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AimsNovel prognostic markers are warranted for gastrointestinal stromal tumours. Caveolin-1 is a multifunctional protein that proved to be associated with outcome in multiple tumour types. Aim of this study was to investigate Caveolin-1 expression and prognostic efficacy in a series of gastrointestinal stromal tumours.MethodsCaveolin-1 expression was assessed by immunohistochemistry in a retrospective series of 66 gastrointestinal stromal tumours representative of the different molecular subtypes. Correlations with clinical, histopathological and molecular features were investigated. Statistical analyses were performed as appropriate.ResultsThirty-five cases out of 66 (53.0%) expressed Caveolin-1. Presence of Caveolin-1 expression correlated with favourable histopathologic and clinical traits, including a lower mitotic count (p=0.003) and lower relapse rate (p=0.005). Caveolin-1 expression also resulted associated with the presence ofPDGFRAmutations (p=0.010). Outcome analyses showed a favourable prognostic significance of Caveolin-1 expression in terms of relapse-free survival (HR=0.14; 95% CI=0.03 to 0.63) and overall survival (HR=0.29; 95% CI=0.11 to 0.74), even after adjusting for the mutational subgroup (relapse-free survival: HR=0.14, 95% CI=0.04 to 0.44; overall survival: HR=0.29, 95% CI=0.11 to 0.51) and imatinib treatment (relapse-free survival: HR=0.14, 95% CI=0.02 to 0.81; overall survival: HR=0.29, 95% CI=0.17 to 0.48).ConclusionCaveolin-1 represents a novel prognostic marker in gastrointestinal stromal tumours. Further studies are warranted to validate these results and to explore the mechanisms linking Caveolin-1 expression with thePDGFRAoncogenic pathway.

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.

Systemic mastocytosis (SM) is a rare, heterogeneous and progressive disease, characterized by the accumulation of atypical mast cells in various organs, including the gastrointestinal tract. Gastrointestinal symptoms are present in up to 80% of patients with SM, the most common being abdominal pain, diarrhea, nausea and vomiting. Up to 50% of patients with SM do not have classical skin lesions at presentation, and in these patients the diagnosis of SM can be difficult for years. Here we report a case of SM that initially mimicked inflammatory bowel disease, although the patient showed poor response to steroid therapy. The right diagnosis was made only on the surgical specimen obtained after emergency surgery for intestinal obstruction. SM should therefore be considered in the diagnostic approach in patients with gastrointestinal symptoms not attributable to other pathologies and in cases of suspected inflammatory bowel disease with unusual course.

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.

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.

The correlation between the bromodeoxiuridine (BrdU)-labelling index (LI) of plasma cells and a new proliferation marker, the Argyrophilic Nucleolar Organizer Regions (AgNORs), was investigated in 44 myeloma patients at diagnosis. A preliminary analysis was made to verify the reproducibility of the assessment of plasma cell infiltration (PC%) in bone marrow aspirates, used to collect cells for LI determination, and in bone marrow biopsies, used for AgNORs evaluation. Although an overall good correlation was observed between PC% in biopsies and aspirates (r = 0.58, p = 0.001), the ratio between PC% in biopsies and in aspirates ranged from 0.35 to 7.5. Only 17 patients (38.6%) were within the 0.5–1.5 range. A positive correlation between LI and AgNORs was observed in these patients (r = 0.68, p = 0.003), whereas the correlation was lost in patients with higher ratio between PC% in biopsies and in aspirates (r = 0.08, p = 0.69). The prognostic significance of AgNORs was confirmed by survival analysis, showing a reduced survival for patients with high (>4.4) AgNOR counts (14 months vs 35 months, p = 0.004). The AgNORs analysis therefore allows the simultaneous evaluation of myeloma cell infiltration, degree of differentiation and kinetics of growth in bone marrow biopsies. AgNOR counts deserve to be included in the procedures for diagnosis and prognostic evaluation of myeloma patients.

A 55-year-old man with a history of acute myeloid leukaemia treated with hematopoietic stem cell transplantation and with a 5-year history of bisphosphonate-related osteonecrosis of the jaws, following 12 cycles of intravenous zoledronic acid therapy, presented in December 2009 with a history of increasingly severe unilateral lower jaw pain. Oral examination revealed, as previously, exposed bone in the left mandible, but also a new exophytic mass on the lower-left buccal mucosa. Biopsy confirmed a diagnosis of oral squamous cell carcinoma. To the best of our knowledge, this is the first report of an oral squamous cell carcinoma that appeared adjacent to an area of osteochemonecrosis.

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.

To identify markers of non-response to neoadjuvant chemotherapy (NAC) that could be used in the adjuvant setting. Sixteen pathologists of the European Working Group for Breast Screening Pathology reviewed the core biopsies of breast cancers treated with NAC and recorded the clinico-pathological findings (histological type and grade; estrogen, progesterone receptors, and HER2 status; Ki67; mitotic count; tumor-infiltrating lymphocytes; necrosis) and data regarding the pathological response in corresponding surgical resection specimens. Analyses were carried out in a cohort of 490 cases by comparing the groups of patients showing pathological complete response (pCR) and partial response (pPR) with the group of non-responders (pathological non-response: pNR). Among other parameters, the lobular histotype and the absence of inflammation were significantly more common in pNR (p

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.

The time-honoured use of formalin, both as a preserver and fixative for histological processing is encountering increasing criticisms because of toxicity and environmental concerns. Moreover, the declaration recently issued by the International Agency for Research on Cancer [1] which classified formaldehyde as a class 1 carcinogen has definitely increased the request by health authorities, technicians and practicing pathologists to reduce exposure. Such requests contrast with the considerable advantages offered by this safe, chip, reliable fixative. Although it should be acknowledged that in modern pathology laboratories, visitors are not any more met by the permeating flavour of a stingy scent because formalin processing is mostly carried out under aspiration hoods, a critical passage is still represented by the transfer of tissues from the surgical theatre to the pathology lab. Apart from the small biopsies, which are directly collected into pre-filled containers and cause no concern, problems are encountered with the immersion of large specimens and organs into large boxes to be filled with formalin. A list of such problems follows

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.

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.

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.

The prognostic significance of the argyrophilic nucleolar organizer regions (AgNORs) in tumour pathology is still a matter of debate. A prospective study was performed in a series of renal cell carcinomas to clarify the prognostic value of AgNOR counting. Sections from 21 renal cell carcinomas were stained in 1990 with the method of Ploton. Black dots within the nucleus from 200 tumour cells were counted: the mean AgNOR count for the whole series was 6.13, the median 5.94 and the SD 1.78. Patients were then followed up for at least 6 years or to death: at the time of the survival analysis (June 1996), 13 patients were alive without evidence of recurrence or metastasis, 6 had died of the disease and 2 of myocardial infarction. All the patients with 5.94 AgNORs per cell or fewer were alive at 6-year follow-up, while only 60% of patients with more than 5.94 AgNORs per cell survived (p=0.01). In the multivariate analysis, only AgNOR count (p=0.015) retained an independent prognostic significance. With the limitation due to the small number of cases, this prospective study clearly indicates that AgNOR count has a significant prognostic role, at least in renal cell carcinoma.

Background and aim: Translational data suggest that nucleoside transporters, in particular human equilibrative nucleoside transporter 1 (hENT1), play an important role in predicting clinical outcome after gemcitabine chemotherapy for several types of cancer. The aim of this study was to retrospectively determine patients' outcome according to the expression of hENT1 in tumoral cells of patients receiving gemcitabine-based therapy. Materials and Methods: The immunohistochemistry analysis was performed on samples from thirty-one patients with unresectable biliary tract cancer (BTC) consecutively treated with first line gemcitabine-based regimens. Results: Positive hENT1 staining patients were 21 (67.7%); negative hENT1 staining patients were 10 (32.3%). Statistical analysis revealed no association between baseline characteristics, toxicities and tumor response to gemcitabine and hENT1 levels. In the univariate analysis, HENT1 expression was significantly correlated with time to progression (TTP) (p=0.0394; HR 2.902, 95%CI 1.053-7.996). The median TTP was 6.33 versus 2.83 months, respectively in patients with positive versus negative hENT1 staining. Moreover, patients with positive hENT1 expression showed a longer median overall survival when compared with patients with low hENT1 expression (14 versus 7 months, respectively), but this difference did not reach the statistical significance (p=0.128). Conclusions: Therefore, hENT1 may be a relevant predictive marker of benefit from gemcitabine-based therapies in patients with advanced BTC. © 2011 Bentham Science Publishers Ltd.

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.

A ganglioneuroma (GN) is the rarest and most benign of the neuroblastic tumors and originates from neural crest cells wherever sympathetic nervous tissue exists, such as in the retroperitoneum and adrenal gland. The diagnosis can be very challenging, given the rarity and asymptomatic presentation of this neoplasia, and can be achieved only by means of histological evaluation. Although benign, a few cases of metastatic GNs have been reported in the literature. The prognosis, however, seems to be excellent after surgical resection. We describe a rare case of multifocal retroperitoneal GN, diagnosed incidentally in a 46-year-old woman, with para-aortic and adrenal localizations. After intraoperative pathological diagnosis was made, complete excision of all the visible masses was performed. The postoperative period was uneventful and she was recurrence free 3 months after surgery. To our knowledge, this is the first case report of a multifocal retroperitoneal GN. Among the broad differential diagnoses of adrenal incidentalomas, an adrenal location of neuroblastic tumors should not be forgotten.

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 proliferative activity of four malignant cellular blue nevi (MCBN) was assessed in routinely fixed, paraffin-embedded material using staining for the argyrophilic nucleolar organizer regions (AgNORs), immunohistochemical staining for proliferating cell nuclear antigen (PCNA [PC10]), and DNA flow cytometry. The objective was to determine whether the evaluation of proliferative activity could represent a useful diagnostic parameter. Four cellular blue nevi (CBN), 10 melanocytic nevi (MN), four common blue nevi (BN), and 10 conventional malignant melanomas (MMs) were selected as controls. In the MCBN the mean AgNOR number, evaluated on the basis of 100 tumor cells, was 8.33 +/- 0.83; NORs were small and dispersed throughout the nucleus; the mean PCNA score was 31.93% +/- 4.4; and two of the cases were aneuploid and two diploid. In the CBN the AgNOR count was 3.69 +/- 0.56; NORs were large and mainly grouped in a central cluster; the mean PCNA score was 3.53% +/- 1.28; and three of the cases were diploid and one aneuploid. The AgNOR counts in the MCBN were significantly different from those in the CBN (P = .0002), MN (3.04; P = .00001), and BN (2.93; P = .00006), whereas they were not significantly different from those in the conventional MMs (7.64; P = .58). The PCNA (PC10) scores in the MCBN were significantly different from those in the CBN (P = .00003), MN (2.05%; P = .00001), and BN (5.06%; P = .00002), whereas they were not significantly different from those in the conventional MMs (28.9%; P = .49). In all the cases a linear relationship between AgNOR counts and PCNA scores was observed (r = .94, P = .00001). Our results indicate that AgNOR analysis and PCNA immunostaining can be regarded as useful additional parameters for the diagnosis of MCBN.

In the present study, we investigated the association of PDGFRA and KIT mutations as well as PDGFRA immunohistochemical expression with clinicopathologic features and prognosis in a series of gastrointestinal stromal tumors (GISTs). Tumor DNA from 40 GISTs was sequenced for the presence of mutations in KIT exons 9, 11, 13 and 17, and in PDGFRA exons 12 and 18. Tissue sections were stained with polyclonal anti-PDGFRA antibody. KIT mutations occurred in 26 cases. There were 13 deletions, 6 substitutions, 3 deletion-substitutions, 3 duplications and 1 insertion. Tumors with KIT deletions/insertion were large with a high mitotic index (MI), and were associated with a high rate of symptoms at diagnosis, invasion into adjacent organs, distant metastasis, relapse and a short disease-free survival (DFS). PDGFRA mutations occurred in 6 gastric GISTs. There were 4 deletions and 2 substitutions. Tumors with PDGFRA mutations were small, with a low MI and Ki67 score, and were associated with a very low rate of symptoms at diagnosis, invasion into adjacent organs and distant metastasis. PDGFRA immunopositivity was found in 23 cases: a peculiar 'dotlike' staining was found in 5 out of 6 PDGFRA mutated cases. Patients with positive PDGFRA immunostaining had a longer DFS than those with negative staining. Our data confirm that the type of KIT mutation is associated with various clinicopathologic features of GISTs, and indicate that PDGFRA mutations are associated with rather indolent tumors. PDGFRA immunopositivity reflects PDGFRA mutational status and is associated with a favorable outcome.

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.

The selection of proper tissues from formalin-fixed and paraffin-embedded tumors before diagnostic molecular testing is responsibility of the pathologist and represents a crucial step to produce reliable test results. The international guidelines suggest two cut-offs, one for the percentage and one for the number of tumor cells, in order to enrich the tumor content before DNA extraction. The aim of the present work was two-fold: to evaluate to what extent a low percentage or absolute number of tumor cells can be qualified for somatic mutation testing; and to determine how assay sensitivities can guide pathologists towards a better definition of morphology-based adequacy cut-offs. We tested 1797 tumor specimens from melanomas, colorectal and lung adenocarcinomas. Respectively, their BRAF, K-RAS and EGFR genes were analyzed at specific exons by mutation-enriched PCR, pyrosequencing, direct sequencing and real-time PCR methods. We demonstrate that poorly cellular specimens do not modify the frequency distribution of either mutated or wild-type DNA samples nor that of specific mutations. This observation suggests that currently recommended cut-offs for adequacy of specimens to be processed for molecular assays seem to be too much stringent in a laboratory context that performs highly sensitive routine analytical methods. In conclusion, new cut-offs are needed based on test sensitivities and documented tumor heterogeneity.

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.

Cell proliferative activity has been extensively investigated in head and neck tumors. Ki67/MIB-1 immunostaining, tritiated thymidine or bromodeoxyuridine labeling indices, DNA S-phase fraction, proliferating cell nuclear antigen expression, potential doubling time and analysis of the nucleolar organizer region associated proteins (AgNORs) have shown significant correlation with prognosis in 4806 cases of tumors of the oral cavity, salivary glands, pharynx and larynx. However, this was not observed in 2968 other reported cases. Discrepancies may depend on various factors: the heterogeneity of the series, which include tumors from various anatomic sites and patients treated with different therapy, and the lack of standardization of methods for assessing cell proliferation. Furthermore, none of the methods currently applied can by themselves define the actual proliferative activity, as it depends both on the proportion of cells committed to the cycle (growth fraction) and the speed of the cell cycle. Indeed, the actual proliferative activity of a tumor could well be measured by the equation [PA = Ki67 or MIB-1 scores x AgNORs], as we did in pharyngeal carcinoma. Provided that large and homogeneous series are evaluated by standardized methods, cell proliferative activity can still be regarded as an inexpensive and reliable prognostic factor in head and neck tumors.

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 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.

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.

Overexpression of the p53 protein has been retrospectively investigated in 90 surgically resected thymomas at diagnosis, using the monoclonal antibody DO7 on routinely processed specimens. p53 accumulation was compared with tumour clinicopathological features, DNA flow cytometry content and cell proliferative activity, assessed by the counts of the argyrophilic nucleolar organizer regions (AgNORs). The purpose was to verify whether the overexpression of the p53 protein could offer additional prognostic information. p53 accumulation was detected in 48 cases (53.4%). No association was found between p53 overexpression and thymoma clinicopathologic features, although p53 protein tended to be more frequently expressed in invasive (61.4%) than noninvasive cases (45.7%, p=0.19) and in DNA aneuploid (71.4%) than diploid cases (48.7%, p=0.15). A strong association was found between p53 immunostaining and AgNOR counts: p53 positive cases had higher AgNOR counts (6.34) than p53 negative thymomas (5.36, p=0.012). In univariate analysis, the 10 year survival rates were significantly higher (83%) for p53 negative than for p53 positive patients (52%, p=0.019). AgNOR counts, tumour DNA content, histological subtypes of the American classification, clinical stage and invasion were also strongly associated with survival. In the multivariate analysis, only tumour stage (p

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.

The cell proliferative activity of the clinico-pathologically heterogeneous non-Hodgkin's lymphomas (NHL) included in the intermediate grade F category of the Working Formulation (WF) was investigated. S-phase fraction with flow cytometry on cell suspensions, and Ki67 on frozen tissue sections were performed in 42 F NHL. An avidin-biotin immunocomplex method was used and 1000 cells from 10 representative fields were counted. DNA content, S-phase and Ki67 were also detected in 194 NHL covering the whole spectrum of the WF. DNA content anomalies were found in 52 of 194 NHL. Their incidence, like that of S-phase fraction and Ki67 positive cells, progressively increased from low- to high-grade. A linear correlation was found between Ki67 and S-phase (r = .59). Using the median value of proliferating cells obtained with both procedures as a cut off, two very different groups of lymphomas could be distinguished within a series of 42 F-intermediate NHL: with low and high proliferative cell activity (p < .0001) that were termed F(low) and F(high), respectively. A intermediate group was placed between them. It differed significantly from the others if Ki67 was used but only from the F(high) group if the S-phase fraction analysis was applied. No significant differences were seen when comparing F(low) with the single categories of low-grade NHL and F(high) with H high-grade NHL; no significant differences were found between F(high) and G, and between G and H categories. The existence of distinct groups of NHL in the F category, as defined by biological parameters assessing the cell proliferative activity, indicates that this category includes biologically heterogeneous lymphoma subtypes with different grades of aggressiveness. The results also indicate that the G intermediate category displays proliferation indices similar to those of H high grade category.

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.

p53 overexpression and proliferative activity were investigated in 28 squamous cell carcinomas of the pyriform sinus of the hypopharynx prior to therapy, using DO1 and MIB-1 monoclonal antibodies in routinely processed biopsies. MIB-1 scores were associated with tumour histological grade (35.4% for grade 3 versus 23.8% for grade 2 cases; p=0.008) and survival (the median of survival was 23 months for cases with MIB-1 scores less than or equal to 33.8% but 11 months only for cases with MIB-1 scores33.8%; p0.001). p53 scores were associated with tumour histological grade (56.5% for grade 3 versus 37.1% for grade 2 cases; p=0.02) and survival (median of survival 20 months for cases with p53 scores less than or equal to 56.2% versus 11 months for cases with p53 scores56.2%; p=0.002). Tumour histological grade was also correlated with prognosis (median of survival 50 months for grade 2 versus 14 months for grade 3 cases; p=0.03). In the multivariate analysis, only MIB-1 (p=0.001) and p53 scores (p=0.003) had an independent prognostic significance. A linear relationship between p53 and MIB-1 scores was observed (r=0.54; p=0.012). With the limitation due to the small number of cases, our findings indicate that p53 overexpression correlates with proliferative activity and survival in squamous cell carcinomas of the pyriform sinus, and suggest the use of p53 and MIB-1 immunostainings in the pretherapeutic assessment of the tumour aggressiveness.

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.