Usefulness of polyclonal antibodies raised against P65 oncofetal protein in immunohistochemical diagnosis of ductal breast cancer.

Paraffin-embedded infiltrating ductal breast cancer tissue slides (135) were analyzed by immunohistochemistry with the use of rabbit polyclonal anti-P65 oncofetal protein and mouse monoclonal anti-estrogen/progesterone receptor (ER, PR) antibodies. Analysis with anti-P65 antibody revealed the positive cytoplasmic reaction in 83 cases, 98 showed the nucleic reaction and 3 were immunologically negative. Among the analyzed cases 49 revealed both cytoplasmic and nucleic reactions. For the whole group of cancers the correlation was found between ER or PR level and P65 cytoplasmic reaction (r = 0.77 and 0.66, respectively) and low inverse correlation with nucleic localization of P65 protein. The percentage of positive cells with cytoplasmic expression of P65 was significantly higher in more histologically differentiated cancers (grade I and II according to Bloom and Richardson) than in grade III. Opposite tendency was observed for the nucleic expression of P65 protein. The percentage of immunopositive nuclei grew with the advance of the disease and was the highest in poorly-differentiated (grade III) tumors. The tumors with P65 cytoplasmic reaction were mainly small (T1, T2), without metastases to lymph nodes (N0) and distant metastases (M0). The dependence between P65 protein localization and clinical stage of disease (TNM classification) was evaluated statistically. The straight dependence existed between P65 nucleic reaction and tumor size (p = 0.0002), metastases to lymph nodes (p = 0.0032) and distant metastases (p = 0.0006). The obtained results suggest that the transfer of P65 protein from cytoplasm to nuclei of the breast cancer cells is connected with more clinically advanced stages and worse prognosis for the patients.

Expression of C-ERBB2 and P65 genes and their protein products in follicular neoplasms of thyroid gland.

C-ERBB2 and P65 gene expression was investigated by reverse transcriptase polymerase chain reaction method (RT-PCR) in thirty follicular thyroid cancers and twenty follicular adenomas. Additionally, the cancers and adenomas were stained by immunohistochemistry for the expression of their protein products. We did not observe P65 gene expression in any of the analyzed follicular cancers (n=30) but it was observed in 13 out of 20 (65%) follicular adenomas. The presence of C-ERBB2 gene expression was found in 18 (90%) follicular adenomas but not in cancers. There were 10 (50%) adenomas and 11 (36.7%) cancers with positive staining for C-ERBB2 protein and 15 (75%) adenomas and 2 (6.7%) cancers with positive staining for P65 protein. We conclude that expression of C-ERBB2 and P65 genes is associated with follicular adenoma but not with cancer of thyroid gland.

Uptake of radiolabelled alpha-fetoprotein by experimental mammary adenocarcinoma and adenoma: in vivo and in vitro studies.

The biodistribution of iodine-labelled alpha-fetoprotein ( I-AFP) in experimental mammary tumours was studied. C3H mice with subcutaneously transplanted mammary adenocarcinoma and Sprague-Dawley rats treated with -methyl- -nitrosourea for mammary adenoma induction were used as animal models. The accumulation of labelled I-AFP in mouse mammary adenocarcinoma was significantly higher than that in rat mammary adenoma. The tumour/muscle radioactivity ratios increased with time and, 48 h after intravenous injection, were estimated as 23.4 and 6.7, respectively. For experiments, extracts from both mammary tumours were prepared. The extracts were subjected to sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE), transferred to polyvinylidene difluoride (PVDF) membranes and incubated with I-AFP. A single major AFP-binding protein with a molecular weight of about 30 kDa was detected in both extracts. The amount of AFP-binding protein was clearly higher for adenocarcinoma than for adenoma. In the presence of cross-linking reagent, I-AFP formed a complex (about 100 kDa) with adenocarcinoma proteins.

Lymphocyctes Tgammadelta in clinically normal skin and peripheral blood of patients with systemic lupus erythematosus and their correlation with disease activity.

Human Tgammadelta lymphocytes constitute from 1 to 15% of all peripheral blood lymphocytes. Recent work has demonstrated that this population plays a major role in the pathogenesis of infectious and immune diseases. Increased numbers of gammadelta T cells have been found in affected skin from systemic sclerosis and chronic cutaneous lupus erythematosus patients. In our study, we have determined the numbers of Tgammadelta lymphocytes and their subpopulations in peripheral blood from 29 patients with systemic lupus erythematosus (SLE) and in 19 healthy volunteers using flow cytometry and specific monoclonal antibodies. The same cells in uninvolved skin from SLE patients and human controls using immunohistochemical analysis were estimated. T-Cell receptor (TCR) delta chain gene rearrangement was identified with primers for Vdelta1, Vdelta2 and Vdelta3 by the polymerase chain reaction. Statistical analysis showed a significantly decreased number of gammadelta T cells in SLE patients (26.4+/-16.9/microl) compared with the control group (55.3+/-20.6/microl (p < 0.001). The number of Vdelta2 TCR+ and Vgamma9 TCR+ subpopulations was also lower in SLE patients than in healthy persons. No statistical correlation between disease activity and the number of gammadelta T cells was demonstrated. The percentage of Tgammadelta lymphocytes in clinically normal skin from SLE patients was twice (22.0+/-9.4%) that found in the skin from healthy persons (11.1+/-5.5%) (p < 0.002). Higher percentages of the Vdelta2 TCR+ and Vgamma9 TCR+ subpopulation of lymphocytes were found in the skin from SLE patients. We have also found positive correlation between the percentage of Tgammadelta lymphocytes in skin and the activity of SLE (r=0.594, p < 0.001), and between subpopulation Vdelta3 TCR+ and disease activity (r=0.659, p< 0.001). In conclusion, the results of our studies demonstrate that, in patients with SLE, accumulation of Tgammadelta lymphocytes can be seen in clinically normal skin, and the percentage of these cells correlates with the activity of the disease.

Altered expression of nuclear non-histone protein (p44/46) in different stages of B-chronic lymphocytic leukemia.

Our previous data have shown some differences in electrophoretic characteristics of proteins from cellular fractions (nuclear, mitochondrial, microsomal and cytosolic) isolated from peripheral blood mononuclear cells of B-cell chronic lymphocytic leukemia (B-CLL), acute lymphoblastic leukemia (ALL) patients and healthy donors. The main differences were found in electrophoretic patterns of nuclear proteins from normal and leukemia cells, especially in the nuclear mass regions of 36-52, 58-85, and 120-180 kDa. Electrophoretically-specific nuclear non-histone protein in the molecular mass zone 44/46 kDa of cells obtained from the peripheral blood of a B-CLL patient was used to produce rabbit polyclonal antiserum. SDS-polyacrylamide gel electrophoresis as well as immunological techniques (Western blot and immunocytochemistry) indicate that the nuclear protein with a molecular mass of 44/46 kDa is specifically expressed in mononuclear cells from B-CLL patients. The expression of this particular nuclear protein seems to correlate with the progression of the leukemia.

Low-grade non-Hodgkin's lymphoma in a patient with systemic lupus erythematosus.

Coexistence of systemic lupus erythematosus (SLE) with low-grade non-Hodgkin's lymphoma (LGNHL) has been described occasionally in the literature with the potential pathogenetic role of monoclonal B CD5+/CD19+ cells. We report a case of LGNHL which developed 18 months after diagnosis of SLE. The monoclonal population of lymphocytes in the peripheral blood and bone marrow was CD5/CD19 negative but CD19/CD22 positive. The SLE responded well to treatment with prednisone and the course of the LGNHL was stable and cytotoxic treatment was not required.

[Prognostic significance of selected oncogene and suppressor gene expression in follicular thyroid carcinoma]. / Prognostyczne znaczenie ekspresji wybranych onkogenów i genów supresorowych w raku pecherzykowym tarczycy.

Oncogene and suppressor gene expression (cyclin D, p21WAF1, nm23-H1, Rb1, p16INK4A, and p53) was evaluated in 23 follicular thyroid carcinomas diagnosed in 20 women and 3 men operated or reoperated in Institute of Oncology in Gliwice in years 1992-1999. Positive reaction with p16INK4A, Rb1 and cyclin D1 antibodies was observed in all tumors, with nm23-H1 in 22 cases. The presence of p21WAF1 was stated in 8 cases (34.8%) and p53 in 7 cases (30.4%). A simultaneous presence of expression of p53 and lack of expression of p21WAF1 was stated three times and in two cases were accompanied by distant metastases. This pattern of expression was only rarely observed in minimally invasive follicular cancer. The prognostic significance of simultaneous immunohistochemical analysis of p53 and p21WAF1 in follicular thyroid carcinoma is suggested and has to be proved in further studies.

Some oncogene and tumour suppressor gene protein products expression in B-cell chronic lymphocytic leukaemia.

The expression of Bcl-2, P53 proteins and known markers of proliferation, namely proliferating cell nuclear antigen (PCNA) and Ki67, in 29 patients with B-cell chronic lymphocytic leukaemia (B-CLL) was investigated. All leukaemic patients were classified, and immunophenotyped by the two-colour immunofluorescence method with the use of fluorocytometry. B-CLL was heterogeneous in the range of biological parameters of tumour cells. B-CLL patients manifested 34% positive Ki67 and 61% PCNA expression, whereas Bcl-2 and P53 positivity was 81% and 42%, respectively. The level of intracellular expression of Bcl-2 and P53 proteins did not depend on the stage of disease estimated by routine methods. Ki67 and PCNA expression was significantly higher in B-CLL patients with more advanced stages of the disease. A statistically significant correlation was established between their mutual expression.

[Significance of angiogenesis in laryngeal cancer]. / Wartosc oceny angiogenezy w raku krtani.

Angiogenesis as multistage process, which is required for forming of new vessels and is essential for the growth of solid tumors and making metastasis. Sixty patients with laryngeal cancer were analyzed. They were treated in ENT Department of Medical University in Lodz and follow-up period was not shorter than 5 years. Intensity of neovascularisation was examined by means of immunohistochemical techniques carried out on paraffin sections using selected antibody against antigens of vessel cells. The value of the prognostic factors of angiogenesis intensity were evaluated against: dissected nodal metastasis, tumor recurrences and metastasis observed in prospective follow-up and after cancer treatment live time. Morphological properities of tumor or hospital treatment of the patients with laryngeal cancer were analyzed as well. Taking into consideration the results, we can make the conclusion that investigation of angiogenesis can be forthcoming prognostic factor for the laryngeal cancer.

[Assessment of cell proliferation antigen Ki-67, protein p53 related to apoptosis and angiogenesis in laryngeal cancer]. / Ocena ekspresji antygenu proliferacji komórkowej Ki-67, bialka zwiazanego z apoptoza p-53 i intensywnosci angiogenezy w raku krtani.

Kinetics (growth fraction of tumour cell populations), death process of cancer cells (apoptosis and necrosis) and neovascularisation in tumour (angiogenesis) have influence on the growth of cancer. Sixty patients with laryngeal cancer treated in ENT Department of Medical Academy of Lodz were analysed. Proliferation activity of cancer cells was examined by means of selection appropriate antigen (Ki-67) characteristic for cell cycle utilising immunohistochemical techniques carried out on laryngeal cancer paraffin samples. Expression of selected protein connected with apoptosis (p-53) and intensity of angiogenesis were examine using selected antibody (anti-CD34) aimed against epithelial antigens. Above-mentioned markers were correlated with: stage of cancer progression, recurrences and metastasis of laryngeal cancer and follow-up of the patients. The morphological properties were examined as well. The researches on apoptosis, angiogenesis and proliferation of cancer cells can be used as prognostic factors for the patients with laryngeal cancer.

Spontaneous apoptosis of Reed-Sternberg and Hodgkin cells; clinical and pathological implications in patients with Hodgkin's disease.

The experiments were designed to study correlation between frequency of apoptosis of Reed-Sternberg/Hodgkin (R-S/H) cells, EBV infection of these cells, expression of the key proteins involved in regulation of apoptosis and cell cycle in R-S/H cells, the patients' pretreatment markers and the clinical outcome. One hundred and ten Hodgkin's disease (HD) patients were studies, of which 69 obtained complete remission (CR) after first-line treatment and 41 did not respond. The time of follow-up was from 18 to 242, median 69.7, months. Apoptosis was evaluated by TUNEL technique (TdT-mediated dUTP nick end labeling) and the presence of EBV-latent membrane protein 1 as well as expression of Bcl-2, tumor suppressor p53, p21WAF1, MDM-2, Rb1, PCNA, p27KIP1 and caspase-3, was detected immunocytochemically on paraffin-embedded lymph node specimens obtained at diagnosis. Positive TUNEL reaction was found in 43 patients with apoptotic index (AI) in this group varying between 10% and 60%. In the remaining 57 patients AI of R-S/H cells was below 10%. In 62 patients the cells surrounding R-S/H cells were also TUNEL-positive; their frequency was variable. The expression of LMP1 protein on R-S/H cells was found in 38 patients, without any correlation with the presence or frequency of apoptosis. No significant difference was seen between the AI and both clinical stage and histological type of the disease. However, the mean AI in non-responding patients was significantly higher than in CR group (p=0.015); the high frequency of apoptosis was also negatively correlated with the progression free survival time (p=0.031) and the overall survival (p=0.042). The expression of PCNA, p21WAF1, p53 protein and caspase-3 also showed positive correlation with frequency of apoptosis (p=0.011, p=0.036, and p=0.001, respectively). On the other hand, no statistically confirmed correlation was found between AI and expression of bcl-2, MDM-2, Rb1, and p27KIP1 on R-S/H cells. These data provide evidence that tumor cells in HD undergo spontaneous apoptosis regardless of EBV infection. High pretreatment AI correlates with poor response to the treatment, and may be considered as a potential negative prognostic factor in HD.

The expression of products of oncogens c-erbB2 and EGFR and proliferating antigens Ki67 and PCNA in primary invasive ductal cancer of female breast.

The aim of the study was to examine the consecutive series of primary ductal invasive tumours and find out: a) the expression of some biological cellular parameters as proliferating antigens Ki67 and PCNA and products of gene EGFR, erbB2; b) correlation between the levels of expression of those factors and classical prognostic factors, such as diameter of tumour, status of axillary lymph nodes, status of steroids receptors, the degree of histological differentiation. We found that: 1. The presence of the expression of oncoproteins c-erbB2 and EGFR, high index IP PCNA, Ki67 and low levels of steroids receptors correlates with high histological malignancy (Bloom III0); 2. The lack of expression of oncoproteins c-erbB2, EGFR and low index IP PCNA, Ki67 correlates with high levels of steroids receptors; 3. The estimation of high levels of index IP PCNA, IP Ki67 can be helpful for separate tumours of high proliferating activity; 4. The expression of oncoprotein c-erbB2 and EGFR does not correlate with the diameter of tumour as well as with the involvement of axillary lymph nodes. It seems that the estimation of proliferating antigens together with the expression of oncoproteins might have greater prognostic value than the estimation of one of these factors.

Immunohistochemical analysis of expression of a 65 kDa oncofetal protein (p65), epidermal growth factor receptor (EGFR), oncogene c-erb B2 and tumor suppressor gene p53 protein products in breast cancer patients.

Paraffin-embedded tissue slides from 88 infiltrating ductal breast carcinoma were examined by immunohistochemistry technique with the use of monoclonal antibody against human p65 antigen and polyclonal antibody against p65-like protein present in fetal bovine serum. Immunohistochemical analysis of expression of growth factor receptors (EGFR), protein product of oncogene c-erb B2 as well as protein product of mutated anti-oncogene p53 was also done. It was established that there is no correlation between p65 and c-erbB2, EGFR or p53 expression. In low differentiated tumors (grade III) high p53 index and high EGFR and c-erbB2 expression was connected with low p65 expression. The lack of c-erbB2 and EGFR and low p53 expression was combined usually with high p65 oncoprotein levels.

Prognostic factors in Hodgkin's disease: multivariate analysis of 327 patients from a single institution.

On the basis of a retrospective study of 327 patients with Hodgkin's disease (HD), the prognostic significance of several factors, accepted previously and recently proposed, has been analyzed with regard to response to treatment and the survival time. Multivariate regression analysis strongly decreased the number of potentially prognostic parameters. The only independent, pretreatment factors negatively influenced by either time of survival or response to treatment were the following: age at diagnosis of more than 45 years, advanced (IIIB/IV) clinical stage, poor clinical status according to Karnofsky's scale (score less than 70), presence of systemic symptoms, mixed cellularity/lymphocyte depletion histological type, multisite peripheral nodal localization of the disease, abdominal lymphadenopathy, and large primary tumor mass (bulky disease). Short time to achieve complete remission (during the first four courses of chemotherapy) has proven to be significantly positive predictive factor. Cumulative dose of cytostatics lower than programmed was a significantly negative prognostic factor that correlated with a shorter time of survival. Lack of or a too-low dose of radiotherapy had the same predictive value. High activity of serum lactate dehydrogenase correlated moderately with poor response to the first-line treatment but did not influence the survival time. Other clinical, morphological, and biochemical parameters influenced neither the prognosis nor the response to treatment. Additionally, immunohistochemical examinations for proliferating cell nuclear antigen and the protein products of the p53 and bcl-2 genes were performed on the lymph nodes obtained from the patients with HD. High expression of proliferating cell nuclear antigen, p53, and BCL-2 correlated with poor response to the treatment and/or short time of survival. Statistical analysis has led us to the conclusion that the pretreatment expression of these oncoproteins can be taken into consideration as a new prognostic factor in HD.

Evaluation of selected parameters of the cytokine system in burn wounds in children.

Using immunohistochemical methods, we studied cellular infiltrations of burn wounds in children. The relative number of cells with cluster of differentiation 3 (CD3) CD68 molecules, cells with receptors for interleukin-2 (IL-2) and its receptor (IL-2R), IL-3, IL-3R, IL-6, IL-6R, tumor necrosis factor-alpha (TNF-alpha), TNF-alphaR, and cells with TNF-alpha molecules on their surface was studied. The relationships between these values were determined. The following results were observed: (1) A positive correlation between the relative numbers of CD68 cells and the percentage of cells with receptors for IL-2 (IL-2R) and TNF-alpha (TNF-alphaR); (2) A negative correlation between the relative numbers of CD3 cells and cells with receptors for IL-2 and TNF-alpha; (3) A strong negative correlation between the relative numbers of CD3 and CD68 cells; (4) A positive correlation between the percentage of cells with receptors for TNF-alpha and the size of the burn wound; and (5) A positive correlation between the relative numbers of cells with the TNF-alpha molecule on their surfaces and studied receptors for interleukins, i.e., IL-2R, IL-3R, IL-6R, and TNF-alphaR. These results indicate a key role for TNF-alpha and its receptor within the cytokine system in the course of the inflammatory reaction in burn wounds.

Mechanism of impaired immunologic response to bacterial antigens in burn wounds in children.

Material was obtained from necrotic tissue excised from burn wounds in 60 children and examined immunohistochemically for the percentages of CD3, CD4, CD8, CD20, and CD68 cells. The results were then correlated with the burn surface area (BSA). Bacteriologic studies revealed the presence of bacteria in only 22 wounds with BSA greater than 10% of total body surface area. In this group, the proportion of CD3, CD4, CD8, CD20, and CD68 cells was correlated with the severity of infection, measured as the number of bacteria per g tissue. The results showed a positive correlation between the BSA and the percentage of CD8 cells (P < 0.001) and a negative correlation between CD4 cells and BSA (P < 0.02). The correlation between CD4/CD8 index and BSA was significantly negative. Likewise, a significant negative correlation was also noted between the percentage of CD4 and CD8 cells within the wound (P < 0.001). The severity of infection (bacteria/g tissue) was also positively correlated with the percentage of CD8 cells (P < 0.05) and negatively with the percentage of CD4 cells (P < 0.05). The correlation between CD4/CD8 index and intensity of infection was also highly significant. The results obtained show that in extensive burn wounds bacterial antigens may not be recognized properly due to the decreased percentage of CD4 cells and increased percentage of CD8 cells, which enhances bacterial growth in these wounds.

[Evaluation of CD44 adhesion molecule, nm23 gene product expression and intensity of angiogenesis in patients with laryngeal cancer]. / Ocena ekspresji czasteczki adhezyjnej CD44, produktu genu nm23 i intensywnosci angiogenezy u chorych z rakiem krtani.

One of the most important factor in prognosis of the patients with laryngeal cancer is presence of the metastases in lymph nodes of the neck. The main purpose of the paper was the evaluation of CD34 and FVIII antigens as angiogenesis markers, and nm23 protein and CD44 antigen expression as metastasis potential markers and description of their role in the tumour progression and making metastasis in the patients with laryngeal cancer. Paraffin-embedded tissue sections from 89 patients with laryngeal cancer were stained with a monoclonal antibody raised against CD34 and FVIII antigens, against nm 23 protein and against CD44 antigen. Measuring the density of the microvasculature in tumour was investigated. We found significant dependence between intensity of angiogenesis (IA) and pT, nodal metastasis, histological grading and survival. There were also significant correlation between nm23 protein expression and nodal metastasis, and between CD44 antigen expression and pT, nm23 protein expression and FVIII antigen expression. Evaluation of mentioned markers allowed to asses the aggressiveness of tumour cells and anticipate neck metastasis in the patients with laryngeal cancer.

[Expression of selected markers for apoptosis, proliferation and metastasis in evaluation of laryngeal cancer invasiveness dynamics]. / Ekspresja wybranych wskazników apoptozy, proliferacji i przerzutowosci w ocenie dynamiki rozrostu raka krtani.

Evaluation of the biology of laryngeal cancer cell is connected either with many process inside the cell or reactions between cancer cell itself and extracellular matrix. The main purpose in this paper was the evaluation of p53 protein, bcl-2 protein, Ki-67 antigen and CD44 adhesive molecule expressions in comparison to clinical and histopathological features in patients with laryngeal cancer. Paraffin-embedded tissue sections from 89 patients with laryngeal cancer were stained with a monoclonal antibody raised against p53 and bcl-2 proteins, Ki-67 and CD44 antigens using a peroxidase-labelled streptavidin-biotin kit. There were statistically significant relationships between p-53 protein over-expression and pT, histological grading, survival and Ki-67 and CD44 antigens expressions. There were no correlation between bcl-2 protein expression and clinical and histopathological features. We observed statistically significant correlation between Ki-67 expression and pT, histological grading, recurrences and survival. Expression of CD44 statistically significant correlated only with tumour size. We conclude that comparison of data covering mentioned tumour markers expression gives valuable evaluation of biological activity of cancer cells and may allow to create the immunological panel of tumour markers which simplify the prognosis about nodal metastases, recurrences and survival in patients with laryngeal cancer.

Membrane-bound paracrystalloid structures in alveolar rhabdomyosarcoma.

We report here a case of an alveolar rhabdomyosarcoma in a 25-year-old woman. Ultrastructural examination revealed a few periodical membrane-bound crystalloids, similar to those regarded as specific for alveolar soft-part sarcoma. Our study provides evidence supporting a myogenic origin of those periodic structures, and simultaneously a myogenic origin of alveolar soft-part sarcoma.

Expression of p21 and MDM-2 proteins on tumor cells in responding and non-responding patients with Hodgkin's disease.

Since the prevention of apoptosis can produce resistance to chemotherapy, abnormal expression of oncoproteins engaged in the regulation of this phenomenon in tumor cells may give some prognostic information in patients with neoplasma. To assess this problem in Hodgkin's disease (HD), the cellular expression of two p53 downstreams proteins p21 and MDM-2 was evaluated in the lymph nodes specimens obtained from 68 patients at the time of diagnosis, and compared with some clinical and pathological data. Positive immunoreaction for p21 and MDM-2 on Reed-Sternberg/Hodgkin's (R-S/H) cells was found in a majority of cases (58.8 and 63.2%, respectively). High expression of p21 protein correlated with poor response to the first-line treatment and disease free survival in both univariate and multivariate regression analysis, whereas expression of MDM-2 did not give prognostic information in patients with HD. Expression of p21 and MDM-2 was also compared with p53 protein immunoreactivity of R-S/H cells. The p21+/p53+ immunophenotype occurred to give particularly negative prognostic information in patients with HD. Diversity of p21/MDM-2/p53 patterns of R-S/H cells observed in particular patients may reflect heterogeneity of apoptosis regulatory mechanisms, as well as differences in p53 gene status.