Protein Immunoexpression Research Articles

Prognostic impact of p53 and p63 immunoexpression in oral squamous cell carcinoma

The role of p53 and p63 proteins in the prognosis of oral squamous cell carcinoma (OSCC) is still debatable. Our aim here was to investigate the relationship between the immunoexpression of these proteins with some clinicopathologic parameters of prognostic significance in OSCC. Formalin-fixed paraffin-embedded sections from 106 patients were used for study together with the following data: primary site, histologic differentiation, recurrences, metastasis, disease-free survival and overall survival (OS). In OSCCs, the positive rate for p63 protein immunoexpression (87.8%) was higher than p53 (52.8%). p53 expression correlated with metastasis. Tumors negative for p53 and with strong intensity for p63 expression had a significantly higher OS. p53 overexpression is associated with a larger number of metastases and is correlated with a poor outcome as well as decreased intensity in p63 immunoexpression.

Developmental expression of cell cycle regulators in the baboon fetal adrenal gland

Although the human and the nonhuman primate fetal adrenal glands undergo a highly unique pattern of cortical zone-specific intrauterine growth and development, studies of the regulatory components of the cell cycle responsible for this growth have not been conducted. Therefore, the present study determined expression of the cell cycle regulators, cyclin D1 and cyclin E, and their cyclin-dependent kinases, Cdk2, Cdk4, and Cdk6, and Ki67 a marker of cell proliferation within the baboon fetal adrenal cortex during advancing stages of gestation. Fetal adrenal glands were obtained on days 60 (early), 100 (mid), and 160-170 (late) of gestation (term = 184 days). Mean (+/- s.e.) cyclin D1 mRNA levels, determined by RT-PCR and expressed relative to 18S rRNA, were similar at early (0.85 +/- 0.09) and mid (1.04 +/- 0.08) gestation, then decreased (P < 0.001, ANOVA) approximately 50% by late gestation (0.57 +/- 0.04). Cyclin E mRNA levels were also similar at early (2.03 +/- 0.07) and mid (1.63 +/- 0.31) gestation, and decreased by 70% (P < 0.001) in late gestation (0.53 +/- 0.09). Coinciding with the decrease in cyclin D1 and cyclin E, the percentage of Ki67 positive cells in the definitive zone decreased twofold (P < 0.01) between mid (28.2 +/- 3.6) and late (13.8 +/- 1.7) gestation. The cyclin D1 and cyclin E proteins, determined by immunocytochemistry, were expressed at high levels in the definitive zone of baboon fetal adrenal gland, where they decreased between mid- and late gestation. In contrast, immunocytochemical expression of the functionally important steroidogenic enzyme Delta(5)-3beta-hydroxysteroid dehydrogenase (3beta-HSD) became abundant in the definitive and transitional zones with advancing pregnancy. However, fetal adrenal Cdk2, Cdk4, and Cdk6 mRNA levels and protein immunoexpression were similar in the baboon fetal adrenal at early-, mid-, and late gestation. In summary, expression of cyclin D1, cyclin E, and Ki67 decreased, while 3beta-HSD expression increased, in the fetal adrenal cortex, particularly in the definitive zone, between mid- and late-baboon gestation. We propose that a developmental decline in cellular proliferation permits functional differentiation of fetal adrenal cortical cells, leading to increased production of steroid hormones important for placental estrogen synthesis and maturation of organ systems within the developing fetus.

Low grade malignant peripheral nerve sheath tumor with smooth muscle differentiation

Malignant peripheral nerve sheath tumors are usually Schwann cell derived. Differentiation along mesenchymal lines is uncommon. Herein we present an example with smooth muscle differentiation occurring in the left distal forearm of a 62-year-old male with neurofibromatosis type I. Incisional biopsy of the slowly growing mass demonstrated a low-grade malignant peripheral nerve sheath tumor with strong S-100 protein immunoexpression as well as focal smooth muscle actin staining in atypical neoplastic cells. Immunostains for epithelial membrane antigen, neurofilament protein, glial fibrillary acidic protein, CD57, desmin, and myogenin were negative. The MIB-1 labeling index was 5.4%. Electron microscopy revealed arrays of microfilaments (actin myofilaments) variably associated with fusiform dense bodies, plasmalemmal micropinocytotic vesicles, and foci of external lamina (basement membrane) consistent with smooth muscle differentiation. Smooth muscle should be added to the spectrum of differentiation exhibited by malignant peripheral nerve sheath tumors. Its recognition requires exclusion of the alternative diagnosis of leiomyosarcoma.

Epithelial growth factor receptor status in primary and recurrent ovarian cancer

Success of epidermal growth factor receptor (EGFR) targeting agents in different cancer types is related to EGFR gene mutations and/or copy number gains. We investigated the EGFR gene status and protein expression by DNA mutational analysis, fluorescence in situ hybridization (FISH), and immunohistochemistry in tumor tissues from 80 patients with primary and corresponding recurrent ovarian serous carcinomas. The patients were classified into six groups with ascending EGFR gene copy numbers. EGFR amplification and high polysomy (FISH+) was present in a significant fraction of the primary (20%) and recurrent (22%) ovarian carcinomas. On mutational analysis, only one tumor with a silent EGFR mutation was observed, and this was the only carcinoma with high-level amplification. EGFR protein immunoexpression was seen in 28% of primary and 33% of recurrent carcinomas and correlated to amplification in the primary tumors (P=0.003). In recurrent carcinoma, moderate and strong EGFR expression was associated with amplification (P=0.034). These molecular events potentially have impact on the responsiveness to EGFR targeting agents in ovarian cancer.

Paraffin-Embedded Cell Line Microarray (PECLIMA): Development and Validation of a High-Throughput Method for Antigen Profiling of Cell Lines

The introduction of high-throughput techniques is increasingly providing abundant information on molecular alterations requiring validation at the posttranscriptional level. Protein expression is now efficiently evaluated in large series of tumors included in tissue microarrays. We propose, describe and validate a technique to elaborate paraffin-embedded cell line microarrays (PECLIMA) from fixed cell cultures, which can be processed like standard surgical pathology biopsies prior to immunophenotyping. Our results show a reliable protein immunoexpression profiling in six widely used cell lines under different fixation conditions. This technique permits the simultaneous analysis of multiple antigens in multiple cell lines under different experimental conditions. Additional features of these arrays are long-term storage, their suitability for a variety of techniques including immunocytochemistry and in situ hybridization and their low cost.

Expression of Cell Proliferation and Apoptosis Markers in Papillomas and Cancers of Conjunctiva and Eyelid

Cell proliferation and programmed cell death are considered to be important events in carcinogenesis. The object of our study was to evaluate the expression of the Bcl-2 protein family (Bcl-2, Bak, Bax), p53, proliferating cell nuclear antigen (PCNA), and Ki-67 protein immunoexpression as well as the correlation between the examined markers and some clinicopathological features in papillomas and cancers of conjunctiva and eyelid. Forty-five squamous cell papillomas (SCP), 11 squamous cell cancers (SCC), and 27 basal cell cancers (BCC) were estimated. In the SCP group, p53 protein expression was observed in 30 cases (66.6%), Ki-67 in 14 (31.1%), PCNA in 44 (97.8%), Bcl-2 in 24 (53.3%), Bak in 28 (62.2%), Bax in 31 (68.9%), and Bcl-xl in 11 (100%). In the SCC group, p53 protein expression was evaluated in 8 cases (72.8%), Ki-67 in 2 (18.2%), PCNA in 8 (72.7%), Bcl-2 in 5 (45.4%), Bax and Bak both in 10 (90.9%), and Bcl-xl in 100%. In the BCC group, p53 protein expression was estimated in 23 cases (85.1%), Ki-67 in 13 (48.1%), PCNA in 26 (96.2%), Bcl-2 in 13 (48.1%), Bak in 21 (77.8%), Bax in 22 (81.5%), and Bcl-xl in 23 (85.2%). We observed a correlation between some clinicopathological features and the examined markers of apoptosis and cell proliferation, which seemed to be important events in cancer development.

Fraction of gemistocytic astrocytes and immunoexpression of p53 protein in astrocytomas grade II and III (WHO)

Twenty-two patients with astrocytomas, grade II or III WHO, were studied from 1990 to 1998. In all cases, histopathology showed that the astrocytomas had a gemistocytic component. The aims of this study were to establish the fraction of gemistocytic astrocytes, to investigate p53 protein immunoexpression and to evaluate correlations between these two parameters with the tumour outcome. Tumor cells were quantified at high-power magnification (x400). At least 1000 neoplastic cells (small neoplastic astrocytes plus gemistocytes) were counted in each specimen. The percentage of gemistocytes was defined as the gemistocytic index. Nuclear expression of p53 protein was evaluated in neoplastic astrocytes and gemistocytes. Both the frequency (7/22) as well the p53 immunoexpression indices in gemistocytes, regardless of the grade of the astrocytomas, were inferior from those reported in the literature. No correlation was found between the gemistocytic indices and the p53 immunoexpression.

Differential Expression of nm23 H-1 Protein in Conjunctival Melanoma and Potential Precursor Lesions

Loss of nm23 gene expression is believed to enhance metastatic spread in diverse human tumors, including skin melanoma. The purpose of this work was to determine the pattern and prognostic relevance of nm23 protein immunoexpression in conjunctival melanoma and potential precursor lesion. Formaldehyde-fixed, paraffin-embedded conjunctival specimens comprising 85 melanocytic lesions (nevi, primary aquired melanosis with and without atypia and primary and locally recurrent malignant melanomas) from 73 patients were used. Sections from all specimens were examined by light microscopy to assess diverse prognostic parameters. Additional sections were then immunostained for nm23 H-1 protein and the immunoreactivity was assessed semi-quantitatively. Survival data for all patients were retrieved from the National Causes of Death Registry of Sweden.Nm23 H-1 protein was differentially expressed in conjunctival melanocytic lesions, however loss of immunoexpression was not more common in melanocytic lesions asociated with a high risk of malignant transformation. Also, primary and recurrent conjunctival melanomas showed an essentially similar nm23 expression pattern and we could not associate the pattern of nm23 immunoexpression with an increased risk for malignant transformation or locally recurrent disease. While there was a tentative separation between cause-specific survival curves after excision for low and high nm23 expression conjunctival melanoma, there was no statistically significant association with metastatic death of patients. However, loss of nm23 protein immunoexpression may still be of some importance as a marker for prognosis in conjunctival melanoma because the present study could only detect large differences in survival. Our results suggest that any potential prognostic value of nm23 immunoexpression would be independent of other markers, underlining the importance of further studies.

Changes in the number, proliferation rates, and bcl-2 protein immunoexpression of epithelial and periductal cells from rat epididymis during postnatal development.

To investigate 1) the correlation between the proliferative activity of epididymal epithelium plus myoid cells and the increase in the number of these cells and 2) the role of the basal epithelial cells in the renewal of epididymal epithelium, a quantitative evaluation of the proliferation of both epithelial cells and periductal myoid cells in the different epididymal regions (caput, corpus, and cauda) has been carried out during postnatal development of the rat by immunohistochemical evaluation of BrdU-labeling indices. These data were correlated with cell numbers and counted by the optical dissector method. The presence of bcl-2 protein was immunohistochemically detected and evaluated. No significant differences in BrdU indices were observed among epididymal regions in any stage studied. Cell proliferation decreased from the prepubertal period to adulthood in both epithelial and myoid cells in the three regions of the epididymis, suggesting a close relationship between epithelial and mesenchymal components. The numbers of both cell types were significantly higher in the caput than in the corpus and cauda in all stages studied, suggesting functional differences between regions. A negative linear correlation between proliferative activity and cell numbers was noted that might be related to regulation of the cell population size. Basal cells showed a lower proliferation rate than principal cells, but most of the immunoreactive bcl-2 protein, in pubertal and adult epididymides, was observed in basal cells. Therefore, these cells might comprise a low-proliferating and apoptosis-resistant population.

Cytologic features of synovial sarcoma with emphasis on the monophasic fibrous variant

BACKGROUND Monophasic fibrous synovial sarcoma (SS) is the most common variant of SS but few cytologic studies concerning this entity are available. bcl-2 protein immunoexpression has been described as a characteristic marker of SS and is useful for its differentiation from other sarcomas. METHODS Twelve cytologic specimens from 11 patients with histologically proven SS were reexamined. Ten samples were obtained by fine-needle aspiration and in two cases fluid from cystic tumoral areas was obtained at surgery. Nine cases corresponded to monophasic fibrous tumors and two to biphasic tumors. Immunoexpression of bcl-2 was evaluated in all histologic specimens and in six cytologic samples. bcl-2 protein also was evaluated in 13 soft tissue tumors. RESULTS All cytologic specimens had a similar appearance. Most smears were highly cellular and were comprised of densely packed, tridimensional groups and numerous isolated, round to oval cells. Remarkable findings included cellular monomorphism and vascular structures within the cell groups. Only one case showed cytologic evidence of epithelial differentiation. Immunoexpression of bcl-2 protein was present in 10 of the 11 histologic specimens (90.9%) and in 5 of the 6 cytologic samples (83.3%). A positive result also was observed in malignant fibrous histiocytoma, dermatofibrosarcoma protuberans, infantile hemangiopericytoma, pleomorphic liposarcoma, and malignant solitary fibrous tumor. CONCLUSIONS Although cytomorphologic features of SS are characteristic enough to permit its recognition, clinical correlation is necessary for its correct diagnosis. The immunoexpression of bcl-2 protein is not specific of SS and other soft tissue tumors can show a similar positive reaction. Cancer (Cancer Cytopathol) 1998;84:50-6. © 1998 American Cancer Society.

Cytologic features of synovial sarcoma with emphasis on the monophasic fibrous variant

Monophasic fibrous synovial sarcoma (SS) is the most common variant of SS but few cytologic studies concerning this entity are available. bcl-2 protein immunoexpression has been described as a characteristic marker of SS and is useful for its differentiation from other sarcomas. Twelve cytologic specimens from 11 patients with histologically proven SS were reexamined. Ten samples were obtained by fine-needle aspiration and in two cases fluid from cystic tumoral areas was obtained at surgery. Nine cases corresponded to monophasic fibrous tumors and two to biphasic tumors. Immunoexpression of bcl-2 was evaluated in all histologic specimens and in six cytologic samples. bcl-2 protein also was evaluated in 13 soft tissue tumors. All cytologic specimens had a similar appearance. Most smears were highly cellular and were comprised of densely packed, tridimensional groups and numerous isolated, round to oval cells. Remarkable findings included cellular monomorphism and vascular structures within the cell groups. Only one case showed cytologic evidence of epithelial differentiation. Immunoexpression of bcl-2 protein was present in 10 of the 11 histologic specimens (90.9%) and in 5 of the 6 cytologic samples (83.3%). A positive result also was observed in malignant fibrous histiocytoma, dermatofibrosarcoma protuberans, infantile hemangiopericytoma, pleomorphic liposarcoma, and malignant solitary fibrous tumor. Although cytomorphologic features of SS are characteristic enough to permit its recognition, clinical correlation is necessary for its correct diagnosis. The immunoexpression of bcl-2 protein is not specific of SS and other soft tissue tumors can show a similar positive reaction.

P53 protein immunoexpression in esophageal squamous cell carcinoma and adjacent epithelium.

Immunoreactivity for p53 tumor suppressor gene product is commonly found in human malignancies and some premalignant lesions, but its role in cancer development and its value as a marker of tumor biologic behavior is still unclear. This study was undertaken to assess p53 immunoexpression in esophageal squamous cell carcinoma and attempts to determine its correlation with morphological features associated with tumor behavior. Immunohistochemical study was performed on archival paraffin-embedded tissue of 37 esophageal squamous cell carcinomas and respective adjacent mucosa. Twenty-one tumors (56.8%) demonstrated specific staining for p53. Sixteen areas of dysplasia were present in 14 out of the 35 cases. p53 positivity was found in one low-grade dysplasia and in six high-grade dysplasias. By univariate analysis, p53 immunoexpression correlated positively with local invasion (P = 0.01) and perineural spread (P = 0.04). Multivariate analysis with logistic regression showed that tumor invasion was the only factor that discriminated between p53 positive and p53 negative cases (OR: 15.6, P < 0.02). No relationship was found between p53 expression and tumor grade, DNA nuclear ploidy, and S-phase fraction. These data suggest that p53 dysfunction may be implicated in early, preinvasive, stages of esophageal cancer as well as in the tumor progression related to a more invasive phenotype.

P53 Immunoexpression in Carcinomas Arising in Pleomorphic Adenoma

p53 protein immunoexpression was evaluated in 17 invasive carcinomas arising in pleomorphic adenoma and correlated with the histologic type and tumor grade. Ten tumors had one malignant histologic component: adenocarcinoma NOS (not otherwise specified) (five), undifferentiated carcinoma (two), malignant myoepithelioma (one), epithelial-myoepithelial carcinoma (one), and malignant oncocytoma (one). In the remaining cases, there was a coexistence of areas of adenocarcinoma (seven), adenosquamous (two), epithelial-myoepithelial (two), adenoid cystic (two), undifferentiated carcinoma (one), and low-grade polymorphous adenocarcinoma (one). p53 positivity was found within adenocarcinoma NOS (three) and adenosquamous carcinoma (two) components of four cases. Tumor areas showing low-grade histology, either mono- or bidifferentiated carcinomas, were always negative in this series, in keeping with previous observations on primary neoplasms of the same histologic type. The benign component of the neoplasms was also found to be consistently negative. The results point to a preferential association of the p53 gene dysfunction and its protein accumulation with the malignant transformation of pleomorphic adenomas into salivary adenocarcinomas with features of high-grade malignancy.

Cytoskeletal protein immunoexpression in fetal neural grafts: distribution of phosphorylated and nonphosphorylated neurofilament protein and microtubule-associated protein 2 (MAP-2).

The present study examined the immunocytochemical expression of important cytoskeletal proteins within the neurons of an extended series of neocortical grafts and smaller group of ventral mesencephalic (nigral) grafts. Using antibodies that were directed at all three neurofilament (NF) epitopes, NF-L, NF-M, and NF-H, we attempted to determine whether these neurons would have an altered cytoskeletal profile following the stress of transplantation, because previous studies have shown such changes following ischemia or direct brain injury. We studied phosphorylated NF protein, which is found predominantly in axons, nonphosphorylated NF protein, which is found predominantly in the somata-dendritic compartment, and MAP-2, a specific microtubule marker that is localized exclusively in the somato-dendritic compartment. The results show that in all neocortical grafts examined, both phosphorylated and nonphosphorylated NF immunoexpression was significantly downregulated and appeared only in relatively few axons and somatic profiles, respectively, even though there were numerous Nissl-stained neuronal profiles in the grafts. There was no particular pattern to the immunopositive profiles. At later times occasional neuronal profiles were positive for phosphorylated NF protein, suggesting a reaction to cellular injury. In contrast to neocortical grafts, the cytoskeletal profiles of MAP-2 and phosphorylated NF protein in nigral grafts appeared very similar to age-matched control although the nonphosphorylated NF protein expression did appear somewhat lessened at 1-2 mo postoperative. Because cytoskeletal proteins play important roles in neuronal size, shape, and structural stability, they may subserve key cellular issues in neural grafting. These results show a significant loss of cytoskeletal protein expression in neocortical grafts that does not occur in nigral grafts. These results suggest that fetal neurons from different brain regions (i.e., graft source) may respond differently to the grafting procedure insofar as their cytoskeletal makeup is concerned. In addition, a potential lack of appropriate growth substrates or synaptic contacts may also produce cytoskeletal alterations. As such, the cytoskeletal protein profiles in central nervous system (CNS) grafts may be useful markers for functional performance, perhaps reflecting a degree of cellular injury.

Cytoskeletal protein immunoexpression in fetal neural grafts: Distribution of phosphorylated and nonphosphorylated neurofilament protein and microtubule-associated protein 2 (MAP-2)

The present study examined the immunocytochemical expression of important cytoskeletal proteins within the neurons of an extended series of neocortical grafts and smaller group of ventral mesencephalic (nigral) grafts. Using antibodies that were directed at all three neurofilament (NF) epitopes, NF-L, NF-M, and NF-H, we attempted to determine whether these neurons would have an altered cytoskeletal profile following the stress of transplantation, because previous studies have shown such changes following ischemia or direct brain injury. We studied phosphorylated NF protein, which is found predominantly in axons, nonphosphorylated NF protein, which is found predominantly in the somata-dendritic compartment, and MAP-2, a specific microtubule marker that is localized exclusively in the somato-dendritic compartment. The results show that in all neocortical grafts examined, both phosphorylated and nonphosphorylated NF immunoexpression was significantly downregulated and appeared only in relatively few axons and somatic profiles, respectively, even though there were numerous Nissl-stained neuronal profiles in the grafts. There was no particular pattern to the immunopositive profiles. At later times occasional neuronal profiles were positive for phosphorylated NF protein, suggesting a reaction to cellular injury. In contrast to neocortical grafts, the cytoskeletal profiles of MAP-2 and phosphorylated NF protein in nigral grafts appeared very similar to age-matched control although the nonphosphorylated NF protein expression did appear somewhat lessened at 1-2 mo postoperative. Because cytoskeletal proteins play important roles in neuronal size, shape, and structural stability, they may subserve key cellular issues in neural grafting. These results show a significant loss of cytoskeletal protein expression in neocortical grafts that does not occur in nigral grafts. These results suggest that fetal neurons from different brain regions (i.e., graft source) may respond differently to the grafting procedure insofar as their cytoskeletal makeup is concerned. In addition, a potential lack of appropriate growth substrates or synaptic contacts may also produce cytoskeletal alterations. As such, the cytoskeletal protein profiles in central nervous system (CNS) grafts may be useful markers for functional performance, perhaps reflecting a degree of cellular injury.