Gastrointestinal stromal tumor in Brazil: Clinicopathology, immunohistochemistry, and molecular genetics of 513 cases

The aim of the present study was to evaluate the clinicopathological, immunohistochemical, and molecular genetic features of gastrointestinal stromal tumors in Brazil and compare them with cases from other countries. Five hundred and thirteen cases were retrospectively analyzed. HE-stained sections and clinical information were reviewed and the immunohistochemical expression of CD117, CD34, smooth-muscle actin, S-100 protein, desmin, CD44v3 adhesion molecule, p53 protein, epidermal growth factor receptor, and Ki-67 antigen was studied using tissue microarrays. Mutation analysis of KIT and platelet-derived growth factor receptor-alpha genes was also performed. There was a slight female predominance (50.3%) and the median age at diagnosis was 59 years. The tumors were mainly located in the stomach (38.4%). Immunohistochemistry showed that CD117 was expressed in 95.7% of cases. Epidermal growth factor receptor expression was observed in 84.4% of tumors. p53 protein expression was found only in 2.6% of cases but all belonged to the high-risk group for aggressive behavior according to the National Institutes of Health consensus approach. No CD44v3 adhesion molecule expression was detected. KIT exon 11 mutations were the most frequent (62.2%). The present data confirm that gastrointestinal stromal tumors in Brazilian patients do not differ from tumors occurring in other countries.

Mitotic figure counts are significantly overestimated in resection specimens of invasive breast carcinomas.

Several authors have demonstrated an increased number of mitotic figures in breast cancer resection specimen when compared with biopsy material. This has been ascribed to a sampling artifact where biopsies are (i) either too small to allow formal mitotic figure counting or (ii) not necessarily taken form the proliferating tumor periphery. Herein, we propose a different explanation for this phenomenon. Biopsy and resection material of 52 invasive ductal carcinomas was studied. We counted mitotic figures in 10 representative high power fields and quantified MIB-1 immunohistochemistry by visual estimation, counting and image analysis. We found that mitotic figures were elevated by more than three-fold on average in resection specimen over biopsy material from the same tumors (20±6 vs 6±2 mitoses per 10 high power fields, P=0.008), and that this resulted in a relative diminution of post-metaphase figures (anaphase/telophase), which made up 7% of all mitotic figures in biopsies but only 3% in resection specimen (P<0.005). At the same time, the percentages of MIB-1 immunostained tumor cells among total tumor cells were comparable in biopsy and resection material, irrespective of the mode of MIB-1 quantification. Finally, we found no association between the size of the biopsy material and the relative increase of mitotic figures in resection specimen. We propose that the increase in mitotic figures in resection specimen and the significant shift towards metaphase figures is not due to a sampling artifact, but reflects ongoing cell cycle activity in the resected tumor tissue due to fixation delay. The dwindling energy supply will eventually arrest tumor cells in metaphase, where they are readily identified by the diagnostic pathologist. Taken together, we suggest that the rapidly fixed biopsy material better represents true tumor biology and should be privileged as predictive marker of putative response to cytotoxic chemotherapy.

Pattern of recurrence of early breast cancer is different according to intrinsic subtype and proliferation index.

INTRODUCTION Recurrence risk in breast cancer varies throughout the follow-up time. We examined if these changes are related to the level of expression of the proliferation pathway and intrinsic subtypes. METHODS Expression of estrogen and progesterone receptor, Ki-67, human epidermal growth factor receptor 2 (HER2), epidermal growth factor receptor (EGFR) and cytokeratin 5/6 (CK 5/6) was performed on tissue-microarrays constructed from a large and uniformly managed series of early breast cancer patients (N = 1,249). Subtype definitions by four biomarkers were as follows: luminal A (ER + and/or PR+, HER2-, Ki-67 <14), luminal B (ER + and/or PR+, HER2-, Ki-67 ≥14), HER2-enriched (any ER, any PR, HER2+, any Ki-67), triple-negative (ER-, PR-, HER2-, any Ki-67). Subtype definitions by six biomarkers were as follows: luminal A (ER + and/or PR+, HER2-, Ki-67 <14, any CK 5/6, any EGFR), luminal B (ER + and/or PR+, HER2-, Ki-67 ≥14, any CK 5/6, any EGFR), HER2-enriched (ER-, PR-, HER2+, any Ki-67, any CK 5/6, any EGFR), Luminal-HER2 (ER + and/or PR+, HER2+, any Ki-67, any CK 5/6, any EGFR), Basal-like (ER-, PR-, HER2-, any Ki-67, CK5/6+ and/or EGFR+), triple-negative nonbasal (ER-, PR-, HER2-, any Ki-67, CK 5/6-, EGFR-). Each four- or six-marker defined intrinsic subtype was divided in two groups, with Ki-67 <14% or with Ki-67 ≥14%. Recurrence hazard rate function was determined for each intrinsic subtype as a whole and according to Ki-67 value. RESULTS Luminal A displayed a slow risk increase, reaching its maximum after three years and then remained steady. Luminal B presented most of its relapses during the first five years. HER2-enriched tumors show a peak of recurrence nearly twenty months post-surgery, with a greater risk in Ki-67 ≥14%. However a second peak occurred at 72 months but the risk magnitude was greater in Ki-67 <14%. Triple negative tumors with low proliferation rate display a smooth risk curve, but with Ki-67 ≥14% show sharp peak at nearly 18 months. CONCLUSIONS Each intrinsic subtype has a particular pattern of relapses over time which change depending on the level of activation of the proliferation pathway assessed by Ki-67. These findings could have clinical implications both on adjuvant treatment trial design and on the recommendations concerning the surveillance of patients.

Quantitative analysis of postnatal neurogenesis and neuron number in the macaque monkey dentate gyrus

The dentate gyrus is one of only two regions of the mammalian brain where substantial neurogenesis occurs postnatally. However, detailed quantitative information about the postnatal structural maturation of the primate dentate gyrus is meager. We performed design-based, stereological studies of neuron number and size, and volume of the dentate gyrus layers in rhesus macaque monkeys (Macaca mulatta) of different postnatal ages. We found that about 40% of the total number of granule cells observed in mature 5-10-year-old macaque monkeys are added to the granule cell layer postnatally; 25% of these neurons are added within the first three postnatal months. Accordingly, cell proliferation and neurogenesis within the dentate gyrus peak within the first 3 months after birth and remain at an intermediate level between 3 months and at least 1 year of age. Although granule cell bodies undergo their largest increase in size during the first year of life, cell size and the volume of the three layers of the dentate gyrus (i.e. the molecular, granule cell and polymorphic layers) continue to increase beyond 1 year of age. Moreover, the different layers of the dentate gyrus exhibit distinct volumetric changes during postnatal development. Finally, we observe significant levels of cell proliferation, neurogenesis and cell death in the context of an overall stable number of granule cells in mature 5-10-year-old monkeys. These data identify an extended developmental period during which neurogenesis might be modulated to significantly impact the structure and function of the dentate gyrus in adulthood.

Tumor cell budding and laminin-5 expression in colorectal carcinoma can be modulated by the tissue micro-environment.

Expression of laminin-5 alpha3, beta3 and gamma2 protein subunits was investigated in colorectal adenocarcinomas using immunostaining and confocal microscopy. The laminin-5 heterotrimer was found in basement membranes and as extracellular deposits in tumor stroma. In contrast to the alpha3 subunit, which was under-expressed, the gamma2 and beta3 subunits were detected in the cytoplasm of carcinoma cells dissociating (budding) from neoplastic tubules, suggestive of focal alterations in laminin-5 assembly and secretion. Laminin-5 gamma2 or beta3 subunit-reactive budding carcinoma cells expressed cytokeratins but not vimentin; they did not proliferate and were not apoptotic. Furthermore, expression of laminin-5 gamma2 and beta3 subunits in budding cells was associated with focal under-expression of the E-cadherin-beta-catenin complex. Results from xenograft experiments showed that budding activity in colorectal adenocarcinomas could be suppressed when these tumors grew at ectopic s.c. sites in nude mice. In vitro, cultured colon carcinoma cells, but not adenoma-derived tumor cells, shared the laminin-5 phenotype expressed by carcinoma cells in vivo. Using colon carcinoma cell lines implanted orthotopically and invading the cecum of nude mice, the laminin-5-associated budding was restored, indicating that this phenotype is not only determined by tumor cell properties but also dependent on the tissue micro-environment. Our results indicate that both laminin-5 alpha3 subunit expression and cell-cell cohesiveness are altered in budding carcinoma cells, which we consider to be actively invading. We propose that the local tissue micro-environment contributes to these events.

Human CD8(+) T cells expressing HLA-DR and CD28 show telomerase activity and are distinct from cytolytic effector T cells.

Cycling lymphocytes may express the enzyme telomerase which is involved in maintenance of telomere length and cell proliferation potential. In CD8(+) T cells freshly isolated from peripheral blood, we found that in vivo cycling cells expressed HLA-DR. Furthermore, CD28-positive cells are known to have longer telomeres than CD28-negative T cells. Therefore we used HLA-DR- and CD28-specific antibodies to sort CD8(+) T cells and measure telomerase activity ex vivo. Relatively high levels of telomerase activity were found in HLA-DR/CD28 double-positive cells. In contrast, HLA-DR-negative and CD28-negative cells had almost no telomerase activity. In summary, HLA-DR expression correlates with proliferation, and CD28 expression with proliferative potential. We have previously identified that ex vivo cytolytic CD8(+) T cells are CD56 (NCAM) positive. Here we show that HLA-DR(+) cells were rarely CD56(+) and vice versa. This demonstrates that telomerase-expressing and cytolytic CD8(+) T cells can be separated on the basis of the cell surface markers HLA-DR and CD56. Thus, activated CD8(+) T cells specialize and exert distinct functions correlating with surface molecule expression.

Esôfago de Barrett: aspectos fisiopatológicos e moleculares da seqüência metaplasia-displasia-adenocarcinoma - artigo de revisão

The Barrett's esophagus (BE) is defined as endoscopically visible columnar mucosa at the distal esophagus, of any extension, proved to harbor intestinal metaplasia on biopsy, highlighted by the presence of goblet cells. BE denotes long-standing gastroesophageal reflux disease (GERD) and is an important risk factor for the development of esophageal adenocarcinoma (EAC). Therefore, these patients must be on follow-up, in order to diagnose cancer early. BE patients have frequent alterations in esophageal physiologyc studies. Alkaline duodenogastroesophageal reflux seems to have important role. The development BE occurs in steps, initially with formation of cardiac type mucosa subsequent intestinalization. Futher progression can follow a sequence, from low grade dysplasia, to high grade dysplasia and esophageal adenocarcinoma. Current follow-up is based on the presence of dysplasia. It has limitations, grouping patients heterogeneously. Different steps of carcinogenesis have been studied looking for an ideal prognostic marker. Uncontrolled proliferative activity, apoptosis inhibition, angiogenesis, tissue invasion and metastases formation are all implicated in cancer origin. Some cycle cell molecules have been studied in BE, such as retinoblastoma protein, ciclins, kinase dependent ciclins and cell cycle inhibitors. The P53 protein is one of the most investigated in the metaplasia-adenocarcinoma progression. Growth Factors, apoptotic proteins, telomers and DNA ploidy have also been searched. Increased proliferative activity has been implicated in Barrett's carcinogenesis and the Ki-67 antigen, through imunohistochemical analysis, has become the the method of choice. Present in the nucleus, it is found in proliferative cells only. Some studies suport association between Ki-67 activity and the metaplasia-dysplasia-adenocarcinoma sequence.The results, however, are inconclusive and research should follow this way.

Comparison of nuclear grade and immunohistochemical features in situ and invasive components of ductal carcinoma of breast

PURPOSE:To compare the prognostic and predictive features between in situ and invasive components of ductal breast carcinomas. METHODS:We selected 146 consecutive breast samples with ductal carcinoma in situ (DCIS) associated with adjacent invasive breast carcinoma (IBC). We evaluated nuclear grade and immunohistochemical expression of estrogen receptor (ER), progesterone receptor (PR), human epidermal growth factor receptor 2 (HER2), cytokeratin 5/6 (CK5/6), and epidermal growth factor receptor (EGFR) in both components, in situ and invasive, and the Ki-67 percentage of cells in the invasive part. The DCIS and IBC were classified in molecular surrogate types determined by the immunohistochemical profile as luminal (RE/PR-positive/ HER2-negative), triple-positive (RE/RP/HER2-positive), HER2-enriched (ER/PR-negative/HER2-positive), and triple-negative (RE/RP/HER2-negative). Discrimination between luminal A and luminal B was not performed due to statistical purposes. Correlations between the categories in the two groups were made using the Spearman correlation method. RESULTS:There was a significant correlation between nuclear grade (p<0.0001), expression of RE/RP (p<0.0001), overexpression of HER2 (p<0.0001), expression of EGFR (p<0.0001), and molecular profile (p<0.0001) between components in situ and IBC. CK 5/6 showed different distribution in DCIS and IBC, presenting a significant association with the triple-negative phenotype in IBC, but a negative association among DCIS. CONCLUSIONS: Our results suggest that classical prognostic and predictive features of IBC are already determined in the preinvasive stage of the disease. However the role of CK5/6 in invasive carcinoma may be different from the precursor lesions.

Evaluation of epithelial cell proliferating activity and fibroblast nuclear kariometry in recurrent pterygium treated with mitomycin C

OBJETIVO: Avaliar a eficácia da mitomicina C (MMC) na prevenção da recorrência quando previamente utilizada no transplante autólogo de conjuntiva (TAC). A avaliação da proliferação celular epitelial pelo antígeno Ki-67 e a cariometria do núcleo dos fibroblastos foram usados como auxiliares na avaliação do tratamento. MÉTODOS: Vinte e nove pacientes com pterígio recidivado foram divididos em três grupos: Grupo (G) 1-TAC e colírio placebo (PLA); G2-TAC, MMC 0,015% subconjuntival e PLA; G3-TAC e colírio de MMC 0,02%. A imuno-histoquímica foi realizada no tecido excisado para o antígeno Ki-67, como a cariometria dos núcleos dos fibroblastos (divididos em lado nasal e temporal). A cariometria dos núcleos dos fibroblastos foi avaliada de acordo com os seguintes parâmetros: volume (Vl) e área (Ar) em pelos menos 50 células por paciente. RESULTADOS: A porcentagem das células epiteliais positivas para o antígeno Ki-67 no lado nasal e temporal após o tratamento dos três grupos estudados foi: nasal (3,30% G1, 4,49% G2 e 3,38% G3) e temporal (3,30% G1, 4,46% G2 e 4,14% G3) não mostrando diferença significativa. A cariometria do núcleo dos fibroblastos foi: Vl nasal (792,1 µ3 G1, 605,1 µ3 G2, e 549,9 µ3 G3) e a Ar (100,58 µ2 G1, 83,13 µ2 G2, e 78,41 µ2 G3). Os três grupos mostraram uma diferença significativa p=0,039 e p=0,035, respectivamente do Vl e da Ar no lado nasal. Após seis meses de tratamento, os três grupos apresentaram a seguinte taxa de recidiva: 22,22% G1, 18,18%, G2 e 33,33% G3 respectivamente. CONCLUSÃO: O uso da MMC não interferiu nas células epiteliais positivas para o antígeno Ki-67 no pterígio recidivado, mas acarretou diminuição do volume e área dos núcleos dos fibroblastos no lado nasal do pterígio. As células epiteliais positivas para o antígeno Ki-67 parecem não ter relação com a recidiva do pterígio após seis meses da cirurgia. Outros estudos devem ser realizados para avaliar o papel da proliferação das células epiteliais na recorrência do pterígio.

Computer-assisted analysis of cell proliferation markers in oral lesions

Abnormalities in any component of the cell cycle regulatory machine may result in oral. cancer, and markers of cell proliferation have been used to determine the prognosis of tumor progression. The aim of this study was to determine whether silver-stained nucleolar organizer region (AgNOR) and Ki-67 measurements could improve the assessment of growth rates in oral lesions. Eighty-three oral biopsies were studied, 20 of which were classified as fibrous inflammatory hyperplasia (FIH), 40 as leukoplakia (LKP) and 23 as oral. squamous cell carcinoma (OSCC). Within the LKP group, 22 out of 29 biopsies were diagnosed as non-dysplastic leukoplakia (LK) and 18 as dysplastic teukoptakia (DLK), presenting discrete, moderate and severe dysplasia. Ki-67 immunotabeting of the lesions increased steadily in the following order: FIH, DLK, LK and OSCC, indicating that Ki-67 is a good marker for predicting the protiferative fraction among benign, premalignant and malignant oral lesions. The median values of AgNOR parameters indicate that the morphometric index gives better results regarding the proliferative rate than the numerical one. A series of linear regressions between AgNOR parameters and Ki-67 showed positive associations. We conclude that a combination of Ki-67 and morphometric AgNOR analyses could be used as an aid in the determination of the protiferative status of oral epithelial. cells in oral cancer. (C) 2007 Elsevier GmbH. All rights reserved.

Quantitative cell-cycle protein expression in oral cancer assessed by computer-assisted system

The knowledge of cell-cycle control has shown that the capacity of malignant growth is acquired by the stepwise accumulation of defects in specific genes regulating cell growth. Histologic diagnosis might be improved by a quantitative evaluation of more specific diagnosis biomarkers, which could help to precisely identify pre-malignant and malignant oral lesions. The aim of the present study is to evaluate whether computer-based quantitative assessment of p53, PCNA and Ki-67 immunohistochemical expression, could be used clinically to foresee the risk of oral malignant transformation. This retrospective study was carried out in ninety-five oral biopsies, 27 were classified as fibrous inflammatory hyperplasia, 40 as leukoplakia and 28 as oral squamous cell carcinoma. Sixteen out of the 40 leukoplakia were diagnosed as non-dysplastic leukoplakia, the other 24 being dysplastic leukoplakia, of which 50.0% were classified as moderate to severe dysplasia. Comparison of the four groups of oral tissues showed significant rises in p53 and Ki-67 positivity index, which increased steadily in the order benign, pre-malignant, and malignant. In contrast, it was not possible to relate higher PCNA levels with pre-malignant and malignant oral lesions. We therefore conclude that PCNA immunohistochemistry expression is probably an inappropriate marker to identify oral carcinogenesis, whereas joint quantitative evaluation of p53 and Ki-67, appears to be useful as a tumor marker, providing a pre-diagnostic estimate of the potential for cell-cycle deregulation of the oral proliferate status.

Human papillomavirus and Epstein-Barr virus infection, p53 expression, and cellular proliferation in laryngeal carcinoma

Laryngeal carcinomas are aggressive neoplasms with controversial association with the human papillomavirus (HPV) and Epstein-Barr virus (EBV). So far, the impairment of p53 protein function and its impact on cellular proliferation has not been studied adequately in these tumors. In this work, molecular biologic techniques were used to assess the frequency of HPV and EBV in 110 squamous cell carcinomas of the larynx. In addition, accumulation of p53 and Ki-67 cell proliferation antigen expression in malignant cells was assessed by immunohistochemical analysis. High-grade HPV was found in 37.3% of cases, and none had demonstrable EBV infection. Accumulation of p53 was found in 78.2% of the cases, and it was related to a high Ki-67 labeling index and higher histologic grade. The results demonstrate association of HPV with more than one third of laryngeal carcinomas studied, mainly glottic tumors. Tumors with increased cell proliferation were more frequently high grade, with p53 accumulation and lymph node metastasis. © American Society for Clinical Pathology.

Long-term ethanol consumption promotes hepatic tumorigenesis but impairs normal hepatocyte proliferation in rats

Chronic and excessive alcohol consumption has been related to an increased risk of several cancers, including that of the liver; however, studies in animal models have yet to conclusively determine whether ethanol acts as a tumor promoter in hepatic tumorigenesis. We examined whether prolonged alcohol consumption could act as a hepatic tumor promoter after initiation by diethylnitrosamine (DEN) in a rat model. Male Sprague-Dawley rats were injected with 20 mg DEN/kg body weight 1 wk before introduction of either an ethanol liquid diet or an isoenergic control liquid diet. Hepatic pathological lesions, hepatocyte proliferation, apoptosis, PPARα and PPARγ, and plasma insulin-like growth factor 1 (IGF-1) levels were assessed after 6 and 10 mo. Mean body and liver weights, plasma IGF-1 concentration, hepatic expressions of proliferating cellular nuclear antigen and Ki-67, and cyclin D1 in ethanol-fed rats were all significantly lower after 10 mo of treatment compared with control rats. In addition, levels of hepatic PPARγ protein, not PPARα, were significantly higher in the ethanol-fed rats after prolonged treatment. Although ethanol feeding also resulted in significantly fewer altered hepatic foci, hepatocellular adenoma was detected in ethanol-fed rats at 10 mo, but not in control rats given the same dose of DEN. Together, these results indicate that chronic, excessive ethanol consumption impairs normal hepatocyte proliferation, which is associated with reduced IGF-1 levels, but promotes hepatic carcinogenesis. © 2011 American Society for Nutrition.

Role of adhesion molecules and proliferation hyperplasic, pre neoplastic and neoplastic lesions in canine prostate

E-cadherin and beta-catenin are component of adherens junctions in epithelial cells. Loss of these proteins have been associated with progression of prostatic diseases. We performed immunohistochemistry for E-cadherin, beta-catenin and Ki-67 on canine prostatic lesions. We analyzed the expression of these antibodies in benign prostatic hyperplasia (BPH, n = 22), in pre neoplastic lesions Prostatic Intra-epithelial Neoplasia (PIN), n = 3 and Prostatic Inflammatory Atrophy (PIA), n = 7 and prostate carcinoma (PC, n = 10). In this study, a membranous expression of E-cadherin and beta-catenin and nuclear expression of Ki-67 antigen were demonstrated. The proliferative index was statistically different between carcinomas and BPH and carcinomas and pre-neoplastic lesions. Like in men, the reduction of E-cadherin and increase of Ki-67 expression in neoplastic lesions in dog prostate may be related to the carcinogenic process in this gland. © 2013 Asian Network for Scientific Information.