Flow cytometric DNA analysis of parathyroid benign lesions.

INTRODUCTION: It is difficult to differentiate benign and malignant lesions just by histopathological evaluation due to lack of clear criteria of diagnosis. Moreover, the group of benign pathologies of parathyroids is not homogenous, and recurrence of symptoms of hyperparathyroidism after surgical management was also noted in this group. This complication is not always due to inappropriate surgical technique. The goal of this work was to find the relationship between cellular ploidy and proliferative activity of adenomas and hyperplasia of parathyroids and preoperative levels of calcium and parathormone in the serum of patients surgically treated for primary hyperparathyroidism. MATERIAL AND METHODS: A total of 98 parathyroid glands were tested, of which 81 (82.7%) were from female patients and 17 (17.3%) from male; the age of the patients was from 22 to 82 years, with an average of 58 years. RESULTS: In resected glands pathological evaluation showed the following results: in 53 (54.1%) adenoma was present, and in 45 (45.9%) there was hyperplasia. Sixty-seven of the samples (68.4%) were characterised as diploid and 31 (31.6%) as aneuploid. There is important positive correlation (r = 0.34595; p = 0.011) between the percentage of S-phase cells (% SPF) and calcium levels measured prior to surgical resection of adenoma. The further analysis of patients with adenoma characterised by aneuploidy proved a statistically valid, positive correlation between %SPF and ionised calcium levels in blood serum of patients both before (r = 0.7189; p = 0.003) and after the surgical treatment (r = 0.6313; p = 0.012). CONCLUSIONS: 1. Benign lesions of parathyroid with ploidy indicates their heterogeneity. 2. In aneuploid benign adenomas of parathyroid glands an increased percentage of cells in S phase (% SPF) correlates with a high level of calcium in serum pre- and post-parathyroidectomy.

Effect of Heme Oxygenase-1 on Melanoma Development in Mice-Role of Tumor-Infiltrating Immune Cells.

OBJECTIVE: Heme oxygenase-1 (HO-1) is a cytoprotective, proangiogenic and anti-inflammatory enzyme that is often upregulated in tumors. Overexpression of HO-1 in melanoma cells leads to enhanced tumor growth, augmented angiogenesis and resistance to anticancer treatment. The effect of HO-1 in host cells on tumor development is, however, hardly known. METHODS AND RESULTS: To clarify the effect of HO-1 expression in host cells on melanoma progression, C57BL/6xFvB mice of different HO-1 genotypes, HO-1+/+, HO-1+/-, and HO-1-/-, were injected with the syngeneic wild-type murine melanoma B16(F10) cell line. Lack of HO-1 in host cells did not significantly influence the host survival. Nevertheless, in comparison to the wild-type counterparts, the HO-1+/- and HO-1-/- males formed bigger tumors, and more numerous lung nodules; in addition, more of them had liver and spleen micrometastases. Females of all genotypes developed at least 10 times smaller tumors than males. Of importance, the growth of primary and secondary tumors was completely blocked in HO-1+/+ females. This was related to the increased infiltration of leukocytes (mainly lymphocytes T) in primary tumors. CONCLUSIONS: Although HO-1 overexpression in melanoma cells can enhance tumor progression in mice, its presence in host cells, including immune cells, can reduce growth and metastasis of melanoma.

Molecular prognostic factors in early-stage cervical adenocarcinoma.

Within the past years the proportion of cervical adenocarcinomas has increased, however, there is a shortage of data regarding immunohistochemical and molecular features and their prognostic relevance in early-stage cervical adenocarcinoma (esCAC). Aim of the present study was to evaluate molecular prognostic factors in esCAC patients treated with primary surgery. Analyses of surgical specimens in 59 patients with esCAC were performed on fixed paraffin-embedded sections of tumour tissue. Tumour tissue sections were routinely stained with hematoxylin and eosin followed by microscopic examination. Immunohistochemical analyses (IHC) were performed on paraffin-embedded section. Flow cytometry (FCM) analysis of paraffin-embedded tumor tissue was performed using flow cytometer FACSCalibur equipped with argon laser. DNA histogram analysis was performed with ModFit application. Treatment effectiveness was evaluated using overall 5-year survival. Survival probability was estimated using the Kaplan-Meier method. Overall survival rate estimated using Kaplan-Meier method was 74.6%. Among the IHC and FCM features univariate analysis showed statistical significance of nm23-H1 gene expression and total S-phase fraction ≤ 11.9% (S-TOT). In multi- variate analysis LVSI and parametrial involvement had significant, negative impact on survival (HR = 8.04, p < 0.003 and HR = 4.03, p < 0.017, respectively). However, none of the tested IHC and FCM features had any influence on overall 5-year survival.

The role of histology, grading, location of tumour and ploidy in evaluation of outcome in patients with liposarcoma.

Department of Tumour Pathology, Maria Sklodowska-Curie Memorial Cancer Center and Institute of Oncology, Kraków, Poland The review of literature indicates that several clinico-morphological factors such as location of the primary tumour as well as its size, histologic subtype, and grade or even selected molecular changes may significantly affect survival of liposarcoma (LPS) patients. Data concerning prognostic importance of DNA ploidy status in LPS cells are extremely limited and results of flow cytometry (FCM) studies have never been compiled with the current classification of malignant adipocytic tumours. Based on evaluation of material from 54 liposarcomas which was available for both histological and FCM analysis, we distinguished four prognostic groups of patients. The best prognosis was noticed for diploid and grade G1 well-differentiated or myxoid liposarcomas localised on extremities. None of the patients with lipoma-like WDLPS and myxoid liposarcoma grade 1 metastasised, while metastases were observed among patients with dedifferentiated LPS (70% of 5-year MFS) and cellular myxoid or round cell liposarcoma (20% of 5-year MFS, only). The metastasis-free survival curves for the above mentioned groups of patients differed significantly (p = 0.00001).

Invasive lobular carcinoma of the breast: cytometric and immunohistochemical characteristics of 96 cases.

The aim of the study was to present microscopic, cytometric and immunohistochemical characteristics of a group of 96 invasive lobular carcinomas (ILC) of the breast. Ninety six patients treated surgically at the Department of Surgical Oncology, Centre of Oncology - Maria Sklodowska-Curie Memorial Institute, Cracow Branch, between 1983 and 1996, were included into the study. In 56 (58.3%) cases, a classical pattern of ILC was diagnosed, whereas atypical variants (solid, pleomorphic, pleomorphic with signet ring cells, signet ring cell, and tubulolobular) were recognized in 40 (41.7%) cases. ILC was characterized by lack of E-cadherin expression, high rate of steroid receptor expression, low rate of P53 and c-erb-B2 expressing tumours, low MIB-1 labelling index, and low S phase fraction, as well as high rate of diploid lesions.

Mediastinal parathyroid carcinoma: a case report.

A 72 year-old woman with primary hyperparathyroidism was operated for parathyroid crisis. PTH serum level was 808 pg/mL. During the operation, only two superior parathyroid glands were found. One was normal, and hypertrophy was revealed in the other. After the surgical procedure, PTH serum level was 726.5 pg/mL. Helical computer tomography examination showed a heterogeneous mass in the anterior mediastinum. The tumour was removed via a sternotomy approach. Histopathological examination revealed parathyroid carcinoma. PTH level dropped to 5.74 pg/mL. Cytofluorometric examination revealed diploidy (DI = 1) in both the hypertrophic and the unchanged upper glands, whereas parathyroid cancer was aneuploid. After the initial operation, the woman was discharged from the hospital on the 27th postoperative day. One year after surgical procedures, she is well. She has to take calcium.

Diagnostic, predictive and prognostic verification of DNA flow cytometric measurements performed at diagnosis for non-Hodgkin's lymphoma adult patients.

More than ten years ago we made first attempts at valuating a prognostic power of flow cytometric DNA measurement results for patients with non-Hodgkin's lymphoma. In multivariate overall survival analysis, S-phase fraction (SPF) showed to be the only independent prognostic factor within the group of patients with low grade lymphomas. In this paper, we have tried to check our previous results in a greater group of patients with longer follow-up, within the specific types of B-cell and T/NK-cell lymphomas verified and classified according to criteria of the WHO 2008 classification. The study was performed on the material obtained from biopsies (85% of lymph nodes) of 484 NHL patients. Patients were diagnosed from 1991 to 2007. The medium follow-up time for living patients was 69 months (range: 25-202 months). All specimens were verified histologically and immunohistochemically. Ploidy and SPF were determined by flow cytometry on fresh tissue obtained during the diagnostic procedure. The diagnostic importance of ploidy and SPF has been confirmed. Ploidy had no predictive or prognostic impact in any of the NHL types, whereas SPF was found to be an independent predictive or prognostic factor in B-CLL/SLL, DLBCL and ALCL.

Effects of heme oxygenase-1 on induction and development of chemically induced squamous cell carcinoma in mice.

Heme oxygenase-1 (HO-1) is an antioxidative and cytoprotective enzyme, which may protect neoplastic cells against anticancer therapies, thereby promoting the progression of growing tumors. Our aim was to investigate the role of HO-1 in cancer induction. Experiments were performed in HO-1(+/+), HO-1(+/-), and HO-1(-/-) mice subjected to chemical induction of squamous cell carcinoma with 7,12-dimethylbenz[a]anthracene and phorbol 12-myristate 13-acetate. Measurements of cytoprotective genes in the livers evidenced systemic oxidative stress in the mice of all the HO-1 genotypes. Carcinogen-induced lesions appeared earlier in HO-1(-/-) and HO-1(+/-) than in wild-type animals. They also contained much higher concentrations of vascular endothelial growth factor and keratinocyte chemoattractant, but lower levels of tumor necrosis factor-α and interleukin-12. Furthermore, tumors grew much larger in HO-1 knockouts than in the other groups, which was accompanied by an increased rate of animal mortality. However, pathomorphological analysis indicated that HO-1(-/-) lesions were mainly large but benign papillomas. In contrast, in mice expressing HO-1, most lesions displayed dysplastic features and developed to invasive carcinoma. Thus, HO-1 may protect healthy tissues against carcinogen-induced injury, but in already growing tumors it seems to favor their progression toward more malignant forms.

DNA content of hepatocyte and erythrocyte nuclei of the spined loach (Cobitis taenia L.) and its polyploid forms.

We analyzed the DNA content of hepatocyte and erythrocyte nuclei of the spined loach Cobitis taenia (diploid) and its allopolyploid forms. Twenty triploid females and one tetraploid were used. At least 20,000 hepatocyte and erythrocyte nuclei were acquired and analyzed by flow cytometry. C. taenia erythrocyte nuclei contain 3.15 +/- 0.21 pg of DNA and the hepatocyte nuclei 4.45 +/- 0.46 pg of DNA. Triploid Cobitis have 5.08 +/- 0.41 pg of DNA in erythrocyte nuclei and 6.11 +/- 0.40 pg of DNA in hepatocyte nuclei, whereas the tetraploid erythrocyte and hepatocyte nuclei contained 6.60 and 7.40 pg of DNA, respectively. In general, the DNA contents correlate positively with the ploidy level of the fish investigated. The DNA content variation in the hepatocyte and erythrocyte nuclei may be due to differences in extent of chromatin condensation, which is more pronounced in the erythrocyte than hepatocyte nuclei, or to the several orders of ploidy that occur in the parenchymal liver cells.

Overexpression of heme oxygenase-1 in murine melanoma: increased proliferation and viability of tumor cells, decreased survival of mice.

Heme oxygenase-1 (HO-1), a cytoprotective enzyme, can be induced in tumors in response to anti-cancer therapies. We investigated the role of HO-1 in B16(F10), S91, and Sk-mel188 melanoma cells. Overexpression of HO-1 after transduction with adenoviral vectors increased cell proliferation, resistance to oxidative stress generated by H2O2, and angiogenic potential as determined by induction of endothelial cell divisions. Likewise, cells stably transfected with HO-1 cDNA (B16-HO-1) showed higher proliferation, stress resistance, and angiogenic activity than the wild-type line (B16-WT). HO-1 overexpression in tumors significantly shortened survival of mice after subcutaneous injection of cancer cells (38 and 22 days for B16-WT and B16-HO-1, respectively; P=0.017). This also resulted in development of more packed tumors, with more melanoma cells, and reduced inflammatory edemas. Mice injected with B16-HO-1 had lower levels of tumor necrosis factor and higher serum concentrations of its soluble receptor tumor necrosis factor-RI, whereas tumors overexpressing HO-1 displayed augmented vascularization and stronger production of vascular endothelial growth factor. Finally, B16-HO-1 cells injected intravenously formed more metastases in lungs. Thus, HO-1 overexpression increased viability, proliferation, and angiogenic potential of melanoma cells, augmented metastasis, and decreased survival of tumor-bearing mice, suggesting that induction of HO-1 may be detrimental in anti-cancer therapy of melanoma.

Expression of insulin-like growth factor-I (IGF-I) in alveolar macrophages and lymphocytes obtained by bronchoalveolar lavage (BAL) in interstitial lung diseases (ILD). Assessment of IGF-I as a potential local mitogen and antiapoptotic cytokine.

Little is known about IGF-I expression in the alveolar lymphocytes (AL), and about local role of IGF-I in physiological conditions and in interstitial lung diseases. Bronchoalveolar lavage was carried out in patients with silicosis, asbestosis, idiopathic pulmonary fibrosis (IPF) and sarcoidosis, as well as in control subjects (n = 13, 9, 12, 56, 15, resp). Alveolar macrophages (AM) and lymphocytes (AL) were studied for (1) IGF-I, BCL-2, Fas and Fas Ligand expression and (2) cell cycle (incl. sub-G1 peak of late apoptosis) with propidium iodide (PI). Flow cytometry (FC) and immunocytochemistry were used. AL early apoptosis was detected by Annexin V FITC/PI staining. IGF-I was present in AL of all tested groups. The number of IGF-I positive AL was significantly higher in IPF (52 +/- 6.7%) and in later (II and III) stages of sarcoidosis (39 +/- 7.8 vs 16 +/- 4.0% in controls, p < 0.05). Increased BCL-2 expression in AL was detected in IPF and sarcoidosis. In all tested groups, AL were almost exclusively Fas+ T cells. Generally, a low number of AL entered apoptosis; no significant differences were found between patient groups, except decreased apoptosis rate in sarcoidosis (0.60 +/- 0.17 vs 1.15 +/- 0.33% in controls, p < 0.05). Proportion of AL positive for IGF-I was significantly correlated with parameters reflecting AL and AM cell proliferation and BCL-2 expression (e.g. AL IGF-I+ vs AM in S phase of cell cycle: r(S) = +0.50, p = 0.001), but not with apoptosis. The results show that human alveolar lymphocytes express IGF-I in normal conditions, as well as in ILD. The proportion of IGF-I+ lymphocytes was significantly increased in IPF and at later stages of sarcoidosis. In our material there was no evidence for profibrogenic or antiapoptotic activity of IGF-I. We suggest that IGF-I originating from AL may be locally active as a mitogen for alveolar macrophages and lymphocytes in ILD.

S-phase fraction and menopausal status as the most important prognostic factors of disease-free survival for node negative patients with breast cancer. A prospective study.

The aim of our study was to determine a prognostic value of DNA flow cytometry measurements performed on fresh breast cancer tissues, separately for patients' groups defined by nodal status, with special attention to histological type of tumor. Between 1993 and 1996 samples from 677 patients were analyzed and 457 cases were included in the survival analysis. Two-hundred and nine patients from them were node negative (N0). The median time of follow-up was 74 months. In multivariate analysis of disease-free survival (DFS), S-phase fraction (SPF) and menopausal status were found to be independent prognostic parameters for N0 group. A combination of this factors allowed us to distinguish three groups different in respect of the risk of recurrence. Our results showed that: 1. SPF and menopausal status could be prognostically valuable factors for DFS in N0 breast cancer patients; 2. prognostic value of SPF and ploidy should be evaluated separately for each histological type of breast cancer.

Prognosis of the surgical treatment of patients with non-small cell lung cancer (NSCLC)--relation to DNA ploidy.

OBJECTIVE: The aim of this study was to evaluate prognostic importance of cell ploidy and proliferation activity in non-small cell lung cancers. Survivals were compared according to the following factors: sex, age, histology, grading, DNA ploidy, tumour size, T factor, N factor and operative procedure. METHODS: In a group of 191 patients in whom cytofluorometric examinations had been performed on archival tumour specimens, postoperative recurrences were observed. RESULTS: Postoperative recurrence was observed in 64 (64.6%) of 99 patients with aneuploid tumours and in 35 (38.0%) of 92 with diploid tumours (P<0.001). Overall survival (OS) rates for the group of 92 patients operated for diploid non-small cell lung cancer (NSCLC) at 5 and 10 years were 62 and 51.1%, whereas of other 99, operated for aneuploid tumours 33.3 and 25.9%, respectively (P<0.001). In the former group of patients disease-free survival (DFS) rates at 5 and 10 years were 58.7 and 51.4% but in the latter 29.3 and 26%, respectively (P=0.00014). Significant differences dependent on cell ploidy were also observed in OS and DFS rates of patients operated respectively for SCLC (P=0.0029; P=0.00318) and adenocarcinoma (AC; P=0.0241; P=0.02109). In general, the mean percentage of S-phase cells in non-small cell lung cancers was 14.0% (SD=13.1) in patients who survived 5 years, and 22.4% (SD=15.7) in those who had a recurrence or died (P<0.001). CONCLUSIONS: In our opinion the most important finding of our work is that determination of cell ploidy in NSCLC provides a valuable supplement to the TNM stage when evaluating late results of the surgical treatment. However, the paper demonstrates that aneuploidy, although unfavourable, is not an independent prognostic factor in the group of patients with NSCLC and in the subgroups - both with squamous cell carcinoma and adenocarcinoma. Our results show also that the percentage of S-phase cells is an independent, unfavourable prognostic factor in patients treated surgically for non-small cell lung cancer and in the subgroup with squamous cell lung carcinoma.

The role of CD44v3 expression in female breast carcinomas.

CD44-protein and its isoforms are the multifunctional cell adhesion molecules participating in cell-cell and cell-matrix interactions. In this study we estimated the frequency of CD44-expression as well as two of its variants (CD44v3 and CD44v5) in female breast cancer. Among 75 breast carcinomas studied, 23 (44.2%) presented strong membrane reaction with monoclonal antibody against antigen CD44. The immunocytochemical reaction to CD44v3 and CD44v5 were observed in 16 (21.3%) and 50 (66.75%) cases, respectively. The presence of CD44v3 antigen on the surface of breast cancer cells significantly correlated with ER expression (0.0430) and the lack of p53 protein (p=0.0252), and also with the percentage of T cells in the total population of lymphocytes infiltrating the primary tumor (TILs) (p=0.0248). What is more important, the reaction to CDv3 significantly correlated with the presence of metastases to the lymph nodes (p=0.0385).