s medical history included primary hypertension, type 2 diabetes mellitus, dyslipidemia, obesity and hyperuricemia. Physical examination revealed marked pain in the right hypochondriac region, and laboratory workup was notable for mildly elevated glycemia (125 mg/dL) and pancreatic amylase (60 IU/L). Abdominal ultrasound showed multiple gallstones, a thickened gallbladder wall and mild edema of the perivisceral adipose tissue as well as a hepatic angioma. During surgery, an incidental subserosal nodule of about 1 cm was detected within the gallbladder fundus. After surgery, the clinical course was uneventful and the patient was discharged. Histopathological examination of the subserosal nodule showed multiple dilated vascular channels within a sclerosing matrix, a finding consistent with a cavernous hemangioma. Diffuse chronic cholecystitis was also present. Conclusions Gallbladder hemangiomas represent a rare, likely underdiagnosed condition which can be undetected during the preoperative workup.

Introduction Many types of research have been performed to improve the diagnosis, therapy, and prognosis of oropharyngeal carcinomas (OP-SCCs). Since they arise in lymphoid-rich areas and intense lymphocytic infiltration has been related to a better prognosis, a TREM-1 putative function in tumour progression and survival has been hypothesized. Materials and methods Twenty-seven human papillomavirus (HPV) 16+ OP-SCC specimens have been analyzed to relate TREM-1 expression with histiocytic and lymphocytic markers, HPV presence and patients’ outcome. Results No differences have been shown between intratumoral and stromal CD4+ cells, while intratumoral CD8+ lymphocytes are higher with respect to the tumour stroma (p = .0005). CD68+ cells are more than CD35+ and TREM-1+; their presence is related to CD35± and TREM-1± histiocytes (p = .005 and .026, respectively). Intratumoral CD4+ lymphocytes are higher in p16+ cases (11/27) than in p16− (p = .042); moreover, p16 positivity correlates to a better survival (p = .034). CD4+, CD8+ and CD35+ cells have no impact on survival, while CD68 expression heavily influences progression and bad outcome (p = .037). TREM-1 positivity also leads to worst overall survival (p = .001): peritumoral expression and death-cause relationship are always significant, particularly when the cause is OP-SCC (p = .000). Conclusion While p16 shows to better stratify HPV16+ patients’ outcome, TREM-1+ macrophages suggest their key importance in HPV-related OP-SCCs progression.KEY MESSAGESTREM-1 positivity correlates to the worst overall survival of HPV16-positive OPSCCs-affected patients.p16-positive HPV16 related OPSCCs patients have a better prognosis with respect to p16-negative ones.

Enolase (ENO) 1 is a key glycolytic enzyme and important player in tumorigenesis. ENO1 overexpression has been correlated with tumor progression and/or worse prognosis in several solid malignancies. However, data concerning the impact of ENO1 in cancer conflict. The study correlated local and circulating ENO1 protein levels in esophageal cancer (EC) with clinicopathological data, to assess its potential clinical value. ENO1 expression was analyzed by immunohistochemistry in paired tumor and non-tumor tissue samples from 40 EC cases and mucosal biopsies from 45 Barrett's esophagus (BE) cases, plus in plasma from these patients and 25 matched healthy controls. ENO1 was abnormally elevated in cancer-cell cytoplasm in both EC types, in esophageal squamous cell carcinoma and in adenocarcinoma (EAC), increasing significantly with tumor stage progression and the transition from BE to EAC. EAC patients exhibited significantly lower ENO1 plasma concentrations than normal subjects. Neither local nor systemic ENO1 expression levels were significantly associated with overall survival. These results indicate ENO1 as potential biomarker, delineating a population of patients with Barrett's esophagus at high risk of cancer, and as new therapeutic opportunity in EC patient management. However, further confirmation might be necessary.

Oral lichen planus (OLP) is a common premalignant chronic inflammatory disorder. Optical Coherence Tomography (OCT) provides a real-time, non-invasive, and in-situ optical signature using light of varying wavelengths to examine tissue. Aim of the present study was to assess the possible role of OCT as diagnostic tool for atrophic-erosive OLP by examining OCT scans of healthy buccal mucosa, and comparing their ultrastructural features with those of a buccal mucosa affected by atrophic-erosive OLP, using their histopathological counterparts as the gold standard. Through grayscale (enface scan) and an application in which the vascularization of the tissue is visible (dynamic scan), it was possible to distinguish the healthy from the lichenoid pattern from 20 controls (12 M; 8 F; mean age: 41.32 years) and 20 patients with histologically confirmed atrophic-erosive OLP (7 M; 13 F; mean age: 64.27 years). In detail, mean width of stratified squamous epithelium (EP) and lamina propria (LP) were evaluated. Among controls, EP and LP showed a mean width of 300 (±50) and of 600 (±50) μm respectively; among cases, disruption of membrane basement prevented from any measurement. Furthermore, a differential pattern of EP and LP emerged between the two groups: a light-grayish, hypo-reflective, homogeneous area of EP recurring in controls turned into a hyper-reflective, non-homogeneous area among cases. Dynamic scan showed a differential profile of LP vascularization, varying from a hypo-reflective red area with small blood vessels in the control group, to a hypo/hyper-reflective area, completely overrun by a denser, wider blood flow amid OLP cases. Although histopathological examination remains the gold standard for OLP diagnosis, OCT could be a potentially helpful tool for the clinician and the pathologist, since it allows analysis of the vascularization of the sample without adversely affecting histological processing.

Objectives Veno-occlusive disease after liver transplant has been sporadically reported, and significant uncertainty exists concerning the best treatment and the long-term outcomes. Here, we reviewed our experience to evaluate clinical presentation, treatment, and the long-term outcomes of these patients. Materials and methods Between 2000 and 2015, 2165 patients underwent liver transplant at our center. The incidence of veno-occlusive disease was 0.3% (7/2165). Results Timing of veno-occlusive disease onset (median 4.7 mo; interquartile range, 2.5-11.1 mo) varied widely as did clinical presentation, which was characterized by a variable association of liver failure and portal hypertension and different disease pro-gression rates. In all cases, diagnosis of veno-occlusive disease was confirmed by liver biopsy. Six patients (85.7%) presented with veno-occlusive disease after a previous episode of acute cellular rejection. Three patients died due to veno-occlusive disease (n = 2) or due to hepatocellular carcinoma recurrence (n = 1). Two patients were treated by increasing immunosuppression and with interventional procedures (pleurodesis and transjugular intrahepatic portosystemic shunt, respectively), and 2 had successful retransplants. 5-year patient and graft survival rates were 57.1% and 28.6%, respectively. Conclusions A tailored approach based on clinical features and including retransplant can achieve acceptable long-term survival in patients with veno-occlusive disease after liver transplant.

Objective: Complete separation of upper and lower respiratory tract after total laryngectomy results in permanent effects on nasal cavities and tracheo-bronchial airways. Aim of this study is evaluating nasal and tracheal cytological alterations of mucosa in laryngectomy long-term survivors, analyzing the feasibility of scraping for cytological examination of tracheal mucosa. Methods: Twenty-five laryngectomy patients underwent symptoms’ evaluation, endoscopic fiber optic examination, prick tests, and nasal and tracheal scraping for cytological exam. Twenty-five healthy subjects underwent the same assessment, except for tracheal scraping. Eleven laryngectomy patients accepted inferior turbinate biopsy for histological examination. Results: Nasal cytological analysis demonstrated mucous cell metaplasia in 20% of laryngectomized patients, but it was absent in all healthy subjects; no squamous cell metaplasia was found in both groups. In 15 patients (60%), bacteria were present, without inflammatory infiltrate. Tracheal cytological analysis demonstrated a quite high rate of squamous cell metaplasia (24%), neutrophilic infiltrate (32%), and presence of bacteria (40%). Histological examination of inferior turbinate showed submucosal stromal fibrosis in all patients and submucosal inflammatory infiltrate in 1 case (9%). Conclusion: Nasal cavities and trachea of laryngectomy patients undergo long-term cytological and histological changes of mucosa and submucosa, probably due to airflow modifications.

Supplemental digital content is available in the text., Evaluation of the molecular status of 1p and 19q is a major relevant diagnostic, prognostic, and predictive tool for oligodendroglial brain tumors. Fluorescence in situ hybridization (FISH) is the most commonly used technique for determining 1p and 19q allelic losses, but it lacks fully standardized criteria for analysis. This lack of standardization has led to interinstitutional disagreement in the interpretation of results, thereby contributing to a “gray prognostic zone” that includes codeleted patients with an unexpectedly unfavorable outcome. To optimize the prognostic potential of 1p/19q status determination, we first compared the actual criteria used for FISH reading (i.e. different ratio cutoff values and the percentage of neoplastic nuclei carrying this chromosomal deletion) in a retrospective series of 143 pure and mixed oligodendroglial tumors. We then created a “weighted” FISH reading based on the merged ratio and percentage of neoplastic cells carrying the deletion that was further differentially modulated for 1p and 19q, respectively. This weighted codeletion setting significantly strengthened the favorable prognostic power of 1p/19q losses by reducing the number of poor outcomes from 42% to 12.5% for patients with codeleted tumors. Thus, by identifying as codeleted only those cases with more than 50% of cells having a combined loss of 1p (using 0.7 ratio cutoff) and 19q (using 0.8 ratio cutoff) arms, we created a molecular report that bears higher clinical impact and strengthens the prognostic potential of 1p/19q allelic loss.

A retrospective analysis of 1,792 solid tissues suggestive of lymphoma, submitted over a 12-year period, was carried out and flow cytometry (FC) results were compared with histologic findings. The final histologic diagnosis of cases documented in this report is as follows: 1,270 non-Hodgkin's lymphomas (NHL); 17 composite lymphomas; four NHL plus carcinomas; five post-transplant lymphoproliferative disorders; 105 Hodgkin's lymphomas (HL); eight acute leukemias; 42 tissue cancers; and 341 non-neoplastic diseases. A strong correlation between morphology and FC data was observed among hematological malignancies (1,268/1,304, 97.2%) with the exception of HL. Among B-NHL, FC detection of clonally restricted B-cell allowed the identification of lymphomas that were not histologically clear and the differential diagnosis between follicular lymphoma and reactive hyperplasia. A high correlation level (r = 0.83; P < 0.0001) was obtained in comparing proliferation results obtained by FC and immunohistochemistry. Among T-NHL, FC detection of an aberrant phenotype direct histologic diagnosis in cases having less than 20% of neoplastic cells. In nine cases, FC suggested the need to evaluate a neoplastic population, not morphologically evident. Results show that FC routinely performed on tissue samples suspected of lymphomas is a fundamental adjunct to morphology in the diagnosis of NHL and may enhance the performance of the histologic evaluation so as to achieve the final diagnosis. To the best of our knowledge, this is the first report in the literature of a wide series of tissues also studied by FC.

This report analyzed the outcomes of patients undergoing surgery for oral squamous cell carcinoma (OSCC) to identify the value of prognostic factors.A total of 525 patients were studied who had undergone surgery for oral squamous cell carcinoma (OSCC) between 2000 and 2011, of whom 222 had received postoperative radiation-therapy (PORT) and or chemoradiation-therapy (PORTC). For each patient, personal data, histological findings, treatment and outcome were recorded and analyzed statistically. Survival curves were calculated using the Kaplan-Meier algorithm, and the difference in survival among subgroups was examined.The overall survival (OS) and disease-specific survival (DSS) 5-year survival rate in the 525 patients were respectively 71.38% and 73.18%. The differences in the overall survival and disease-specific 5-year survival were significant (p 0.05) for age 40 years, site of origin, N status, staging, grading, osseous medullar infiltration, and perineural invasion. In patients undergoing radiation therapy, only perineural invasion negatively influenced the survival prognosis. In 150 pT1 cases of tongue and floor-of-mouth cancer, an infiltration depth (ID) 4 mm was statistically correlated with poorer prognosis.The results demonstrate an improvement in the 5-year OS and DSS rates during the past decade compared with the previous decade. Univariate analysis revealed that age, tumor staging, and lymph node involvement, extracapsular spread, grading, perineurial invasion, infiltration depth, and osseus medullary invasion were associated significantly with overall survival and disease-specific survival.

Summary Gastric cancer shows intratumoral heterogeneity for human epidermal growth factor receptor 2 expression. We evaluated whether the number of tissue blocks analyzed or the antibodies used may influence the immunohistochemical results in gastrectomy specimens. Clinicopathologic data from 148 patients receiving gastric surgery for cancer were collected. One tissue block for each of 88 primary tumors and 60 paired primary tumors and metastases was examined for human epidermal growth factor receptor 2 status by immunohistochemistry using 3 different antibodies (HercepTest, CB11, and 4B5) and by fluorescent in situ hybridization. Two additional tissue blocks of the primary tumor were tested by immunohistochemistry if the results were negative on the first tissue block. The concordance among the 3 antibodies was 94.5% (testing 1 tissue block). Two cases showed a clinically significant discrepancy between primary tumor (score 0) and lymph nodes metastases (score 3+). Additional block analysis increased both the sensitivity (from 63% to 83%) and the accuracy (from 91% to 94%) of immunohistochemistry as compared with fluorescent in situ hybridization. The multiblock approach could potentially identify a greater number of human epidermal growth factor receptor 2–positive gastric cancers, particularly those with higher levels of intratumor heterogeneity. In turn, human epidermal growth factor receptor 2 positivity correlated with a worse prognosis ( P = .011) and was an independent variable in multivariate analysis (hazard ratio, 1.57). In conclusion, testing more than 1 tissue block of cancer from specimens of gastric resection provides a more reliable human epidermal growth factor receptor 2 assessment regardless of the antibody used.

BACKGROUND:Neoadjuvant chemo-radiotherapy (CRT) followed by surgical resection is the standard treatment for locally advanced rectal cancer, although complete tumor pathological regression is achieved in only up to 30% of cases. A clinicopathological and molecular predictive stratification of patients with advanced rectal cancer is still lacking. Here, c-Met and YKL-40 have been studied as putative predictors of CRT response in rectal cancer, due to their reported involvement in chemoradioresistance in various solid tumors. MATERIAL AND METHODS:A multicentric study was designed to assess the role of c-Met and YKL-40 expression in predicting chemoradioresistance and to correlate clinical and pathological features with CRT response. Immunohistochemistry and fluorescent in situ hybridization for c-Met were performed on 81 rectal cancer biopsies from patients with locally advanced rectal adenocarcinoma. All patients underwent standard (50.4 gy in 28 fractions + concurrent capecitabine 825 mg/m2) neoadjuvant CRT or the XELOXART protocol. CRT response was documented on surgical resection specimens and recorded as tumor regression grade (TRG) according to the Mandard criteria. RESULTS:A significant correlation between c-Met and YKL-40 expression was observed (R = 0.43). The expressions of c-Met and YKL-40 were both significantly associated with a lack of complete response (86% and 87% of c-Met and YKL-40 positive cases, p< 0.01 and p = 0.006, respectively). Thirty of the 32 biopsies co-expressing both markers had partial or absent tumor response (TRG 2-5), strengthening their positive predictive value (94%). The exclusive predictive role of YKL-40 and c-Met was confirmed using a multivariate analysis (p = 0.004 and p = 0.007 for YKL-40 and c-Met, respectively). TRG was the sole morphological parameter associated with poor outcome. CONCLUSION:c-Met and YKL-40 expression is a reliable predictor of partial/absent response to neoadjuvant CRT in rectal cancer. Targeted therapy protocols could take advantage of prior evaluations of c-MET and YKL-40 expression levels to increase therapeutic efficacy.

Most follicular lymphomas (FLs) have a phenotype consistent with the origin from CD5-, CD10+, bcl-6+ follicular center cells and can progress to diffuse large B-cell lymphoma (DLBCL). CD5 is expressed in about 10% of DLBCLs, showing prognostic value, whereas expression is rare in FL. We present 6 cases with coexisting features of CD5+ FL and CD5+ DLBCL, supporting a follicular origin for some CD5+ DLBCLs. The follicular areas showed a meshwork of CD21+ follicular dendritic cells that were lacking in the DLBCL areas. All cases showed a clonal CD19+, CD20+, CD5+, and CD10+ population in both follicular and diffuse areas. Molecularly, 4 of 6 cases demonstrated immunoglobulin heavy chain rearrangements and 1 case, a bcl-2/immunoglobulin heavy chain gene rearrangement. Somatic hypermutations were high in all 4 cases, in keeping with their germinal center origin. Four of five patients died of disease within 42 months, consistent with the proposed prognostic value of CD5 expression in DLBCL. Our data describe an aggressive variant of CD5+ FL suggesting the follicular origin of some CD5+ DLBCLs.

Summary Histological and cytological criteria in predicting clinical outcomes in patients with oncocytic poorly differentiated carcinoma (PDC) of the thyroid were investigated. In a set of 102 PDC patients, we performed a computer-assisted evaluation of cell size based on two different methods. Univariate analysis showed that cell size was a discriminant prognostic parameter in oncocytic PDC (30 cases) but not in the non-oncocytic carcinoma cases (72 cases). Patients with oncocytic PDC with small-medium cell size had a significantly increased risk of death ( P = .029) and a decrease of disease-free survival ( P = .014). This correlation was absent in cases of non-oncocytic PDC, where age and extensive vascular invasion were significant indicators of progression. The proposed morphological signature shows a robust discriminatory ability when tested on the oncocytic PDC group, and cell size assessment could thus be proposed as an inexpensive and readily evaluable parameter for predicting prognosis and planning therapy in this tumor type.

Most follicular lymphomas (FLs) are genetically defined by the t(14;18)(q32;q21) translocation that juxtaposes the BCL2 gene to the immunoglobulin heavy chain (IgH) 3' regulatory regions (IgH-3'RRs). Despite this recurrent translocation, FL cases are heterogeneous in terms of intratumoral clonal diversity for acquired mutations and variations in the tumor microenvironment. Here we describe an additional mechanism that contributes to inter- and intratumoral heterogeneity in FLs. By applying a novel single-molecule RNA fluorescence-based in situ hybridization (FISH) technique to detect mRNA molecules of BCL2 and IgH in single cells, we found marked heterogeneity in the number of BCL2 mRNA transcripts within individual lymphoma cells. Moreover, BCL2 mRNA molecules correlated with IgH mRNA molecules in individual cells both in t(14;18) lymphoma cell lines and in patient samples. Consistently, a strong correlation between BCL2 and IgH protein levels was found in a series of 205 primary FL cases by flow cytometry and immunohistochemistry. Inter- and intratumoral heterogeneity of BCL2 expression determined resistance to drugs commonly used in FL treatment and affected overall survival of FL patients. These data demonstrate that BCL2 and IgH expressions are heterogeneous and coregulated in t(14;18)-translocated cells, and determine the response to therapy in FL patients.

BACKGROUND Pleural malignant mesothelioma (PMM) is a rare and highly aggressive tumor, whose development is strictly related to occupational exposure to asbestos. The prognosis of PMM is generally poor (median survival, 4–12 months), but a few have a relatively long survival. The objective of this study was to evaluate the use of the cell cycle–related proteins p27kip1 and MIB-1 as prognostic indicators of survival in PMMs. METHODS Of 621 PMMs, the authors selected 27 cases with a relatively long-term survival (> 24 months) and a control group of 36 PMMs having a shorter (usual) survival (< 24 months). RESULTS The expression of the p27kip1 was significantly higher in the long-term survival group compared with the control (short survival) group (81.41% vs. 31.94%; P < 0.0001). The PMMs of epithelioid histotype had a significantly higher p27kip1 immunoreactivity compared with those of biphasic type (59.24% vs. 38.94%; P = 0.02). In agreement with the data in the literature, the proliferative activity (as detected by MIB-1 immunoreactivity) was significantly higher in short than long survival PMMs (43.53% vs. 14.11%; P < 0.0001) and in the biphasic histotype than in the epithelioid type (43.19% vs. 26.02%; P = 0.006). CONCLUSIONS The combined expression of high/low p27kip1 and low/high Ki-67 values identified with 100% specificity and sensitivity long versus short survivors. p27kip1 represents a reliable additional predictive factor for PMMs and a useful marker to identify patients having a more favorable prognosis. Cancer 2001;92:1245–50. © 2001 American Cancer Society.

The importance of the analysis of the silver-stained nucleolar organizer regions (AgNORs) for prognostic purposes in tumor pathology has been reviewed. Current available data from the literature demonstrate that the evaluation of the quantity of interphase AgNORs is an independent prognostic factor in several types of human tumors. Results of our investigations indicate that AgNORs are the most powerful variable predicting survival in patients with pharyngeal carcinoma, multiple myeloma, male breast and prostate carcinoma. The combination of AgNOR counts and histologic pattern allows the stratification of patients with multiple myeloma, pharyngeal and prostate carcinoma into low- and high-risk groups, which could benefit from different therapy. Moreover, AgNOR analysis predicts response to treatment in adult patients with acute myelogenous leukemia, and appears as an independent prognostic factor in a prospective study on renal cell carcinoma. Therefore, AgNOR analysis is a really important prognostic factor for several human neoplasias. The experimental and theoretical justifications for AgNORs as a prognostic factor are also reviewed, in particular the strict correlation between AgNOR quantity and tumor cell doubling time. Lastly, the lack of prognostic significance of AgNOR analysis in some circumstances is critically discussed.

BACKGROUND Histologic grade of differentiation is a strong prognostic factor for prostate carcinoma. However, most tumors fall in the intermediate group. Nuclear and nucleolar morphometry and analysis of the argyrophilic nucleolar organizer regions (AgNORs) were performed to improve prognosis, especially for patients with intermediate histologic grade tumors. METHODS Core needle biopsies from 65 patients with primary prostate carcinoma at diagnosis were studied. Patients received only hormone therapy. Formalin fixed and paraffin embedded sections were stained with the method of Ploton. The mean AgNOR count was calculated in 100 tumor cells for each case. Nuclear and nucleolar areas from 100 cells were measured with an automated image analyzer. One-way analysis of variance and uni- and multivariate survival analyses were used for statistical evaluation. RESULTS In the whole series, World Health Organization (WHO) tumor grade, nuclear and nucleolar areas, and AgNOR counts were correlated with survival time. By multivariate analysis, only AgNOR counts retained independent prognostic significance. In WHO Grade 2 carcinoma, the 5-year survival rate for patients with AgNOR/cell ≤ 7.84 was 77%, but was only 12% for those with higher counts (P < 0.0001). These survival rates were similar to those obtained when patients with WHO Grade 1 carcinoma and Grade 2 carcinoma plus low AgNOR counts were compared with patients with Grade 3 carcinoma and Grade 2 carcinoma plus high AgNOR counts. In Gleason intermediate Grade 6 and 7 carcinomas, the 5-year survival rate for patients with AgNOR/cell ≤ 7.84 was 71%, but was only 7% for those having higher counts (P = 0.0001). CONCLUSIONS Nuclear and nucleolar areas, as well as AgNOR counts, supplement histologic grading in the prognostic assessment of prostate carcinoma in patients receiving only hormone therapy. AgNOR count also is a prognostic factor for patients with intermediate grade tumors. The combination of histologic grade and proliferative activity allows the stratification of patients into low and high risk groups. Cancer 1997; 79:1956-63. © 1997 American Cancer Society.

Renal cell carcinoma (RCC) follows a variable clinical course related to disease stage and metastatic spread (including to bone). Molecular and genetic factors bear prognostic significance in RCC, including proteins involved in extracellular matrix invasion. Among these, bone sialoprotein (BSP) and osteopontin (OPN) are physiologically implicated in bone metabolism, and have a prognostic role in several tumors. BSP expression was also predictive of bone spread propensity in lung and prostate carcinoma. In RCC, no data are available for BSP, while OPN has been correlated with tumor stage, grade and survival. We aimed to define the predictive (of bone spread) and prognostic role of BSP and OPN immunohistochemical expression in clear cell RCC.from a series of 305 renal tumors resected between 1993 and 2002, 75 surgically resected clear cell RCCs with tissue material, clinical data and follow-up information available, were selected for the preliminary series; a second group of 126 chemo-naïve, radically-resected, consecutive RCCs was collected as a validation series. Immunohistochemical expression of BSP and OPN on paraffinized samples was evaluated by H-score [=Σ (intensity × percentage of positively stained cells)].In the preliminary series, BSP and OPN reactivity was found in 85% and 77% of cases, respectively. No predictive role of bone spread propensity of RCC was identified. Conversely, both BSP and OPN were significantly associated with shorter survival considering median (p=0.002) and upper quartile (p=0.03) expression values, respectively. In the validation group, a prognostic role was confirmed for BSP only (p=0.008), while OPN showed a trend of association with poorer survival (borderline p-value of 0.058).BSP was shown for the first time to be an independent parameter associated with poor prognosis in RCC. Its coexpression with OPN identifies a subgroup of RCC having the worst outcome.

Thymic epithelial tumors include several entities with different biologic behavior. Chemotherapy is indicated in advanced disease, but limited data exist on gene expression correlation with the response to chemotherapeutic agents.A series of 69 thymic neoplasms (7 A-, 6 AB-, 6 B1-, 10 B2-, 14 B3-thymomas, 22 carcinomas and 4 combined tumors) was collected to assess gene expression of thymidylate synthase (TS), excision repair cross complementing-1 (ERCC1), ribonucleotide reductase subunit 1 (RRM1), topoisomerase 2α (TOP2A) and mTOR.A strong linear correlation between TS gene and protein expression was observed (P0.0001, R=0.40). TS expression was significantly lower in pure A-thymomas and thymic carcinomas (P0.0001) and progressively decreasing from B1-type to thymic carcinomas (B1B2B3C; P0.0001). RRM1 and TOP2A mRNA expression levels were significantly correlated with TS levels (both P=0.03) with a similar trend of expression among histotypes. RRM1 and TOP2A high levels were significantly correlated with high TS (P=0.03) and low tumor stages (I-II) (P0.0001 and P0.01, respectively). No relevant changes of ERCC1 and mTOR were detected.Low TS and, to a minor extent, RRM1 and TOP2A expression were detected in aggressive thymic tumors. These findings should be prospectively considered in selecting the most appropriate chemotherapy.

DNA flow cytometry and the monoclonal antibody D07 were applied in formalin-fixed, paraffin-embedded specimens from 34 primary male breast carcinomas to verify whether DNA ploidy and p53 expression were associated with survival and proliferative activity. They were compared with tumor clinicopathologic features, sex steroid hormone receptors and cell proliferative activity, assessed by the counts of the argyrophilic nucleolar organizer regions (AgNORs), the monoclonal antibody PC10 against the proliferating cell nuclear antigen and the monoclonal antibody MIB-1. A significant correlation was found between survival and tumor ploidy (median survival, 77 months for diploid but only 38 months for aneuploid cases; P = .03) and p53 expression (median survival, 95 months for cases with p53 scores ⩽ 14.06% versus 33 for cases with p53 scores > 14.06%; P = .0004; median survival, 99 months for p53 negative vs 39 for positive cases; P = .007). Tumor histological grade ( P = .006), AgNOR counts ( P = .0001), PC10 scores ( P = .002), and MIB-1 scores ( P = .001) were also associated with prognosis. In the multivariate analysis, only p53 scores ( P = .001) or p53 immunopositivity ( P = .003) and AgNOR counts ( P = .022) retained an independent prognostic significance. Aneuploid tumors had higher AgNOR counts ( P = .002), PC10 ( P = .007), MIB-1 ( P = .006), and p53 scores ( P = .01) than diploid cases. A linear relationship was observed between p53 scores and AgNOR counts ( r = .41; P = .014), PC10 ( r = .46; P = .005), and MIB-1 scores ( r = .44; P = .011). These results indicate that DNA ploidy and p53 expression are associated with survival and cell proliferative activity in male breast carcinoma. Quantitative parameters, such as DNA ploidy, p53 scores, AgNOR counts, PC10, and MIB-1 scores substantially improve the prognostic significance of the traditional parameters in male breast carcinoma.

Aim Routine prophylactic inguinal irradiation in anal cancer may cause significant toxicity associated with overtreatment bias. The aim of this study was to determine the risk of regional node metastases in anal carcinoma by identifying predictive molecular biomarkers. Method Clinicohistopathological data from 50 pretreatment anal carcinoma biopsies were collected. Immunohistochemical analyses with antibodies against Ki67, p53, epidermal growth factor receptor (EGFR) and YKL-40 were performed. Statistical correlations between biomarkers and clinicopathological features and outcomes were studied. Sentinel lymph node biopsy was performed in a subset of 36 patients. Results All patients had undergone synchronous radiochemotherapy; tumour recurrence had developed in 26%, and 16% had died. YKL-40 tumour expression correlated with lymph node metastasis, whereas no inguinal node metastases were found in any of the (14%) patients presenting with a YKL-40/EGFR-negative tumour. YKL-40 expression and node metastasis were both significantly associated with shorter overall and disease-free survival. Tumour grade significantly correlated with disease-free survival only. HIV, tumour histological type, Ki67, p53 and EGFR were not associated with outcome. Conclusion YKL-40 expression in anal carcinoma is correlated with a poor outcome and can predict lymph node metastases. The combined absence of YKL-40 and EGFR expression in a first biopsy of anal carcinoma reliably selects a subset of patients without inguinal metastases. Such patients could be spared sentinel lymph node biopsy and/or inguinal radiotherapy.

AIMS--To verify the correlation between MIB-1, Ki67, and proliferating cell nuclear antigen (PCNA-PC10) scores and S-phase fraction in intermediate grade non-Hodgkin's lymphomas (Working Formulation F); and their reliability in differently processed tissues. METHODS--Forty one non-Hodgkin's lymphomas were classified as (F) intermediate grade malignant lymphomas according to the Working Formulation; mitotic counts and percentage of large cells were assessed for each case. Sections from formalin fixed, paraffin wax embedded tissues were stained with anti MIB-1 monoclonal antibody, after microwave oven processing, and anti-PCNA (PC10) monoclonal antibody using an avidin-biotin immunoperoxidase (ABC) method. One thousand cells from 10 representative fields were scored. Frozen sections from surgical specimens were stained with Ki67 monoclonal antibody using the ABC method; the fraction of Ki67 positive cells was calculated scoring 1000 cells. Flow cytometry analysis (FCM) was performed on cell suspensions from fresh tissues. Correlations between data were estimated using linear regression. RESULTS--A linear correlation was found between MIB-1 and Ki67 scores (r = 0.92; p < 0.00001); between MIB-1 and PCNA scores (r = 0.79; p < 0.00001); and between MIB-1 score and S-phase fraction (r = 0.51; p = 0.0006). A linear correlation was also found between Ki67 and PCNA scores (r = 0.85; p < 0.00001); between Ki67 score and S-phase fraction (r = 0.6; p = 0.0002); and between PCNA score and S-phase fraction (r = 0.74; p < 0.00001). A correlation was found between mitotic counts and MIB-1 (r = 0.56; p = 0.0001), PCNA (r = 0.51; p = 0.0007), or Ki67 scores (r = 0.47; p = 0.002); between the percentage of large cells and MIB-1 (r = 0.49; p = 0.0009), PCNA (r = 0.6; p = 0.00003), and Ki67 scores (r = 0.53; p = 0.0003) and S-phase fraction (r = 0.55; p = 0.0002). CONCLUSION--MIB-1, Ki67, and PCNA (PC10) scores and S-phase fraction are highly correlated and equally well represent the proliferative activity of intermediate grade non-Hodgkin's lymphomas in differently processed material. MIB-1 and PCNA stains can be applied even on small biopsy specimens. MIB-1 produces homogenous staining without background; it also strongly stains mitotic figures. It can be performed on routinely processed tissues, permitting the simultaneous evaluation of the morphology and tumour cell kinetics. The wide standard deviations of the proliferative indices found for intermediate grade NHL suggest that this category probably includes various degrees of malignancy.

Objectives: The aim of this prospective study was to assess the clinical and histologic outcomes obtained with calcium sulfate (CS) used as a filler material in fresh premolar and molar postextraction sockets. Materials and Methods: Sixty premolar or molar postextraction sockets were filled with CS. Among the 60 grafted sockets, after 3 months, 50 underwent implant placement and clinical assessment. The removal of a sample core of newly generated intrasocket tissue was performed in 19 sockets. Collected samples were sent for histologic examination. The percentage of vital bone, nonvital bone, residual CS, amorphous material, and connective areas in every sample was calculated and recorded. Results: Fifty postextraction regenerated sockets that underwent implant placement 3 months after tooth removal were included in this study. A partial postoperative exposition of the graft was observed in 12 of 50 sockets. At the surgical reentry, the augmented extraction sockets were completely filled by a hard material with an adequate alveolar crest in 41 cases. Histologic examination of the cores revealed that 63.16% of the intrasocket tissue was new vital bone, 2.1% was nonvital bone, 4.74% was fibrous tissue, and 30% was amorphous material. No residual CS was identified in bone cores. Conclusions: This study confirmed that CS is an ideal grafting material. The clinical adequacy aspect of filled sockets at surgical reentry seemed to be indicative of a qualitatively better bone regeneration. Postoperative exposition of graft material after a first intervention seemed to constitute an important risk factor for a worse bone regeneration.

Merkel cell carcinoma of the skin is a malignant neuroendocrine tumour, whose prognostic criteria are a matter of dispute. Specifically, no predictor is presently available in stage I–II tumours. We collected clinical and follow-up data from 70 Merkel cell carcinomas of the skin. The same cases were studied for p63 expression by immunohistochemistry, by reverse-transcription PCR (RT-PCR) and TP63 gene status by FISH and for presence of Merkel cell polyomavirus by PCR. Stage emerged as a significant prognostic parameter (P¼0.008). p63 expression, detected in 61% (43/70) of cases by immunohistochemistry, was associated with both decreased overall survival (Po0.0001) and disease-free survival (Po0.0001). Variable expression patterns of the different p63 isoforms were found only in cases immunoreactive for p63. In these latter lesions, at least one of the N-terminal p63 isoforms was detected and TAp63a was the most frequently expressed isoform. TP63 gene amplification was observed by FISH in only one case. Presence of Merkel cell polyomavirus DNA sequences was detected in 86% (60/70) of Merkel cell carcinomas and did not emerge as a significant prognostic parameter. Merkel cell carcinoma cases at low stage (stage I-II) represented over half (40/70 cases, 57%) of cases, and the clinical course was uneventful in 25 of 40 cases while 15 cases died of tumour (10/40 cases) within 34 months or were alive with disease (5/40 cases) within 20 months. Interestingly, a very strict correlation was found between evolution and p63 expression (Po0.0001). The present data indicate that p63 expression is associated with a worse prognosis in patients with Merkel cell carcinoma, and in localised tumours it represents the single independent predictor of clinical evolution.

Supratentorial ependymomas account for a minority of intracranial ependymomas, which still have uncertain prognostic markers. Among them, epidermal growth factor receptor (EGFR) overexpression correlates with a poor prognosis. In glioblastoma cells, EGFR function has been reported to be regulated by its migration from cell membrane infoldings called caveolae and by its colocalization with the caveolae-associated protein caveolin-1 (cav-1). Therefore, we decided to investigate cav-1 expression and coexpression with EGFR in a series of adult intracranial ependymomas. We analyzed 22 adult supratentorial ependymomas and compared tumor grades as determined by the WHO classification and patient survival rates with the expression of EGFR, cav1, and p53 and the values of the proliferation marker Ki-67, all tested by immunohistochemistry; in addition, we investigated the mutational profile of cav-1. The results demonstrate that the tumor grade is directly correlated with EGFR, Ki-67, and cav-1 expression only, whereas (by univariate analysis) the expression of all the studied markers, as well as the tumor histological grade, significantly correlated with the patient’s overall survival (OS). By multivariate analysis using the Cox proportional hazards model, among all variables considered, cav-1 was the only independent prognostic marker related to OS (relative risk 5 13.92; P 5 .013). Among grade II ependymomas, only cav-1 correlated with poor OS (P 5 .011), distinguishing 2 distinct subgroups of tumors with different outcomes despite sharing identical grading. All the patients studied carried wild-type cav-1 sequences, demonstrating that cav-1 overexpression is not driven byactivating mutations, as previously reported in other tumor types. Interestingly, after stratifying all cases into 4 distinct groups according to cav-1 and EGFR expression (cav-11/EGFR1 ,c av-1 2/EGFR2, cav-11/EGFR2, and cav-12/EGFR1), the coexpression of cav-1 and EGFR identified a subset of patients with definitively poor prognoses. Further studies are needed to support this evidence on a larger scale and to clarify how cav-1 and EGFR interaction can influence tumor aggressiveness.

The prognostic significance of argyrophilic nucleolar organizer regions (AgNORs) has been evaluated in bone marrow trephine biopsies from 64 patients with multiple myeloma (MM) prior to therapy. The univariate Kaplan-Meyer survival analysis showed a significant correlation between survivals and AgNOR counts (median of survival 51.3 months for cases with 4.62 AgNORs per plasma cell (PC) versus 16 months for cases with >4.62 AgNORs per PC; P=0.0000) or AgNOR distribution in PC nucleus (AgNOR configuration) (median of survival 71.67 months for cases with tightly grouped AgNORs. 16.26 for partially grouped and 11.74 for dispersed AgNORs; P=0.001). Significant prognostic correlations were also found for monoclonal immunoglobulin type (P=0.008), platelet counts (P=0.0078). serum creatinine level (P=0.0001), Durie's clinical stage (P=0.02). percentage of plasma cells in bone marrow biopsies (BMPCX) (P= 0.005), pattern of medullary involvement (P=0.003) and PC atypia (P= 0.009). Borderline result was detected for the percentage of PCs in aspirates (P=0.06). No significant correlation was found between prognosis and patients age, sex, haemoglobin level, serum albumin or calcium level, marrow cellularity and excess of haemosiderin. Multivariate survival analysis showed that only two variables were significantly correlated with prognosis: AgNOR counts (P=0.003) and AgNOR configuration (P

Caveolin 1 (cav-1) is the basic component of the flask-shaped membrane microdomains known as caveolae that are involved in various cell functions. Caveolin 1 can be overexpressed in tumors,suggesting a proneoplastic role, or it can be downregulated. We previously reported that cav-1 expression increases with tumor grade in astrocytomas. Here, we studied cav-1 immunoreactivity in brain umors with an oligodendroglial component to determine the prognostic value of cav-1 expression and to correlate it with 1p/19q deletions. Fifty-four oligodendrogliomas, 26 mixed oligoastrocytomas,and 7 glioblastomas with an oligodendroglial component were assessed for cav-1 expression by immunohistochemistry and for 1p/19q status by fluorescence in situ hybridization. Caveolin-1 was detected in a minority of cases (22%) and was associated mostly with Grade III mixed oligoastrocytomas and glioblastomas with anoligodendroglial component; cav-1 expression was significantly correlated with the absence of a 1p/19q deletion (p = 0.0002). In the 63 cases in which survival data were available, cav-1 expression was also significantly associated with shorter survivals, whereas 1p/19q deletion was associated with longer survivals. Among high-grade tumors, cav-1 expression was the only factor that retained a statistical significance after multivariate analysis for the prediction ofa short survival (p G 0.015). These data are the first evidence that cav-1 immunohistochemistry is an independent prognostic marker in tumors with an oligodendroglial component regardless of the 1p/19q status.

Background. The aim of this study was to deter- mine whether the aberrant expression of cell-cycle or immune- response markers together with human papillomavirus (HPV) positivity impacts patient survival in different head and neck squamous cell carcinoma (HNSCC) subsets. Methods. A total of 59 HNSCC specimens were analyzed for expression of cell cycle and proliferation markers, and macro- phage infiltration. HPV was evaluated by polymerase chain reac- tion and DNA sequencing. Results. The HPV presence in oropharynx carcinoma was associated with survival advantage. Low Ki67 expression was associated with favorable outcome in oropharynx and oral cavity carcinoma. A more favorable outcome was associated with low cyclin E expression in larynx carcinoma and with low p53 expression in squamous cell carcinoma (SCC) of the oral cavity. A direct correlation between macrophage infiltration and tumor proliferation index was observed irrespective of the tumor subset. Conclusions. The assessment of proliferation, viral, and immunologic profiles may be crucial to finding beneficial treat- ments for the different HNSCC subsets. V C 2008 Wiley Periodi- cals, Inc. Head Neck 31: 318-327, 2009

We hypothesized that the potent neovascularization marker endoglin (CD105), by differentially highlighting a subset of microvessels (MV) in esophageal cancer (EC), could provide better prognostic/therapeutic information than the panendothelial marker CD34, which also highlights MV.Endoglin messenger ribonucleic acid (mRNA) expression in normal, malignant, and adjacent nontumorous esophagus tissue was quantified by real-time reverse-transcription polymerase chain reaction (RT-PCR). Sections of formalin-fixed, paraffin-embedded tissues were analyzed immunohistochemically for CD105 and CD34. MV density was counted following a standard protocol. Circulating soluble endoglin levels were determined in patient and control sera, and compared with clinical outcome.CD105 mRNA was upregulated by a median factor of 2.89 in ECs versus controls. In 28% of patients, CD105 mRNA was upregulated by a median factor of 2.65 in adjacent non-tumorous versus normal tissue. In tumor tissues, CD105 was stained negatively or positively only in a subset of MV. CD34 always showed positive extensive MV staining. In adjacent nontumorous esophagus, CD105 rarely showed diffuse MV staining, while CD34 stained blood-vessel endothelial cells in all non-neoplastic tissue. CD105 expression was high in residual highly dysplastic Barrett's-type mucosa associated with some adenocarcinomas. No statistically significant difference in endoglin serum levels appeared between patients and normal subjects. Correlation with clinicopathological data showed higher intra-tumor MV-CD105+ scores at more-advanced clinical stages. High-scoring MV-CD105+ patients had significantly shorter disease-free and overall survival; MV-CD34+ density was not survival related. Diffuse CD105 expression in adjacent nontumorous esophagus predicted poorer disease-free and overall survival.Our findings could help identify EC patients who may benefit from targeted anti-angiogenic therapies.

PurposeBone metastases (BM) in non–small-cell lung cancer (NSCLC) may be detected at diagnosis or during the course of the disease, and are associated with a worse prognosis. Currently, there are no predictive or diagnostic markers to identify high-risk patients for metastatic bone dissemination.Patients and MethodsThirty patients with resected NSCLC who subsequently developed BM were matched for clinicopathologic parameters to 30 control patients with resected NSCLC without any metastases and 26 patients with resected NSCLC and non-BM lesions. Primary tumors were investigated by immunohistochemistry for 10 markers involved in bone resorption or development of metastases. Differences among groups were estimated by χ2test, whereas the prognostic impact of clinicopathologic parameters and marker expression was evaluated by univariate (Wilcoxon and Mantel-Cox tests) and multivariate (Cox proportional hazards regression model) analyses.ResultsThe presence of bone sialoprotein (BSP) was strongly associated with bone dissemination (P < .001) and, independently, with worse outcome (P = .02, Mantel-Cox test), as defined by overall survival. To evaluate BSP protein expression in nonselected NSCLC, a series of 120 consecutive resected lung carcinomas was added to the study, and BSP prevalence reached 40%. No other markers showed a statistically significant difference among the three groups or demonstrated a prognostic impact, in terms of both overall survival and time interval to metastases.ConclusionBSP protein expression in the primary resected NSCLC is strongly associated with BM progression and could be useful in identifying high-risk patients who could benefit from novel modalities of surveillance and preventive treatment.

Intrasinusoidal infiltration (ISI) is a pattern of invasion that is rarely found on bone marrow (BM) biopsies, and is considered as a hallmark of splenic marginal zone cell lymphoma (SMZL). We analysed BM biopsies showing intrasinusoidal infiltration from 54 consecutive patients with different types of lymphoma to verify if ISI quantity was a diagnostic criterion for SMZL. There were 35 primary splenic lymphoma (PSL) and 19 non-PSL; 28 SMZL, three non-splenic MZL, six mantle cell, six small lymphocytic, four follicular, four diffuse large B cell, one peripheral T cell, one lymphoplasmacytic and one anaplastic large-cell lymphoma. The quantity of BM infiltrate was assessed on CD45, CD20 and CD3 stained sections. The mean percentage of total (TI) and intrasinusoidal (ISI) lymphocytes was calculated in 10 areas for each case. TI quantity was 21.57 in PSL and 35.05 in non-PSL (P = 0.04). ISI quantity was 5.23 in PSL and 7.62 in non-PSL (P = 0.08), 5.83 in SMZL and 2.83 in other types of PSL (P = 0.12), 4.46 in non-splenic MZL and 8.21 in other types of non-PSL (P = 0.28). No difference in ISI quantity was found among the lymphoma subtypes, either in PSL (P = 0.74) or non-PSL (P = 0.3). The data demonstrate that ISI quantity in BM biopsies is not a reliable diagnostic parameter for SMZL.

Crohn's disease (CD) and ulcerative colitis (UC), the two major forms of chronic inflammatory bowel disease (IBD), are characterized by mucosal immune cell activation that is driven by a cytokine imbalance. Several cytokines involved in IBD act through the activation of the signal transducers and activators of transcription (STAT) family. We investigated the activation of STAT3 in the mucosa of CD and UC patients, and evaluated whether this event is specific for IBD patients. Using immunofluorescence and immunoblotting, total and phosphorylated STAT3 levels were assessed in biopsy specimens, isolated lamina propria mononuclear cells, and peripheral blood mononuclear cells from patients with CD, UC, other forms of intestinal inflammation, and control subjects. Immunoblotting revealed phosphorylated STAT3 in mucosal biopsy specimens from patients with CD, UC, celiac disease, and acute self-limited colitis, but not in the normal mucosa of control subjects. In IBD patients, STAT3 activation was confined to actively inflamed areas. Accordingly, activated STAT3 was detected in isolated lamina propria mononuclear cells from inflamed IBD tissues, but not in peripheral blood mononuclear cells from control subjects or IBD patients. Immunofluorescence demonstrated that the sources of activated STAT3 were macrophages and T lymphocytes, but not neutrophils. STAT3 activation also was detected in T cells infiltrating the duodenal mucosa of celiac disease patients. We conclude that STAT3 signaling occurs in both CD and UC, where it is strictly confined to areas of active inflammation and is limited to infiltrating macrophages and T cells. The occurrence of STAT3 signaling in other acute and chronic intestinal inflammatory conditions suggests that, rather than a specific feature of IBD, it represents a fundamental signaling pathway that is shared by multiple forms of gut inflammation.

BACKGROUND The term poorly differentiated (PD) carcinoma was proposed 20 years ago to define aggressive, follicular-derived thyroid carcinomas with behavior intermediate between follicular/papillary and anaplastic carcinomas. Among the variable histologic patterns recognized in such tumors, trabecular-insular-solid (TIS) areas usually are predominant. Conversely, some authors pointed out that PD carcinomas are characterized by unequivocal, high-grade histology with atypias, high mitotic counts, and necrosis rather than by a specific growth pattern. METHODS The clinicopathologic features of a series of 183 thyroid carcinomas with predominant (n = 165 tumors) or focal (n = 18 tumors) TIS growth patterns were studied by univariate and multivariate overall survival analyses and were compared with clinical outcomes. Subgroups included tumors with predominant oxyphilic features (n = 66 tumors) and (residual) papillary carcinoma features (n = 24 tumors). Control groups of papillary (n = 68 tumors), follicular (n = 71 tumors), and anaplastic (n = 35 tumors) carcinomas also were included for overall survival analysis. RESULTS TIS carcinomas had an intermediate behavior between papillary/follicular and anaplastic carcinomas (P 45 years (P = 0.007), the presence of necrosis (P 3 per 10 high-power fields (P = 0.01) were associated with poor outcome. A simplified scoring system based on statistically significant parameters allowed the identification of three prognostic subgroups (P < 0.0001). CONCLUSIONS PD TIS carcinomas overall followed a more aggressive course compared with differentiated thyroid carcinomas, irrespective of the extent of the TIS component. However, a numeric scoring system applied to specific clinicopathologic parameters further may identify three prognostic categories of patients who have significantly different survival rates at 5 years and 10 years. Cancer 2004;100:950–7. © 2004 American Cancer Society.

The expression of p53, c-erbB-2, bcl-2 and c-myc proteins was compared to the quantity of the nucleolar organiser regions (AgNORs) and MIB-1 antigen to elucidate the relationship between oncogene expression and rapidity of cell proliferation and tumor growth fraction. Sections from 50 male breast carcinomas (MBC) and 62 superficial papillary bladder neoplasias were stained with the standardised AgNOR method and monoclonal antibodies MIB-1, DO7, CB11, bcl-2 124 and 9E11. p53 immunopositivity was associated with high AgNOR quantity and MIB-1 scores both in MBC and bladder neoplasm. c-erbB-2 expression was associated with high AgNOR quantity in bladder neoplasm. bcl-2 expression was associated with low AgNOR quantity in MBC. c-myc expression was associated with high AgNOR quantity in MBC. MBC patients with low AgNOR quantity, and p53, c-erbB-2 and c-myc immunonegativity had the longest overall survival. Patients with bladder neoplasia with low AgNOR quantity, negative p53 and positive c-erbB-2 immunostaining had the longest disease-free survival time. Our results indicate that p53 overexpression reflects both the rapidity of cell proliferation, as assessed by AgNOR quantity, and tumor growth fraction, as assessed by MIB-1 scores, while c-erbB-2, c-myc and bcl-2 expression mainly reflects the rapidity of cell proliferation. The combination of AgNOR quantity and oncogene expression may stratify patients into different risk groups.

Background: Esophageal replacement with gastric tube is a well-established reconstruction of the alimentary tract after esophagectomy in cancer patients. The resulting molecular events in the transposed gastric tube and residual esophagus have yet to be investigated. Stem cell factor (SCF) was recently shown to be critical for signaling in gastrointestinal motility. SCF expression is here correlated with changes in mucosal morphology, acid and biliary reflux, and motility in the residual esophagus and gastric tube. Methods: Thirteen patients surgically resected for squamous esophageal carcinoma with gastric tube replaced by esophagogastric anastomosis underwent upper endoscopy, esophageal manometry, 24-hour pH monitoring, and bile reflux detection. Esophageal and gastric mucosa samples were examined for SCF expression by immunohistochemical and semiquantitative reverse transcriptase-polymerase chain reaction analysis and for SCF serum levels by enzyme-linked immunosorbent assay. Results: All patients showed severe residual esophagus hypoperistalsis and no gastric tube motor activity. The 24-hour pH monitoring was positive in most; 24-hour bile detection was mostly negative. SCF levels in the residual esophageal and gastric tube mucosa were dramatically decreased compared with those of normal subjects. The correlation between SCF and slow-wave activity was positive. Conclusions: Hypomotility of the residual esophagus and gastric tube seems closely associated with disruption of the SCF/c-kit signaling pathway. However, the absence of notable relations between mucosal changes after chronic exposure to acid, biliary gastric content, and SCF expression indicates that this analysis cannot be considered part of endoscopic follow-up.

BACKGROUND Recurrence of transitional cell carcinoma of the bladder cannot be predicted accurately by traditional criteria alone. This study examined the value of cell proliferative activity, morphometry, and expression of p53, c-erbB-2, and bcl-2 oncogenes in predicting recurrence of superficial papillary urothelial neoplasms of low malignant potential (LMP) and Grade 1 (G1) papillary carcinomas of the bladder. METHODS Sixty-two patients (mean age, 62 years) with newly diagnosed superficial pTa bladder tumors (19 LMP, and 43 G1) were analyzed retrospectively. All patients underwent transurethral resection (TUR). Median follow-up was 69 months. Serial sections from formalin-fixed, paraffin-embedded material at initial TUR were stained with monoclonal antibodies (MoAbs) DO7, CB11, and bcl-2-124. Cell proliferation was assessed by MIB-1 MoAb, the quantity of argyrophilic nucleolar organizer region-associated proteins (AgNORs), and mitotic count. RESULTS Of the 62 patients, 42 (67.7%) had one or more recurrences. Recurrence rates were higher in MIB-1 (P < 0.0001) and p53 immunopositive cases (P = 0.02), when the mitotic count was greater than 5 (P = 0.004), and in G1 carcinomas (P = 0.04). In univariate analysis, the disease-free period was shorter for MIB-1 (P < 0.0001) and p53 immunopositive (P = 0.0001) cases, for cases with high AgNOR quantity (P = 0.04), mitotic count greater than 5 (P = 0.01), and in G1 carcinomas (P = 0.002). In multivariate analysis, only MIB-1 immunoreactivity retained independent prognostic significance. CONCLUSIONS Despite the small cohort, the results confirm the prognostic value of cell proliferation and p53 expression in patients with bladder neoplasms. The results also indicate that MIB-1 immunopositivity is the most significant predictor of recurrence and disease-free survival in superficial LMP and G1 papillary bladder carcinomas. Cancer 2002;95:784–90. © 2002 American Cancer Society. DOI 10.1002/cncr.10733

We investigated the expression of oncogenes p53, c-erbB-2, and bcl-2 and cell proliferative activity in 62 newly diagnosed superficial pTa papillary bladder tumors. Based on the 1998 World Health Organization/International Society of Urological Pathology (WHO/ISUP) and 1999 WHO classifications, 19 were urothelial neoplasias of low malignant potential (LMP) and 43 low-grade (grade 1) papillary carcinomas. All the patients underwent transurethral resection and were followed up to 97 months; 42 had recurrences. Initial biopsies were tested for p53, c-erbB-2, and bcl-2 proteins using DO7, CB11, and bcl-2 124 monoclonal antibodies. Cell proliferation was assessed by MIB-1 mAb and mitotic count. LMP had significantly lower MIB-1 (p = 0.002) and p53 immunopositivity (p = 0.03), mitotic count (p = 0.006), and recurrence rates (p = 0.04) than did grade 1 cases, whereas no difference was observed for c-erbB-2 and bcl-2 expression. The median disease-free survival for LMP was 76 months but only 15 months for grade 1 cases (p = 0.002). Although the cohort is small, the results indicate that the distinction between LMP and low-grade (grade 1) papillary urothelial neoplasias, as proposed by the 1998 WHO/ISUP and 1999 WHO classifications, reflects different biologic activity and clinical behavior; however, a long-term follow-up is advisable also for patients with LMP.

PURPOSE: To investigate the prognostic value of biomarkers in male breast carcinoma (MBC). PATIENTS AND METHODS: Fifty patients (mean age, 62.2 years) with invasive ductal carcinoma were retrospectively studied. All patients received surgery; 35 had adjuvant postoperative therapy. The median follow-up was 59 months (range, 1 to 230 months). c-myc, c-erbB-2, p53, and bcl-2 proteins were immunohistochemically detected on sections from formalin-fixed, paraffin-embedded tissues using 9E11, CB11, DO7, and bcl-2 124 monoclonal antibodies (mAbs). Estrogen, progesterone, and androgen receptors were detected using specific mAbs. Cell proliferation was assessed by MIB-1 mAb. RESULTS: In univariate analysis, c-myc, c-erbB-2, and p53 protein overexpression was significantly correlated with prognosis. The median survival was 107 months for c-myc–negative and 52 months for c-myc–positive patients (P = .01), 96 months for c-erbB-2–negative and 39 months for c-erbB-2–positive patients (P = .02), and 100 months for p53-negative and 33 months for p53-positive patients (P = .0008). Tumor histologic grade (P = .01), tumor size (P = .02), patient age at diagnosis (P = .03), and MIB-1 scores (P = .0004) also had prognostic value. In multivariate analysis, only c-erbB-2 and p53 immunoreactivity retained independent prognostic significance. All nine patients who did not express c-erbB-2 and p53 proteins were alive after 58 months, whereas none of the 14 patients expressing both proteins survived at 61 months follow-up (P = .0002). CONCLUSION: Overexpression of c-myc, c-erbB-2, and p53 proteins may be regarded as an additional prognostic factor in MBC. The combination of c-erbB-2 and p53 immunoreactivity can stratify patients into different risk groups.

The relationship between therapy and overall survival was retrospectively investigated in 50 patients with primary male breast carcinoma. Forty-five had radical or modified radical mastectomy and 5 simple mastectomy. Thirty-five received adjuvant post-operative therapy, including radiation, hormone and chemotherapy, given separately or in combination. The mean follow-up period was 67 (range, 1-230) months. The median survival was 33 months for patients receiving surgery alone and 86 months for those who also had adjuvant therapy (p=0.003). No difference in survival was found between simple or radical/modified radical mastectomy, nor among the various types of adjuvant therapy. Adjuvant therapy was most effective in large size, node positive and poorly differentiated tumors, and retained independent prognostic significance in multivariate analysis. With the limitation due to the small number of cases, our data suggest that adjuvant therapy may improve survival in males with cancer of the breast.

To investigate the prognostic value of nuclear morphometry in male breast carcinoma (MBC), histological samples from 50 patients (mean age 62.2 years) were retrospectively analyzed by computerized nuclear morphometry. All patients received surgery; 35 had multiple combinations of adjuvant therapies. Mean follow-up was 67 months (range 1-230). In each case, 100 tumor cells were measured, and the mean nuclear area (MNA), standard deviation of the nuclear area (SDNA), mean nuclear perimeter (MNP), standard deviation of the nuclear perimeter (SDNP) and shape factor (SHF) were calculated. Morphometric features were compared with tumor histological grade, size, nodal status, DNA ploidy evaluated by flow-cytometry and cell proliferative activity assessed by the quantity of argyrophilic nucleolar organizer region-associated proteins (AgNORs), monoclonal antibody (MAb) PC 10 against proliferating cell nuclear antigen and MAb MIB-I. Comparison was also made with the immunohistochemical detection of p53, bcl-2, c-erbB-2 and c-myc proteins. Significant association was found between nuclear morphometric parameters and tumor grade, DNA content and cell proliferation indices. SDNA was greater in p53-positive and bcl-2-negative cases; SDNP was greater in p53-positive cases; SHF was lower in p53- and c-myc-positive cases. Overall survival was shorter in carcinomas with high MNA, SDNA, MNP and SDNP and low SHF. In multivariate analysis, performed by testing nuclear morphometric parameters, histological grade, tumor size, nodal status and p53 immunostaining in the Cox model, p53 over-expression and histological grade retained independent prognostic significance. When p53 was excluded, only SDNP appeared as an independent prognostic variable. Our results indicate that nuclear morphometric parameters can identify an aggressive tumor phenotype and provide additional prognostic information for patients with MBC.

PURPOSE The analysis of the nucleolar organizer regions (AgNORs) was performed in patients with acute myelogenous leukemia (AML) to verify the role of cell proliferation in predicting complete remission (CR) and survival. MATERIALS AND METHODS Bone marrow biopsies from 40 adult patients with AML were stained with the argyrophilic method. The mean AgNOR number (AgNOR count) was calculated for each case. After induction therapy, patients who achieved CR received intensive consolidation; two underwent autologous and four allogeneic bone marrow transplantations (BMT). RESULTS The mean AgNOR count for the whole series was 6.6 (SD = 1.35); it was higher in CR patients than in resistant ones (P = .02). The median duration of CR was 26 months for patients with an AgNOR count greater than 6.6, but only 6 months for those with lower counts (P = .01). Sixteen patients who achieved a CR relapsed and 14 reached a second CR; the median duration of second CR was 16 months for patients with AgNOR count greater than 6.6, but only 5 months for those with lower counts (P = .01). The median survival time for the whole series was 14 months, with 30% of patients alive and in continuous CR at 103 months. Survival was longer for patients with an AgNOR count greater than 6.6 (33 months) than for those with lower counts (6 months; P = .0009). In multivariate analysis, when CR was excluded from the model, AgNOR count appeared as an independent prognostic variable (P = .005). CONCLUSION AgNOR analysis is a suitable method to assess cell proliferation in bone marrow biopsies and can predict CR, remission duration, and survival in AML patients.

We have analysed the expression of bcl-2 protein retrospectively in 34 primary male breast carcinomas (MBC), using the monoclonal antibody bcl-2 in formalin-fixed, paraffin-embedded tissues. Bcl-2 expression was compared with tumour clinicopathological features, sex steroid hormone receptors, DNA content, p53 immunoreactivity and cell proliferative activity assessed by counts of the argyrophilic nucleolar organizer regions (AgNORs), the monoclonal antibody PC10 against proliferating cell nuclear antigen and the monoclonal antibody MIB-1. Most (28, or 82.3%) of the 34 cases of MBC were bcl-2 positive. No association was found with clinicopathological features of the tumours, although bcl-2 tended to be more frequently expressed in small tumours (P=0.09) and in cases without necrotic areas (P=0.1). Nor was any association found with hormone receptor status, p53 immunoreactivity, DNA content, cell proliferative activity or patient survival. In multivariate analysis, only proliferative activity (expressed by AgNOR counts) and p53 immunoreactivity had independent prognostic significance. Our results indicate that MBC differs from FBC in that in MBC bcl-2 protein is not related to an oestrogen-dependent transcription pathway and bcl-2 alone is not sufficient to induce increased proliferation. These characteristics, together with the high prognostic value of cell proliferation and the lack of prognostic significance for hormone receptor status, support the hypothesis that MBC is biologically different from FBC.

We investigated the histologic pattern and the cell proliferative activity of myeloma cells by the analysis of the nucleolar organizer regions (AgNORs) in bone marrow biopsy specimens from 150 multiple myelomas at diagnosis. The objective was an attempt to define risk groups of myeloma patients. On univariate analysis, the percentage of bone marrow plasma cells (BMPC%), the pattern of infiltration, the degree of plasma cell (PC) atypia, the marrow fibrosis, and the number of AgNOR/PC were correlated with survival time. On multivariate analysis, only AgNOR counts and pattern of infiltration retained independent prognostic significance. At 4-year followup, all patients with BMPC%or = 20, interstitial pattern of invasion, and well-differentiated (G1) PC plus AgNOR/cellor = 3.32 were alive, while no patient with BMPC%50, diffuse pattern of infiltration, and poorly differentiated (G3) PC plus AgNOR/cell5.15 survived (p0.0001). In conclusion, the histologic pattern and proliferative activity of myeloma cells, evaluated by AgNOR counts, are reliable predictors of survival in myeloma. Both parameters can be easily assessed in the same biopsy specimen, are reproducible, and permit identification of a group of patients with favourable outcome at 4-year followup. Thus, bone marrow biopsy should always be included in the diagnostic procedures for myeloma patients.

We performed p53 immunohistochemistry, DNA flow cytometry and analysis of the argyrophilic nucleolar organizer regions (AgNORs) in formalin-fixed, paraffin-embedded sections from 46 non-invasive thymomas and correlated the results with the traditional clinicopathologic features of the tumor. p53 immunopositivity was detected in 21 of 46 cases; it was not associated with any clinicopathologic features nor DNA content but significantly correlated with AgNOR counts. On univariate analysis, 10-year survival rates were 100% for p53-negative cases but only 71% for p53-positive cases and 93% for patients with low AgNOR counts but only 77% for patients with high AgNOR counts. Age, sex, histologic type, myasthenia gravis and DNA content did not correlate with survival. Our results indicate that p53 staining and evaluation of proliferative activity allow assessment of prognosis in non-invasive thymomas, when all of the other parameters are insufficient. Furthermore, the high rate of p53 expression in non-invasive thymomas suggests that abnormal p53 immunoreactivity may occur early in the neoplastic process.

We performed DNA flow cytometry and analysis of the argyrophilic nucleolar organizer regions (AgNORs) in formalin-fixed, paraffin-embedded sections from 60 surgically resected thymomas. The results were correlated with histologic pattern, stage, associated clinical features, and survival to assess which parameters could best predict prognosis. On univariate analysis, the 10-year survival rates were 86% for predominantly lymphocytic type but only 42% for predominantly epithelial, mixed lymphoepithelial, or spindle cell thymomas (p = 0.006); survival rates were 85% for noninvasive but only 34% for invasive thymomas (p = 0.0002); 73% for diploid but only 38% for aneuploid cases (p = 0.005); 88% for thymomas with 5.75 AgNORs per cell or fewer but only 34% for thymomas with more than 5.75 AgNORs per cell (p < 0.0001). On multivariate survival analysis, tumor stage (p < 0.001) and AgNOR counts (p = 0.009) retained independent prognostic significance. The 16 patients with predominantly lymphocytic type and 5.75 AgNORs per cell or fewer were all alive at the end of the observation period. In conclusion, the histologic type of the American classification and the proliferative activity evaluated by AgNOR analysis are the best predictors of long-term survival for patients with thymoma. Both predictors can be easily evaluated in the same histologic section, are highly reproducible, and permit identification of a group of patients with a favorable outcome regardless of other clinicopathological features.

BACKGROUND The prognosis of thymoma is related mainly to the tumor stage. The prognostic value of argyrophilic nucleolar organizer regions (AgNORs) has been demonstrated in several human neoplasias. Ninety primary thymomas were investigated retrospectively to assess whether AgNOR analysis could offer additional prognostic information. METHODS Sections from surgically resected thymomas, routinely fixed in formol and embedded in paraffin, were stained with the argyrophilic method of Ploton. The mean number of AgNORs in the nuclei of 100 tumor cells (AgNOR counts) was calculated for each case. The association between AgNOR counts and survival was assessed by means of uni- and multivariate survival analyses. RESULTS On univariate analysis, AgNOR counts were associated significantly with 5- and 10-year survival rates (95% and 90%, respectively, for thymomas with 5.58 or fewer AgNORs per cell, but only 55% and 44%, respectively, for tumors with more than 5.58 AgNORs per cell; P < 0.0001). Histologic subtypes of the American classification (P = 0.0006) and clinical stage (P < 0.0001) also were correlated with prognosis. The multivariate survival analysis showed that AgNOR counts (P = 0.001), clinical stage (P < 0.001), and age (P = 0.011) were independent prognostic variables. AgNOR counts were associated with histologic subtypes in the American (P = 0.0001) and European (P = 0.005) classifications and with the clinical stage (P < 0.0001). Moreover, thymoma cells and intermingling lymphocytes showed different numbers of AgNORs and patterns of AgNOR distribution. CONCLUSIONS Analysis of argyrophilic nucleolar organizer regions provides useful prognostic information for patients with thymomas and offers an exact evaluation of the proliferative activity of the neoplastic cells even for thymomas with prominent lymphocytic infiltration.

Argyrophilic nucleolar organizer region (Ag-NOR) analysis, proliferating cell nuclear antigen (PC-NA/PC10) and MIB-1 immunohistochemistry, nuclear morphometry and DNA flow cytometry have been performed on formalin-fixed, paraffin-embedded biopsies from 50 patients with transitional cell carcinoma of the urinary bladder. The mean AgNOR count was 6.01 for the 17 grade 1 (G1), 7.59 for the 21 G2 and 13.33 for the 12 G3 carcinomas (p0.001). The mean PCNA score was 15.03% for G1, 24.04% for G2 and 40.01% for G3 cases (p0.001). The mean MIB-1 score was 11.31% for G1, 17.09% for G2 and 34.47% for G3 carcinomas (p0.001). The mean nuclear area was 35.53 microns2 for G1, 38.65 microns2 for G2 and 83.62 microns2 for G3 cases (p0.001). Aneuploidy rates were significantly higher (91.7%) in G3 than in G2 (42.9%, p0.01) or G1 cases (47.1%, p0.05) but not different for G1 versus G2 cases (p = 0.94). While many overlaps of values were seen between G1 and G2 tumours, no overlaps were found between G3 and G1/G2 tumours. Significant differences of values were also found between pTa and invasive tumours (p0.0001 for AgNOR count and PCNA score; p0.001 for MIB-1 score and mean nuclear area; p0.01 for DNA ploidy); however many overlaps were seen. Our findings indicate that the quantitative parameters obtained with different methods are associated with histological grade of bladder urotheliomas and may improve the grading reproducibility. In addition, the absence of overlaps between G3 and G2/G1 carcinomas supports the tendency to classify bladder urotheliomas in only two categories of malignancy.

The argyrophilic nucleolar organizer regions (AgNORs) were analysed in bone marrow biopsies from 80 patients with multiple myeloma (MM) at presentation. The mean AgNOR number per MM cell (AgNOR counts) and their distribution within the nucleus (configuration) were assessed. AgNOR counts were significantly associated with several recognized prognostic factors: Durie and Salmon clinical staging system (p = 0.02), percentage of plasma cells (PCs) in aspirates (p = 0.01) and in bone marrow biopsies (p = 0.0000), pattern of bone marrow involvement (p = 0.0003), calcaemia (p = 0.0005) and creatininaemia (p = 0.0003). AgNOR counts were also associated with the degree of PC differentiation (p = 0.0000). A single central cluster of 2-3 large-sized AgNORs (configuration A) was evident in most G1 MM; one cluster of 4-5 medium-sized dots or two clusters of 2-4 dots (configuration B) were seen in most G2 MM; many small-sized, scattered dots were present in G3 MM (configuration C). AgNOR counts and configuration were related to the prognosis: in the univariate analysis, the 5 year survival rate was 7% for cases with4.5 AgNORs/cell and 46% for cases withor = 4.5 AgNORs/cell (p = 0.01), 53% for configuration A, 12% for configuration B and 0% for configuration C (p = 0.0000). AgNOR counts (p = 0.02) and configuration (p = 0.000) were independent prognostic variables in the multivariate analysis. The AgNOR counts were significantly higher in "fulminant myeloma" than in less aggressive cases (p = 0.002). The plasma cell labelling index (LI%), evaluated in 44 MM patients, showed significant correlation with prognosis: the 5 year survival rate was 51% for LI%or = 1 and 17% for LI%1 (p = 0.02). More than 70% of patients with low LI% had low AgNOR counts and more than 70% of patients with high LI% had high AgNOR counts (p = 0.007). AgNOR counts and configuration reflect the myeloma cell mass, the degree of differentiation and the kinetics of the myeloma cells. They offer an exact evaluation of the tumour characteristics and can be useful additional parameters for MM prognosis.