The epigenetic modifiers 5-aza-2'-deoxycytidine and trichostatin A influence adipocyte differentiation in human mesenchymal stem cells.

Epigenetic mechanisms such as DNA methylation and histone modification are important in stem cell differentiation. Methylation is principally associated with transcriptional repression, and histone acetylation is correlated with an active chromatin state. We determined the effects of these epigenetic mechanisms on adipocyte differentiation in mesenchymal stem cells (MSCs) derived from bone marrow (BM-MSCs) and adipose tissue (ADSCs) using the chromatin-modifying agents trichostatin A (TSA), a histone deacetylase inhibitor, and 5-aza-2'-deoxycytidine (5azadC), a demethylating agent. Subconfluent MSC cultures were treated with 5, 50, or 500 nM TSA or with 1, 10, or 100 µM 5azadC for 2 days before the initiation of adipogenesis. The differentiation was quantified and expression of the adipocyte genes PPARG and FABP4 and of the anti-adipocyte gene GATA2 was evaluated. TSA decreased adipogenesis, except in BM-MSCs treated with 5 nM TSA. Only treatment with 500 nM TSA decreased cell proliferation. 5azadC treatment decreased proliferation and adipocyte differentiation in all conditions evaluated, resulting in the downregulation of PPARG and FABP4 and the upregulation of GATA2. The response to treatment was stronger in ADSCs than in BM-MSCs, suggesting that epigenetic memories may differ between cells of different origins. As epigenetic signatures affect differentiation, it should be possible to direct the use of MSCs in cell therapies to improve process efficiency by considering the various sources available.

The epigenetic modifiers 5-aza-2'-deoxycytidine and trichostatin A influence adipocyte differentiation in human mesenchymal stem cells

Epigenetic mechanisms such as DNA methylation and histone modification are important in stem cell differentiation. Methylation is principally associated with transcriptional repression, and histone acetylation is correlated with an active chromatin state. We determined the effects of these epigenetic mechanisms on adipocyte differentiation in mesenchymal stem cells (MSCs) derived from bone marrow (BM-MSCs) and adipose tissue (ADSCs) using the chromatin-modifying agents trichostatin A (TSA), a histone deacetylase inhibitor, and 5-aza-2′-deoxycytidine (5azadC), a demethylating agent. Subconfluent MSC cultures were treated with 5, 50, or 500 nM TSA or with 1, 10, or 100 µM 5azadC for 2 days before the initiation of adipogenesis. The differentiation was quantified and expression of the adipocyte genes PPARG and FABP4 and of the anti-adipocyte gene GATA2 was evaluated. TSA decreased adipogenesis, except in BM-MSCs treated with 5 nM TSA. Only treatment with 500 nM TSA decreased cell proliferation. 5azadC treatment decreased proliferation and adipocyte differentiation in all conditions evaluated, resulting in the downregulation of PPARG and FABP4 and the upregulation of GATA2. The response to treatment was stronger in ADSCs than in BM-MSCs, suggesting that epigenetic memories may differ between cells of different origins. As epigenetic signatures affect differentiation, it should be possible to direct the use of MSCs in cell therapies to improve process efficiency by considering the various sources available.

Neoadjuvant therapy affects tumor growth markers in early stage non-small-cell lung cancer.

INTRODUCTION: While adjuvant therapy of early-stage non-small-cell lung cancer (NSCLC) is widely accepted, literature data concerning neoadjuvant treatment provide contradictory results with both improved and unaffected survival rates. Also, data concerning potential effects of neo-adjuvant therapy on cellular level are scarce. OBJECTIVE: The aim of present study was to analyze the effect of chemotherapy followed by surgical resection on several key biological markers of tumor growth (TGF-beta, VEGF), apoptosis (sAPO-1/Fas/CD95) and invasiveness (TIMP-1) assessed in the sera of NSCLC early-stage patients (IB-IIIA). - MATERIAL AND METHODS: Measurements were performed by ELISA method in blood serum from 24 NSCLC patients (I-IIIA) collected prior therapy, one day before surgery and 3 days after. RESULTS: TGF-beta serum concentrations were significantly lower after both chemotherapy (P<0.05) and surgery (P<0.01) in comparison to the baseline. VEGF levels decreased following NEO therapy with subsequent significant up-regulation after surgery (P<0.001). Interestingly, post-surgery serum VEGF strongly correlated with TGF-beta concentration (r = 0.52, P = 0.014). No significant differences were observed for serum sAPO-1/CD95/FAS as well as TIMP-1 concentrations at any of three evaluated time-points. CONCLUSION: Neoadjuvant treatment of early-stage NSCLC affects mostly mechanisms responsible for tumor growth and vascularization. Its effect on cancer cells apoptotic activity needs further evaluation.

Neuroendocrine phenotype of non-small cell lung carcinoma: immunohistological evaluation and biochemical study.

AIMS AND METHODS: The prevalence and distribution of neuroendocrine differentiation in non-small cell lung cancer (NSCLC) was estimated by assays for synaptophysin (SYN), chromogranin A (CgA), Leu7 and neuron-specific enolase (NSE). Serum NSE and CgA were determined in parallel to find the values of these markers for distinguishing neuroendocrine differentiation in NSCLC. Fifty-eight resected NSCLC specimens and 34 sera of NSCLC patients entered the study. Neuroendocrine differentiation was graded according to the percentage of neuroendocrine tumor cells as NE0--0%, NE1-NE4--1%->76%. Serum NSE <12.5 ng/mL and serum CgA <46 U/L were taken as cutoff levels. RESULTS: 63.8% (37/58) of NSCLC were scored as NE1-NE4 according to the SYN, CgA and Leu7 levels; 34.5% as NE1; 29.3% as NE2-NE4. 56.8% of tumors were positive for SYN, 34.4% for CgA, 22.4% for Leu7, and 79.3% for NSE. A significant relationship was found between tumor SYN and tumor CgA expression, and between tumor SYN expression and tumor stage. Adenocarcinomas showed a significantly higher rate of neuroendocrine differentiation than squamous cell carcinomas. All normal serum CgA levels corresponded to a lack of CgA expression in the tumors. The increased serum NSE levels presented by 26% of NSCLC patients (mainly <16 ng/mL) did not correlate with tumor NSE expression. CONCLUSIONS: The prevalence of neuroendocrine differentiation in NSCLC varies and depends on the immunohistochemical criteria used; this warrants standardization of the immunohistochemical criteria for neuroendocrine differentiation in NSCLC. NSE expression in the tumor and a mild increase in serum NSE are poor markers for distinguishing neuroendocrine differentiation in NSCLC.

[Invasive aspergillosis in autopsy material of patients treated at the Institute of Tuberculosis and Chest Diseases during the years 1993-2000]. / Inwazyjna aspergiloza w materiale autopsyjnym u chorych leczonych w Instytucie Gruzlicy i Chorób Pluc w latach 1993-2000.

The aim of this paper is an analysis of clinical documentation and results of autopsy of 21 patients (pts) who died of invasive aspergillosis (IA) in the Institute of Tuberculosis and Chest Diseases in years 1993-2000 and the assessment of predisposing factors for IA. In 17 pts IA was the main and in other 4 only an accessory cause of death. All pts were treated with corticosteroids and/or cytostatic drugs--because of lung cancer (11 pts), cancer in other site (2 pts), haematologic disorders (2 pts), Wegener's granulomatosis (1 pt), polymyositis (1 pt), idiopathic pulmonary fibrosis (1 pt) and other diseases (3 pts). In 15 out of 21 pts granulocytopenia was revealed (from 0.008 x 10(9)/L to 0.82 x 10(9)/L) on an average one month before death. In 15 pts IA was limited to the lungs, in 6 others there were also fungal lesions in brain, kidneys, liver, spleen and heart. Pts with disseminated form of IA had significantly lower granulocyte count and were treated with higher doses of corticosteroids than others. Immunosuppressive drugs and granulocytopenia can be regarded as predisposing factors. Fatal course of IA depended also on the late diagnosis.

[Alveolar hemorrhage caused by exposure to pesticides]. / Krwawienie pecherzykowe wywolane ekspozycja na pestycydy.

17 years old boy was admitted because of cough, hemoptysis and mild fever. These symptoms appeared a day after exposure to Decis-pesticide of relatively low toxicity for people. In hospital respiratory failure (pO2 48.5 mmHg) and alveolar haemorrhage (the presence of bloody fluid with hemosiderin loaded macrophages, the signs of alveolar filling in chest HRCT scan and elevation of diffuse capacity) were recognised. All symptoms completely disappeared after 5 month of corticosteroids therapy.

Fatal infection in patients treated for small cell lung cancer in the Institute of Tuberculosis and Chest Diseases in the years 1980-1994.

The study was performed to explore the frequency of infections present at death and infection as the main cause of death (fatal infection - FI) in 845 consecutive patients (pts) treated for small cell lung cancer (SCLC) at the Institute of Tuberculosis and Chest Diseases in Warsaw, in the period 1980-1994. Diagnosis of infection was based on clinical signs and symptoms, the presence of new lesions on the chest X-ray, microbiological tests and/or autopsy examination. All cases of fungal infection, Pneumocystis carinii pneumonia (PCP) and tuberculosis were proved by autopsy and microscopic examination (including special staining). FI was diagnosed if no progression of cancer was noted and no other complications occurred. Infection was present at the time of death in 116 patients (13.7%) and FI was the cause of death in 39 of them (4.6%). Nine patients died from fungal infection, eight from bacterial infection, seven from PCP and two from tuberculosis. In 13 cases the aetiology of infection found at autopsy was not determined. All FI patients received chemotherapy and corticosteroids, 16 of them also had radiotherapy on the tumour and mediastinum. Thirty-two out of 35 patients had leucopenia. The risk of death from infection was greater in patients above 60 years of age. Patients in bad performance status died of infection significantly earlier than others (P<0.05).

[Granulomatous lung lesions after occupational exposure to glass fibers]. / Ziarniniakowe zapalenie pluc po ekspozycji zawodowej na wate szklana.

39 years old man with granulomatous lesions in both lungs caused by occupational contact with glass fibers was described. He has been working as an bricklayer-plasterer for 18 years and was in contact with lime, cement, plaster, asbestos, dust of coal and wood and with glass fibers. For the last two years before admission in 1993 he has had frequent bronchial infections. On admission he was in good general condition, his spirometric examination and blood gases were within normal limits. On chest x-ray disseminated lesions were found. Those lesions were of the round shapes on chest CT. Many sputum cultures for tubercle bacilli were negative. ANA and ANCA were not found in the serum. ACE was within normal limits. No precipitins to environmental antigens were found. Cancer metastases were suspected and lung biopsy during videothoracoscopy was done. Many foreign body type granulomas were found throughout the specimen. The character of the lesions was not typical for tuberculosis, sarcoidosis, extrinsic allergic alveolitis, silicosis or asbestosis. There are some reports concerning the possibility of development of such lesions after the exposition to glass fibers. We suspect that case is an example of such pathology. His occupational exposition was stopped in 1993 and he was observed without treatment. During the 5 years of observation (up till 1998) he was in good health with stable chest x-ray picture and results of respiratory system function.

[The relation between lymphocyte profile in BALF and the course of sarcoidosis based on short term observation]. / Zaleznosc miedzy zawartoscia limfocytów w BALF a krótkotrwala obserwacja przebiegu sarkoidozy.

The aim of this study was to assess how the extent of the number and percentage of lymphocytes in BALF and also the CD4 to CD8 ratio can help to predict the short outcome in sarcoidosis. Material consisted of 74 patients, 39 men and 35 women in the age from 23 to 58 years. 11 patients had chest lesions in stage I, 43 in stage II and 20 in stage III. Clinical markers of activity (fever, erythema nodosum) were present in 22 cases. Extrathoracic lesion were present in 31 and abnormal pulmonary function in 30. In all patients BAL was done before treatment and lymphocyte count, percentage and CD4/CD8 ratio was calculated. 50 patients were treated with corticosteroids and 24 were observed without treatment. After 6-12 month of observation regression of sarcoid lesions was observed in 46 of 50 patients treated with corticosteroids and in 17 out of 24 patients observed without treatment. There were no differences in lymphocyte count and percentage in BALF and in the short term outcome between group treated with corticosteroids and without treatment. The patients in whom regression of lesions was observed have however significantly higher CD4/CD8 ratio than others.

[The use of monoclonal antibodies in the detection of small cell lung cancer metastases in bone marrow]. / Próba zastosowania przeciwcial monoklonalnych do wykrywania przerzutów drobnokomórkowego raka pluca w szpiku.

Expression of a number of antigens associated with small cell lung cancer (SCLC) have been proposed as a marker of malignancy and the diagnostic tool for the staging procedures and important prognostic factor. Since the bone marrow (BM) was described as a frequent site for SCLC metastases, we have decided to assess clinical importance of cancer cells detection in BM, using immunofluorescence with MAC-1, MAC-31, NSE and anti-Fucosyl-GM1 (PF3) antibodies. The group of 32 patients with SCLC was assessed using our panel of antibodies. Control group consisted of 5 patients with other malignancies (3 patients with malignant lymphoma, 1 with chronic lymphocytic leukaemia and 1 with non-SCLC). The study revealed no correlation between the expression of SCLC markers in patients BM and the cancer treatment outcome measured as a response for treatment, time to progression, and survival time, and no significant difference was found between the patients and control group.

Frequency and clinical significance of beta-subunit human chorionic gonadotropin expression in non-small cell lung cancer patients.

Human chorionic gonadotropin (HCG), a classic trophoblastic marker, has been found recently in many nontrophoblastic tumors. Previously we have found elevated serum betaHCG levels in 14% of small cell lung cancer patients. The aim of the present study was to assess the frequency and clinical significance of betaHCG expression in non-small cell lung tumors and in the sera of patients. 153 non-small cell lung cancer patients entered into this study. The control group consisted of 85 patients with benign lung diseases. Serum betaHCG elevation exceeding 5 mIU/ml was found in 3.5% of patients with benign lung diseases and in 12% of lung cancer patients (p = 0.03). Tumor analysis revealed the presence of betaHCG positivity in 28% of resected lung specimens. betaHCG positivity was found more often in adenocarcinoma than in squamous cell lung carcinoma both in tissue and in serum, the differences being not significant. Elevated serum betaHCG values were found more frequently in stage IV patients than in the remainder (p = 0.03). Response to chemotherapy (partial or minor response) was obtained more often in the patients with normal serum betaHCG than in those with serum betaHCG elevation (p = 0.03). We suppose that the ability to produce betaHCG is a rare but important biologic feature of lung carcinomas combined to some extent with chemoresistance.

[Usefulness of analyzing ANCA changes for diagnosis of Wegener's granulomatosis]. / Przydatnosc oznaczania miana ANCA dla diagnostyki ziarniniakowatosci Wegenera.

UNLABELLED: ANCA were described in 1982 as sensitive and specific markers for active Wegener's granulomatosis (WG). We analysed the results of ANCA test performed in 298 patients hospitalized in Institute of Tuberculosis and Lung Diseases in the period 1990-1998 because of different symptoms and syndromes of respiratory system which could be symptoms of WG. Presence of c-ANCA, p-ANCA and other not well defined types of ANCA in the titer greater than 1:40 in the serum was regarded as positive result of test. We found it in 60 patients. In 47 WG pts ANCA were present in 40 of 42 pts with active disease and 1 of 5 in remission. Further 19 positive results were found in a group of 251 patients with other diseases: 1 of 4 pts with Churg-Strauss syndrome, 1 of 2 with pulmonary renal syndrome, 5 of 28 with connective tissue diseases, 1 of 21 with tuberculosis, 1 of 23 with sarcoidosis, 1 of 6 with histiocytosis, 3 of 11 with hypersensitivity pneumonitis, 1 (lymphoma) of 34 with neoplasms, 1 of 20 with pulmonary fibrosis, 1 of 8 with cardiac failure, 1 of 5 with pleural fluid, 1 of 10 pneumoconiosis and toxic reactions (after furagin), 1 of 6 with BOOP. Sensitivity of ANCA test in our material is 87%, specificity = 95%, and positive prevalence accuracy is 68%. It means that 32% of the patients with positive results could be inappropriate treated as WG. CONCLUSION: ANCA test could be not used as a screening test. Results of ANCA test alone cannot be used as basis for treatment. ANCA test is a helpful tool in diagnosing of WG.

[Infection as a main or additional cause of death in patients treated for small cell lung cancer]. / Zakazenia jako glówna lub dodatkowa przyczyna zgonu u chorych leczonych z powodu drobnokomórkowego raka pluca.

The aim of this study was to analyse the frequency of infection as a cause of death in small cell lung cancer (SCLC) patients. Our material consisted of 845 unselected SCLC patients, 246 women and 599 men, aged 29-78 years, treated between 1980-1994 in the Institute of Tuberculosis in Warsaw. 479 patients had limited and 366 extensive disease. 530 were in good (0-2) and 315 in bad (3-4) performance status. 784 patients died. Autopsy was done in 211 patients. Infection was regarded as a main cause of death in 39 patients (4.6%) and as a coexistent cause in 77 (9.1%). At the time of death from and/or with infection in 16 patients complete remission and in 27 partial remission of cancer was confirmed. The risk of death from and/or with infection was not related to the age and sex or to the performance status of patients and to extension of cancer. The risk of death from and/or with infection in the first 3 months of treatment was however greater for patients in bad performance status and with extensive disease and later (after 3rd months) for patients in good performance status and with limited disease.

[Cytostatic treatment of patients with advanced non-small cell lung cancer]. / Leczenie cytoredukcyjne chorych na zaawansowanego niedrobnokomórkowego raka pluca.

92 patients with advanced non-small cell lung cancer were treated with cisplatin 80 mg/m2 day 1 and etoposide 120 mg/m2 on days 1-3. In 58 of them vinblastine 5 mg/m2 was also applied on days 1 and 3. In 25% of all cases partial response and in another 26% minimal regression was found after 2 courses of chemotherapy, independently to treatment modality. Partial regression was observed significantly more often in patients with adenocarcinoma, but survival time was significantly shorter in this group. Median survival time was 8 months for all patients, 10 months for stage IIIB and 6 months for stage IV. This difference was significant.

[The value of diagnostic methods used to assess the response to treatment in patients with limited small cell lung carcinoma]. / Wartosc metod oceniajacych odpowiedz na leczenie u chorych na ograniczona postac drobnokomórkowego raka pluca.

Tumor response is used as a main criterion whether or not the treatment yields an anticancer activity. The tumor response criteria are defined by WHO recommendation but little is known about the tests must be used. The aim of this paper was to compare the degree of response to the treatment of 268 patients with limited small cell lung cancer, using independently 3 methods: radiological, bronchoscopic and cytological of bronchial material. Particular categories of response (CR, PR NR and presence or absence of carcinomatous cells) were related to survival time of patients independently to method of assessment. Multivarinte Cox analysis selected 3 parameters related to 3 different methods as independent survival risk factors. We conclude that each of diagnostic method (chest x-ray, bronchoscopy, cytological examination of bronchial material yield independent information correlated with survival risk of particular patient.

[Chemotherapy versus chemoradiotherapy in patients with limited small cell lung carcinoma]. / Chemioterapia versus naprzemienna chemio-radioterapia u chorych z ograniczona postacia drobnokomórkowego raka pluca.

62 patients with a limited small cell lung cancer were randomly qualified into two groups. 32 patients of the first group were treated only with the chemotherapy regimen, consisted of three drugs (Carboplatine, Etoposide and Vincristine administered in 6 courses, on regular, 3-weeks basis). The second group of 30 patients had been treated with the identical chemotherapy schedule, but alternatively combined with a primary site irradiation in a total dose of 40Gy, applied in parts after the chemotherapy courses 2, 3, and 4. The significantly higher proportion of a complete remission results was observed in the alternate-treatment group: 14/30 (46.7%), compared with the chemotherapy-only group: 10/32 (31%). Alternate chemoradiotherapy resulted both in the increased median remission duration time, and the increased median survival time. Only in the alternate chemotherapy group, in 14/30 patients (46.7%) the pneumotoxicity symptoms appeared, whilst no differences in other organ-specific treatment-induced toxic effects were noted.

[Soft tissue sarcomas as a rare cause of pleural effusion]. / Miesaki tkanek miekkich jako rzadka przyczyna wysiekowego zapalenia oplucnej.

Malignant disease is the cause of about 24% of all pleural effusions. They are caused mainly by lung and breast cancer. Three cases of pleural effusion caused by very rare neoplasm, soft tissues sarcoma are presented. In two of them the lesion found in the leg was observed for 4-14 month and not connected with the presence of pleural effusion. Difficulties in the histologic diagnosis of pleural sarcoma and of differentiating this tumour from mesothelioma are also presented.

Neuron-specific enolase (NSE) serum level in non small cell lung cancer--can it be an indicator of tumour chemosensitivity?

It is believed that the tissue or serum expression of neuroendocrine markers in non small cell lung cancer patients can implicate better prognosis in cases undergoing chemotherapy. The aim of the study was to assess the value of NSE serum level for anticipation of the tumor response to chemotherapy. We found elevated serum level of NSE in 34 of 60 patients (56.7%) at the time of diagnosis of inoperable non small cell lung cancer, significantly more often in those presenting stage IV of disease. In 21.7% partial response and in another 21.7% minimal regression was found after chemotherapy. Treatment results revealed no significant differences in respect to NSE serum level, however 77% of patients achieving partial response had elevated serum level of NSE.

The clinical value of Cyfra 21-1 estimation for lung cancer patients.

Cytokeratin-19, one of the cytoskeletal proteins, is expressed both in bronchial epithelium and in lung cancer cells. The aim of our study was to establish the value of serum cytokeratin-19 soluble fragment (Cyfra 21-1) measurement in lung cancer patients. Cyfra 21-1 levels were estimated in 35 patients (pts) with benign lung diseases and in 116 lung cancer patients: 55 pts with squamous cell lung cancer, 38 pts with small cell lung cancer and 23 pts with adenocarcinoma. The cutoff level was set at 4 ng/ml with a specificity of 94% and a sensitivity of 40%. Elevated Cyfra 21-1 values were found in 44% of squamous cell lung cancer, 39% of adenocarcinoma and 34% of small cell lung cancer pts (the difference was not significant). In squamous cell lung cancer and in adenocarcinoma elevated Cyfra 21-1 values were observed more often in patients with advanced disease than in patients with limited disease. There was no significant correlation between the initial Cyfra 21-1 level and the response to chemotherapy. Cyfra 21-1 was not a prognostic indicator, although in operable squamous cell lung cancer the proportion of survivors in the second year of observation was higher among the patients with normal preoperative Cyfra 21-1 levels.

[The value of determining neuron specific enolase for diagnosis of complete remission during treatment of small cell lung cancer]. / Wartosc oznaczania neuroswoistej enolazy NSE dla rozpoznania remisji calkowitej w przebiegu leczenia drobnokomórkowego raka pluca.

The aim of this study was to assess whether the lowest serum NSE obtained during treatment of small cell lung cancer patients can be helpful in the diagnosis of complete remission (CR). The material consisted of 68 patients with small cell lung cancer, treated in the Institute o Tuberculosis and Lung Diseases from 1.III.1993 to 15.II.1995. In the course of treatment CR was obtained in 13 patients, partial remission (PR) in 37 and no remission (NR) in 18. The distribution and median of the lowest NSE serum levels were the same in CR and RP patients. NSE serum levels remained above normal, that is above 12.5 ng/ml, in two CR patients and in 4 PR patients. 3 patients (2 with CR and I with PR) are still living for 26, 27 and 41 months in spite of NSE serum levels 14.3, 15.6 and 13.6 ng/ml respectively. In those patients in whom NR was obtained the lowest NSE level above 20 ng/l was connected with bad prognosis. We conclude that the estimation of the lowest NSE serum level in the course of treatment can not help to differentiate CR from PR.