Concerning cellular defense in carcinoma of the larynx.

In the search of microscopic manifestations of cellular defense in tumor disease, larynx specimens removed surgically for carcinoma of the larynx and neighboring lymph nodes were studied morphologically. Blastic transformation of lymphocytes from the lymph nodes was also investigated. Activity of nuclear chromatin of these cells was assessed on the basis of its sensitivity to acid hydrolysis in the reaction of Feulgen and incorporation of labeled 3H-actinomycin D. The results indicate existence of cellular defense reaction in carcinoma of the larynx, around the primary tumor as well as in neighboring lymph nodes. Intensity of the reaction in lymph nodes increases with growth of the primary tumor.

Flow cytometric DNA ploidy and cell proliferative activity in colorectal adenomatous polyps.

The retrospective flow cytometric analysis of 54 colorectal adenomatous polyps resected endoscopically from 43 patients was performed. The aim of the study, with the use of flow cytometry was to determine, DNA ploidy and proliferative activity of epithelial cells in a series of adenomas and to compare the results with classical histopathological criteria. Overall 30 tubular and 19 tubulo-villous/villous adenomas with different grade of dysplasia as well as 5 adenomatous polyps with carcinoma in situ (CIS) were examined. DNA aneuploidy was found in 4 of 54 polyps (7.4%), all with histological features of CIS. Proliferation rate increased significantly with the degree of dysplasia (9.0 +/- 3.1%; 16.3 +/- 4.3%; 22.1 +/- 4.1% in adenomas with mild, moderate and severe dysplasia respectively; p < 0.05). No significant differences in cell proliferative activity were found between groups of adenomas as compared to histological type and size. The results show that cell proliferative activity in colorectal adenomatous polyps depends strongly on the grade of dysplasia and does not show such a direct association with respect to histological type or size of adenomas. It may be concluded as well that DNA aneuploidy precedes histological changes of invasive carcinoma in colorectal adenomas and therefore flow cytometric DNA analysis may be of great value in defining more accurately the biological stage of neoplasmatic process in individual cases.

[Renal gene expression of renin and transforming growth factor Beta 1 in children with glomerulonephritis]. / Nerkowa ekspresja genów reniny i transformujacego cznnika wzrostu beta 1 u dzieci z klebuszkowymi zapaleniami nerek.

Increase of renal expression of transforming growth factor beta 1 (TGF-beta 1) gene caused by activation of the local renin-angiotensin system plays an important role in the pathogenesis of glomerulonephritis (GN). The aim of the present study was to measure the expression of renin and TGF-beta 1 genes (own modification of the RT-PCR method) in the isolated renal glomeruli or in the homogenates of renal biopsy specimens in children with various types of glomerulonephritis. The study enrolled 13 children with glomerulonephritis and 3 boys with Wilm's tumour (control group). The expression of the studied genes was presented using arbitrary units defined as multiplicity of the GAPDH gene. No significant difference was found in expression of mRNA renin in the biopsy specimens of the kidney between GN group and control group. Expression of the TGF-beta 1 gene was found in biopsy specimens in all patients from the control group, and only in one GN child, the sole one who was not treated with converting-enzyme inhibitors. No transcripts of the studied genes were found in all RNA samples obtained from the renal glomeruli using the microdissection method. The RT-PCR method applied in the present study allows evaluation of renal expression of renin and TGF-beta 1 genes. The authors would like to point out that storage of biopsy specimens at -80 degrees C would not prevent the total degradation of RNA during microdissection.

[The value of DNA flow cytometry in evaluation of proliferating activity of neoplasm cells in patients with laryngeal cancer]. / Wartosc cytometrii przeplywowej DNA w ocenie zdolnosci proliferacyjnej komórek nowotworowych u chorych na raka krtani.

A study of DNA ploidy was carried out including 84 cases of laryngeal squamous cell carcinoma. Material was fixed during 30 min in 3% paraformaldehyde and embedded in low-melting-point paraffin. The histologic studies were done for diagnosis and general view. Results were as follows. The highest percentage of cancers with DNA diploidy was found in well- and medium-differentiated tumors. Among well-differentiated carcinomas there were 18% diploid versus 5% polyploid tumors. In the medium-differentiated cancers there were 23.5% diploid, 6% polyploid, and 6% hyperdiploid tumors. The highest percentage of DNA hyperdiploidy and poliploido-aneuploidy was found in low-differentiated cancers. Additionally, tumor proliferative activity was evaluated in relation to the percentage of cells in phase S of cellular cycle. Special attention was paid to tumors which revealed the phenomenon of DNA poliploidy-aneuploidy beside DNA diploidy.

[Diagnostic and prognostic value of p53 oncogene and the selected neoplastic markers (Ki67, PCNA, DNA ploidy) of the ultrastructure in patients with laryngeal cancer]. / Wartosc diagnostyczna i prognostyczna onkogenu p53 wybranych markerów nowotworowych (Ki67, PCNA, DNA Ploidii) ultrastruktury u chorych na raka krtani.

A comparison was performed of staining intensity of immunohistochemical proliferating antigens (p53, PCNA, Ki67), DNA flow cytometry and ultrastructure of the carcinoma cells in 120 cases of laryngeal cancer. Clinically very advanced tumors were in majority (T3 - 43%, T4 - 18%). A 5 graded scale was adapted to evaluate the level of immunohistochemical staining of the carcinoma cell nuclei. A positive staining was obtained in 70% for p53, 57% for Ki67 and in 80(2/3) for PCNA. 62% of the cases were DNA diploid and 38% DNA aneuploid. The DNA diploid carcinomas were accompanied by the enlargement of the cell nuclei, preserving of the nuclei's wide margins of heterochromatine, enlargement of the nuclear area and increase of the number of nuclei. In the aneuploid-polyploid cancer the nuclei had a substantial polymorphism with large cleaved nuclei and with significant variation in size, and with nuclear envelope. A frequent finding was euchromatization of chromatine. Dense chromatine appeared in the form of small clumps spread over the whole area of these irregular nuclei. Enlargement and activation of nucleoli occurred. There was a positive correlation (Chi-square) between T- and N-stage and immunohistochemical staining. There was also a positive correlation in staining intensity between p53, Ki67 and PCNA. There is also strong correlation between these markers of proliferative activity and the degree of aggressiveness of the tumour.

[Survival of patients and DNA-ploidy of cells in non-Hodgkin's lymphoma of low-grade malignancy]. / Przezycie chorych a DNA-ploidia w komórkach chloniaków nieziarniczych o niskiej zlosliwosci.

DNA-ploidy in lymphoma cells obtained from lymph nodes of 107 patients with non-Hodgkin's lymphomas of low grade malignancy was studied prior to the treatment introduction. The survival of patients was within the range of 24-126 months (median 63 months). There were 70 hyperdiploidic and 37 diploidic patients. The statistical tendency to prolonged survival was seen among patients with lymphocytic lymphoma (CLL) as compared to other histological types of the disease. There were no statistical differences in the probability of survival (p-P) between diploidic and hyperdiploidic patients either in the whole studied group or in particular histologic types of lymphoma.

[Bone marrow changes in patients with hairy cell leukemia after 2-chlorodeoxyadenosine treatment]. / Zmiany w szpiku chorych na bialaczke wlochatokomórkowa po leczeniu 2-chlorodeoksyadenozyna.

Bone marrow abnormalities were assessed in 6 patients with hairy cell leukemia after 2-chlorodeoxyadenosine treatment. In spite of clinical and haematological remission in all patients, hairy cells and fibrosis were found within the marrow. However, the hairy cells number, the thickness of argentophilic fibres and the extent of fibrosis. It was stressed that in addition to histological analysis with hematoxylin-eosin also other stainings for fibrosis should be applied in the bone marrow evaluation, followed by immunohistochemical and molecular methods which allow to exclude minimal residual disease after leukemia treatment.

[Verification of hematological studies and immunologic phenotype of lymphoma cells in the blood by histological examination of the lymph nodes in non-Hodgkin's lymphoma of low and medium degree of malignancy]. / Weryfikacja badan hematologicznych i fenotypu immunologicznego komórek chloniakowych z krwi badaniem histologicznym wezla chlonnego w chloniakach nieziarniczych o malej i posredniej zlosliwosci.

In 125 patients with non-Hodgkin lymphomas of low or medium malignancy the type of lymphoma determined on the basis of morphological features and immunological phenotype of lymphoma cells in then circulation was verified by histological examination of lymph node. Dissemination of lymphoma cells into blood was found to have occurred in 80% of patients. The highest agreement of histological and haematoimmunological diagnoses was found in lymphocytic lymphoma and lymphoplasmoid (immunocyte) lymphoma, and the lowest one was in centrocytic and centroblastic-centrocytic lymphoma. It is stressed that in a part of the patients histological examination of the lymph node could be abandoned.

[DNA--ploidy in non hodgkin's lymphomas of low malignancy]. / DNA-ploidia w chloniakach nieziarniczych o niskiej zlosliwosci.

Lymphoma cell DNA cytometry in imprints of dissected lymph nodes was performed in 98 patients with low grade non-Hodgkin lymphomas and in 15 patients with reactive lymphadenopathy. The percentage of resting and proliferating cells was also calculated on the basis of computer analysis of DNA amount and cell image. It has been shown that, in contrast to the control group characterized by diploidic DNA amount, DNA aneuploidy was observed in 63.3% of lymphoma patients (DNA hyperdiploidy in 58.2% and DNA aneuploidio-polyploidy in 5.1% of the studied group). The proliferative activity of lymphoma cells was higher in patients with DNA aneuploidy than in DNA diploidic patients and in the control group.

[DNA index and proliferative activity of blastic cells in childhood ALL]. / Indeks DNA i aktywnosc proliferacyjna komórek bialaczkowych w OBL u dzieci.

DNA Index and the pretreatment proliferative activity of blastic cells were assessed in relation to treatment results in 58 children with acute lymphoblastic leukemia. ALL patients were treated with the use of BFM 79 protocol. After the median follow-up time of 32 months p-EFS and p-DFS were 0,425 and 0,495 respectively. There were 35 (56.45%) diploic, 21 (33.81%) hyper-diploic and 2 (3.22%) hypo-diploic patients. There was no statistical difference in p-EFS and p-DFS between diploic and hyper-diploic patients. Patients in I-st remission were characterized by significantly higher percentage of cells remaining in the S-phase of the cell cycle.

Malignant gliomas--ploidy and DNA-content before and after therapy.

DNA ploidy and S-phase percentage from nine malignant gliomas (four glioblastomas, four anaplastic astrocytomas grade 3 and one anaplastic oligoastrocytoma grade 3) have been estimated by single cell cytophotometry on biopsy and necropsy specimens. All gliomas from biopsy material showed, with the exception of two diploid tumours, a polyploid-aneuploid DNA-pattern and stem-lines of different ploidy. The most frequent stem-lines were diploid and hyperdiploid. In necropsy material, following treatment i.e. operation and combined drug and radiation therapy, the heterogeneous nature of all malignant gliomas persisted. From seven aneuploid-polyploid gliomas four showed an elevation and three of them a decrease of DI values. Diploid tumors remained diploid. Because of marked heterogeneity of ploidy patterns and the small number of tumors investigated, ploidy changes could not be used for estimation of therapy efficacy and prognosis. Further studies will be necessary to answer this question.

A comprehensive analysis of selected diagnostic methods with respect to their usefulness in evaluating the biology of neoplastic cells in patients with laryngeal cancer.

The difficult and complicated mechanism of cancer development with little knowledge about the biology of existing cancers can lead to a permanent search for new examination techniques to improve the precision of life expectancy in patients and the selection of the most efficient methods of treatment. The aim of this study was to analyze certain prognostic factors, i.e., p53, Ki67, proliferating cell nuclear antigen (PCNA), DNA ploidy and cell proliferating activity, as well as the degree of morphological differentiation and cell maturity evaluated on an ultrastructural level in patients with laryngeal cancers in connection with data obtained from follow-up examinations and the clinical course of the disease. Neoplastic tissue was taken from 120 patients with laryngeal cancers. All underwent surgical treatment, radiotherapy and combined treatment in the Department of Otolaryngology. Karol Marcinkowski University School of Medical Sciences, Poznan, Poland, and the Department of Otolaryngology-Head and Neck Surgery, Haukeland University, Bergen, Norway. Before beginning treatment all patients underwent histological verification of their neoplastic tissues. Histopathological examination proved that the majority of cases (95%) had a squamous cell carcinoma. The occurrence of changes within the lymph nodes of the neck (N) was significantly correlated with T, S, Ki67, metastases to lymph nodes, DNA ploidy, site and surgery performed. The degree of clinical progression (S) was intercorrelated with T, N, p53, Ki67, PCNA, DNA ploidy, site and laryngectomy. The occurrence of oncoprotein p53 in neoplastic cells was measured by the staining degree of their nuclei and was correlated with T, S, DNA ploidy, metastases to lymph nodes, PCNA and site. The degree of staining of neoplastic cells for the nuclear antigen Ki67 was correlated to T, N, G, S, DNA ploidy, metastases to lymph nodes and surgical treatment. The proliferative antigen PCNA in the examined population of patients was intercorrelated with T, p53, Ki67, metastases to lymph nodes and surgical treatment. The results obtained from DNA flow cytometry could be associated with N, G, p53, Ki67 and metastases to lymph nodes. On the basis of the results obtained, the techniques suggested for the morphological and biological evaluation of neoplastic cells in cancer of the larynx should include TNM classification + G + DNA + p53 + Ki67.

[Comparative cytophotometric and cytomorphometric studies of concave cells and atypical squamous epithelial cells of the cervix uteri]. / Vergleichende zytophotometrische und zytomorphometrische Untersuchungen an Koilozyten und atypischen Plattenepithelzellen der Cervix uteri.

Cytologic slides from 11 women with clinical, cytological and histological signs of a papillomavirus infection of the cervix uteri are investigated by cytophotometry and cytomorphometry. The mean values of DNA content, chromatin density, nuclear area and nuclear circumference of koilocytes were notably higher than for atypical or normal squamous cells. In those patients with concurrent cervical intraepithelial neoplasia the DNA content of koilocytes was inversely related to the degree of atypia. Squamous cells with HPV-induced lesions can be characterized by cytophotometry and cytomorphometry as a distinct cell population forming a continuum with the preneoplastic epithelium.

Blastic transformation of blood lymphocytes in patients with laryngeal carcinoma.

In 40 patients with carcinoma of the larynx transformation of lymphocytes under the influence of PHA was studied. In 20 patients, in addition, venous blood flowing from the tumor, collected during the operation was also examined. In 33 patients the index of blastic transformation was lowered as compared with that in the control group. In 15 of 20 patients, the lymphocytes of venous blood from the vicinity of the tumor showed significantly higher indices of blastic transformation in comparision with those from peripheral blood. It was assumed that difference was due to an increased participation of T-lymphocytes in venous blood flowing from the tumor area. T-lymphocytes might move into the blood from the lymphoid infiltrates which accompany the neoplastic tissue.

DNA cytometric and histologic findings in mouse tumors (BP and S 180) with different response to treatment with tumor necrosis factor.

DNA cytometric and histopathologic investigations were performed in two tumor models (BP and S 180) which differed in their sensitivity to tumor necrosis factor (TNF). TNF induced strong necrosis in both tumors, but only the sarcoma 180 showed total regression. After TNF administration DNA cytometry revealed in the BP tumor an increase of cells in the S-phase, and in the S 180 tumor a loss of aneuploid cell populations. Histologic examination revealed a more obvious effect of TNF on tumor blood vessels in BP tumor, whereas infiltration of inflammatory cells was observed only in the S 180 tumor. We concluded that cell infiltration may be of importance for tumor regression and that aneuploid cell populations are more sensitive to TNF treatment than eudiploid cells.

Effect of recombinant human tumor necrosis factor on tumor and spleen in mice.

The ability of recombinant human tumor necrosis factor (rH-TNF-alpha) to induce regression of sarcoma 180 in vivo was evaluated. The tumor was cured by TNF in the course of 4 weeks. TNF inhibited proliferation of sarcoma 180 cells in vitro, which suggests a direct effect of TNF on tumor cells in vivo. In parallel to the TNF effect on tumor growth, some cell parameters in spleen were investigated. Activation of splenic macrophages was enhanced in vitro. This result suggests that macrophages may participate in the host defense against the tumor. In the first phase of therapy, TNF did not affect the proliferation of splenocytes but increased the transition of G0 into G1 cells. Furthermore, TNF normalized the tumor-induced increase of null cells in tumor-bearing mice. All parameters investigated in spleen reached normal values at the time of tumor regression. Our results suggest that various mechanisms may be involved in TNF-induced regression of sarcoma 180.