How a discerning cytological examination can aid in the diagnosis of infectious diseases: case reports.

Infections caused by uncommon and resistant pathogens in unusual sites have been increasingly reported in medical literature. We describe four cases of rare cytological findings and clinical impact for patients. In the first case, Aspergillus sp and Pneumocystis jirovecii were observed in the bronchoalveolar lavage of a patient with severe systemic lupus. In the second and third cases, we describe the presence of Trichomonas sp and Strongyloides sp larvae in samples of pleural and peritoneal fluid, respectively. The fourth report is about a patient with a wrist subcutaneous nodule whose synovial aspiration and cytology revealed the presence of brown septate hyphae. The early identification of the infectious agent in the cytological examination was essential for the introduction and/or re-adaptation of therapy in the four cases described. Patients in this report were immunocompromised with severe comorbidities, conditions often associated with unfavorable clinical outcomes.

How a discerning cytological examination can aid in the diagnosis of infectious diseases: case reports

Infections caused by uncommon and resistant pathogens in unusual sites have been increasingly reported in medical literature. We describe four cases of rare cytological findings and clinical impact for patients. In the first case, Aspergillus sp and Pneumocystis jirovecii were observed in the bronchoalveolar lavage of a patient with severe systemic lupus. In the second and third cases, we describe the presence of Trichomonas sp and Strongyloides sp larvae in samples of pleural and peritoneal fluid, respectively. The fourth report is about a patient with a wrist subcutaneous nodule whose synovial aspiration and cytology revealed the presence of brown septate hyphae. The early identification of the infectious agent in the cytological examination was essential for the introduction and/or re-adaptation of therapy in the four cases described. Patients in this report were immunocompromised with severe comorbidities, conditions often associated with unfavorable clinical outcomes.

Detection of sputum cofilin-1 as indicator of malignancy.

Cofilin-1 (CFL1), a small protein of 18 kDa, has been studied as a biomarker due to its involvement in tumor cell migration and invasion. Our aim was to evaluate CFL1 as an indicator of malignancy and aggressiveness in sputum samples. CFL1 was analyzed by ELISA immunoassay in the sputum of 73 lung cancer patients, 13 cancer-free patients, and 6 healthy volunteers. Statistical analyses included ANOVA, ROC curves, Spearman correlation, and logistic regression. Sputum CFL1 levels were increased in cancer patients compared to cancer-free patients and volunteers (P<0.05). High expression of sputum CFL1 was correlated to T4 stage (P=0.01) and N stage (P=0.03), tobacco history (P=0.01), and squamous cell carcinoma histologic type (P=0.04). The accuracy of sputum CFL1 in discriminating cancer patients from cancer-free patients and healthy volunteers were 0.78 and 0.69, respectively. CFL1 at a cut-off value of 415.25 pg/mL showed sensitivity/specificity of 0.80/0.70 in differentiating between healthy volunteers and cancer patients. Sputum CFL1 was also able to identify cancer-free patients from patients with lung cancer. The AUC was 0.70 and, at a cut-off point ≥662.63 pg/mL, we obtained 60% sensitivity and 54% specificity. Logistic regression analysis controlled for tobacco history, histologic types, and N stage showed that cancer cell-associated CFL1 was an independent predictor of death. Smoker patients with squamous cell carcinoma, lymph node metastasis and sputum CFL1>1.475 pg/mL showed augmented chance of death, suggesting lung cancer aggressiveness. CFL1 presented diagnostic value in detecting lung cancer and was associated to tumor aggressiveness.

Detection of sputum cofilin-1 as indicator of malignancy

Cofilin-1 (CFL1), a small protein of 18 kDa, has been studied as a biomarker due to its involvement in tumor cell migration and invasion. Our aim was to evaluate CFL1 as an indicator of malignancy and aggressiveness in sputum samples. CFL1 was analyzed by ELISA immunoassay in the sputum of 73 lung cancer patients, 13 cancer-free patients, and 6 healthy volunteers. Statistical analyses included ANOVA, ROC curves, Spearman correlation, and logistic regression. Sputum CFL1 levels were increased in cancer patients compared to cancer-free patients and volunteers (P<0.05). High expression of sputum CFL1 was correlated to T4 stage (P=0.01) and N stage (P=0.03), tobacco history (P=0.01), and squamous cell carcinoma histologic type (P=0.04). The accuracy of sputum CFL1 in discriminating cancer patients from cancer-free patients and healthy volunteers were 0.78 and 0.69, respectively. CFL1 at a cut-off value of 415.25 pg/mL showed sensitivity/specificity of 0.80/0.70 in differentiating between healthy volunteers and cancer patients. Sputum CFL1 was also able to identify cancer-free patients from patients with lung cancer. The AUC was 0.70 and, at a cut-off point ≥662.63 pg/mL, we obtained 60% sensitivity and 54% specificity. Logistic regression analysis controlled for tobacco history, histologic types, and N stage showed that cancer cell-associated CFL1 was an independent predictor of death. Smoker patients with squamous cell carcinoma, lymph node metastasis and sputum CFL1>1.475 pg/mL showed augmented chance of death, suggesting lung cancer aggressiveness. CFL1 presented diagnostic value in detecting lung cancer and was associated to tumor aggressiveness.

Pleural fluid tumour markers in malignant pleural effusion with inconclusive cytologic results.

BACKGROUND: The presence of tumour cells in pleural fluid or tissue defines an effusion as malignant. Cytology analysis of the pleural fluid has about 60% diagnostic sensitivity. Several tests have been proposed to improve diagnosis-among them, the concentrations of tumour markers in pleural fluid. We evaluated whether the concentrations of tumour markers in pleural fluid could improve the diagnosis of malignant pleural effusion (mpe) when cytology is doubtful. METHODS: Lymphocytic pleural fluids secondary to tuberculosis or malignancy from 156 outpatients were submitted for cytology and tumour marker quantification [carcinoembryonic antigen (cea), cancer antigen 15-3 (ca15-3), carbohydrate antigen 19-9 (ca19-9), cancer antigen 72-4 (ca72-4), cancer antigen 125 (ca125), and cyfra 21-1). Oneway analysis of variance, the Student t-test or Mann-Whitney test, and receiver operating characteristic curves were used in the statistical analysis. RESULTS: Concentrations of the tumour markers cea, ca15-3, ca125, and cyfra 21-1 were higher in mpes than they were in the benign effusions (p < 0.001), regardless of cytology results. The markers ca19-9 and ca72-4 did not discriminate malignant from benign effusions. When comparing the concentrations of tumour markers in mpes having positive, suspicious, or negative cytology with concentrations in benign effusions, we observed higher levels of cea, ca15-3, cyfra 21-1, and ca125 in malignant effusions with positive cytology (p = 0.003, p = 0.001, p = 0.002, and p = 0.001 respectively). In pleural fluid, only ca125 was higher in mpes with suspicious or negative cytology (p = 0.001) than in benign effusions. CONCLUSIONS: Given high specificity and a sensitivity of about 60%, the concentrations of tumour markers in pleural effusions could be evaluated in cases of inconclusive cytology in patients with a high pre-test chance of malignancy or a history of cancer.

Effect of temperature and storage time on cellular analysis of fresh pleural fluid samples.

OBJECTIVE: Despite the methodological variability in preparation techniques for pleural fluid cytology, it is fundamental that the cells should be preserved, permitting adequate morphological classification. We evaluated numerical and morphological changes in pleural fluid specimens processed after storage at room temperature or under refrigeration. METHODS: Aliquots of pleural fluid from 30 patients, collected in ethylenediaminetetraacetic acid-coated tubes and maintained at room temperature (21 °C) or refrigeration (4 °C) were evaluated after 2 and 6 hours and 1, 2, 3, 4, 7 and 14 days. Evaluation of cytomorphology and global and percentage counts of leucocytes, macrophages and mesothelial cells were included. RESULTS: The samples had quantitative cellular variations from day 3 or 4 onwards, depending on the storage conditions. Morphological alterations occurred earlier in samples maintained at room temperature (day 2) than in those under refrigeration (day 4). CONCLUSIONS: This study confirms that storage time and temperature are potential pre-analytical causes of error in pleural fluid cytology.

Pleural fluid cytokines correlate with tissue inflammatory expression in tuberculosis.

SETTING: A tertiary care research centre in São Paolo, Brazil. OBJECTIVE: To quantify interleukin (IL) 8, tumour necrosis factor alpha (TNF-alpha), vascular endothelial growth factor (VEGF) and transforming growth factor beta(1) (TGF-beta(1)) in pleural fluid from tuberculous patients, correlating its values with the histopathological patterns in pleural biopsies. DESIGN: Cytokines were quantified in patients with transudates secondary to congestive heart failure (n = 8) and exudates secondary to tuberculosis (TB; n = 39). In parietal pleural biopsies from TB patients, the histological patterns of the inflammatory response were quantified by morphometric analysis (stereological point-counting method). RESULTS: IL-8, TNF-alpha, VEGF and TGF-beta(1) levels were higher in TB than in transudates. A positive correlation existed between components of the fibrinoid exudative phase with pleural fluid IL-8 (R = 0.52, P = 0.004) and VEGF (R = 0.42, P = 0.0021) levels. A negative correlation existed between pleural fluid IL-8 (R = -0.37, P = 0.048) and VEGF (R = -0.44, P = 0.0015) levels with tissue components of fibroproliferation. CONCLUSION: The high pleural levels of TNF-alpha, IL-8, VEGF and TGF-beta(1) suggest the involvement of these cytokines in the TB immunological response. The positive correlation between pleural fluid IL-8 and VEGF with the components of the acute exudative phase and the negative correlation between these cytokines with the fibroproliferative components suggest a temporary inflammatory response in the pleural space.

Cytokine profile in pleural fluid and serum after lung transplantation.

BACKGROUND: Lung transplantation is the procedure of choice in several end-stage lung diseases. Despite improvements in surgical techniques and immunosuppression, early postoperative complications occur frequently. OBJECTIVE: To evaluate the pleural inflammatory response after surgery. PATIENTS AND METHODS: Twenty patients aged 18 to 63 years underwent unilateral or bilateral lung transplantation between August 2006 and March 2008. Proinflammatory cytokines interleukin (IL)-1beta, IL-6, and IL-8 and vascular endothelial growth factor in pleural fluid and serum were analyzed. For cytokine evaluation, 20-mL samples of pleural fluid and blood (right, left, or both chest cavities) were obtained at 6 hours after surgery and daily until removal of the chest tube or for a maximum of 10 days. Data were analyzed using analysis of variance followed by the Holm-Sidak test. RESULTS: All effusions were exudates according to Light's criteria. Pleural fluid cytokine concentrations were highest at 6 hours after surgery. Serum concentrations were lower than those in pleural fluid, and IL-1beta, IL-6, and IL-8 were undetectable at all time points. CONCLUSIONS: There is a peak concentration of inflammatory cytokines in the first 6 hours after transplantation, probably reflecting the effects of surgical manipulation. The decrease observed from postoperative day 1 and thereafter suggests the action of the immunosuppression agents and a temporal reduction in pleural inflammation.

Acute inflammatory response secondary to intrapleural administration of two types of talc.

Intrapleural instillation of talc has been used in the treatment of recurrent pleural effusions but can, in rare instances, result in respiratory failure. Side-effects seem to be related to composition, size and inflammatory power of talc particles. The aim of this study was to evaluate the inflammatory response to intrapleural injection of talc containing small particles (ST) or talc containing particles of mixed size (MT). 100 rabbits received intrapleural talc, 50 with ST (median 6.41 mum) and 50 with MT (median 21.15 mum); the control group was composed of 35 rabbits. Cells, lactate dehydrogenase, C-reactive protein (CRP), interleukin (IL)-8 and vascular endothelial growth factor were evaluated in serum and bronchoalveolar lavage at 6, 24, 48, 72 and 96 h. Lung histology and the presence of talc were also analysed. Statistics were performed using ANOVA and an unpaired t-test. Most of the parameters showed greater levels in the animals injected with talc than in the controls, suggesting a systemic and pulmonary response. Higher serum levels of CRP and IL-8 were observed in the animals injected with ST. Talc particles were observed in both lungs with no differences between groups. Lung cell infiltrate was more evident in the ST group. In conclusion, talc with larger particles should be the preferred choice in clinical practice in order to induce safer pleurodesis.

Evidence that mesothelial cells regulate the acute inflammatory response in talc pleurodesis.

Intrapleural instillation of talc is used to produce pleurodesis in cases of recurrent malignant pleural effusions. The mechanisms by which pleurodesis is produced remain unknown but may involve either injury or activation of the mesothelium. The aim of the current study was to assess the inflammatory response of pleural mesothelial cells to talc in an experimental model in rabbits. A group of 10 rabbits were injected intrapleurally with talc (200 mg.kg(-1)) and undiluted pleural fluid was collected after 6, 24 or 48 h for measurement of interleukin (IL)-8, vascular endothelial growth factor (VEGF) and transforming growth factor (TGF)-beta1. Samples of pleura were studied to assess the inflammatory infiltrate and mesothelial cell viability. The pleural fluid IL-8 concentration peaked at 6 h, whereas VEGF and TGF-beta1 concentrations increased steadily over 48 h. Immunohistochemistry for cytokeratin showed a preserved layer of mesothelial cells despite the intense inflammatory pleural reaction. In conclusion, it is proposed that the mesothelial cell, although injured by the talc, may actively mediate the primary inflammatory pleural response in talc-induced pleurodesis.

Respiratory mechanics and pleural remodelling in pleurodesis induced by barium sulphate.

The aim of this study was to determine whether an intrapleural injection of barium sulphate would produce pleurodesis in rats. Additionally, respiratory mechanics and pleural remodelling were analysed. Single intrapleural injection of barium sulphate (100%) or saline was given to Wistar rats. Respiratory system, lung, and chest wall elastic, resistive and viscoelastic/inhomogeneous pressures were measured by the end-inflation occlusion method at 2 and 30 days after injection. The pleura were examined for gross and histopathological evidence of pleural inflammation and fibrosis, and the underlying lungs were also studied by morphometry. All pulmonary mechanical parameters increased at day 2, but were not different from control at 30 days after injection. Chest wall mechanical parameters did not change. Macroscopic evaluation demonstrated pleural adherence without haemothorax. Histopathologic analysis showed pleural inflammation and fibrosis. There was no alveolar inflammation or fibrosis in both groups. In conclusion, barium sulphate induced pleurodesis with either no changes in respiratory mechanics or lung lesion at day 30.

Acute pulmonary and hematological effects of two types of particle surrogates are influenced by their elemental composition.

Several epidemiological studies have consistently demonstrated significant associations between ambient levels of particulate matter and lung injury and cardiovascular events with increased morbidity and mortality. Particle surrogates (PS), such as residual oil fly ash (ROFA), have been widely used in experimental studies aimed at characterizing the mechanisms of particle toxicity. Since PS composition varies depending on its source, studies with different types of PS may provide clues about the relative toxicity of the components generated by high-temperature combustion process. In this work, we have studied the effects of nasal instillation of increasing doses of different PS in mice: saline, carbon, and two types of particle surrogates. PS type A (PSA) was the ROFA collected from the waste incinerator of our university hospital; PS type B (PSB) was collected from the electrostatic precipitator of a large steel company and thus had an elevated metal content. After 24h, we analyzed hematological parameters, fibrinogen, bronchoalveolar lavage, bone marrow, and pulmonary histology. Nasal instillation of the two types of PS-induced leucopenia. PSB elicited a greater elevation of plasma fibrinogen levels. Bone marrow and pulmonary inflammatory changes were more intense for PSA. We concluded that the PS composition modulates acute inflammatory changes more significantly than the mass for these two types of PS.

Tumour cell and stromal features in metastatic and non-metastatic non-small cell lung carcinomas.

AIMS: Tumour cell behaviour depends on the interactions between nuclear genetic changes in the malignant cells and a stroma favourable for growth, invasion and metastasis. To evaluate such interactions, we studied the relationship between tumour cell and stromal features for proliferative factors, p53, microvessel density and metalloproteinases, controlled for the extent of the primary lesion (T1 to T4), in early (non-metastatic) and late (metastatic) non-small cell lung carcinomas (NSCLC). METHODS AND RESULTS: Variables were examined for differences and correlations in the frequency of p53, AgNOR, CD34 and MMP-9 expression in primary lesions and metastases of NSCLC using a general linear model. The patients included 58 males and 22 females (mean age 62 +/- 9 years) with 19 T1 (23.8%), 40 T2 (50.0%), 14 T3 (17.5%) and seven T4 (8.8%). In late disease, AgNOR and p53 were statistically related to the extent of the primary lesion, whereas in early disease AgNOR tended to be increased in tumours without metastasis, while p53 expression tended to decrease progressively in tumours with metastasis. Microvessel density in late disease was of no statistical significance, whereas in early disease strong CD34 expression was seen in tumours with metastasis, being at its maximum in T3 primary lesions. The best marker for the extent of the lesion and its progression was MMP-9, with greater expression by tumours with metastasis than those without. CONCLUSIONS: Different tumour cell and stromal interactions control metastasis and therefore the biological risk of NSCLC. A panel of molecular markers, such as p53, MMP-9 and CD34 could help to identify subgroups of patients that could benefit from adjuvant therapy.

Low concentration silver nitrate pleurodesis in rabbits: optimal concentration for rapid and complete sclerosing effect.

Pleurodesis is a useful therapeutic tool when local treatment of a recurrent malignant pleural effusion or pneumothorax is needed. We have previously demonstrated that the intrapleural injection of 0.5% silver nitrate (SN) produces a significant pleurodesis, while 0.25% SN has no sclerosing effect in a rabbit model. The objective of this study was to determine the minimum concentration of SN needed to induce pleurodesis in our experimental model. One hundred twenty male New Zealand white rabbits received 0.3, 0.4, or 0.5% SN (40 animals per group) in a total volume of 2 mL instilled intrapleurally. These animals were sacrificed 3, 7, 14 or 28 days after the intrapleural injection (n = 10 animals per group), and the pleural spaces were then assessed grossly for evidence of pleurodesis and microscopically for evidence of fibrosis and inflammation. By 28 days, all concentrations of SN had produced a pleurodesis. There was evidence of a gross pleurodesis 7 days post-injection in animals that received 0.5% SN (score of 2.8 +/- 0.2 on a scale of 0-4). After 14 days, significant pleural adhesions were evident in the groups that received 0.4 or 0.5% SN. We conclude that SN concentrations as low as 0.3% can effectively produce a pleurodesis within 28 days of intrapleural injection. However, the precocious pleurodesis development observed 7 days after the intrapleural injection of 0.5% SN suggests that this concentration may be optimal when a fast result is necessary.

Experimental pleurodesis in rabbits induced by silver nitrate or talc: 1-year follow-up.

STUDY OBJECTIVE: To compare the pleurodesis results from the intrapleural injection of silver nitrate and talc slurry over an observation period of 12 months in rabbits. DESIGN: Rabbits were randomized to receive 2 mL of 0.5% silver nitrate or 400 mg/kg of talc slurry in 2 mL intrapleurally. Ten rabbits in each group were killed at 1 month, 2 months, 4 months, 6 months, 8 months, 10 months, and 12 months after intrapleural injection. The degree of gross pleurodesis and the amount of microscopic pleural fibrosis and inflammation were graded on a scale of 0 to 4. RESULTS: The mean +/- SEM gross pleurodesis score in the 70 rabbits that received silver nitrate was 3.34 +/- 0.08, which was significantly higher than the score of 2.32 +/- 0.09 in the 70 rabbits that received talc. The mean gross pleurodesis score was significantly higher at each of the observation times (p < 0.05), except at 2 months, in the rabbits that received silver nitrate. The pleurodesis was distributed throughout the thorax in the rabbits that received silver nitrate, while it was only in the ventral thorax in the rabbits that received talc slurry. The gross pleurodesis scores showed no tendency to decrease during the 12-month observation period in either treatment group. The persistence of talc in the pleural space did not lead to chronic inflammatory changes because the inflammation scores were similar in both groups at all observation times. The microscopic pleural fibrosis score tended to decrease with time in the silver nitrate group but not in the talc slurry group. CONCLUSIONS: The intrapleural injection of 2 mL of 0.5% silver nitrate produces a better pleurodesis than does the intrapleural injection of 400 mg/kg of talc slurry in rabbits. The pleurodesis induced by silver nitrate persists for at least 1 year. The efficacy of silver nitrate as a sclerosing agent in humans should be evaluated.

Relationship between pleural fluid and serum cholesterol levels.

INTRODUCTION: Since the criteria of Light and colleagues for differentiating transudates and exudates were described, other tests, including the pleural fluid (PF) cholesterol test, have been proposed for the same purpose. However, the factors influencing PF cholesterol levels have not been clearly delineated. PURPOSE: To analyze the relationships among total cholesterol (CHOL), low-density lipoprotein (LDL) cholesterol, high-density lipoprotein (HDL) cholesterol, and triglycerides (TRIG) in serum (S) and PF. METHODS: PF and S from 99 patients (transudates, 13 patients; exudates, 86 patients) were analyzed for CHOL, HDL, LDL, TRIG, apolipoprotein AI, apolipoprotein B, and protein. The relationship between the PF and S level for each of these measurements was analyzed with linear regression and multiple regression using the ratio of PF to S protein for that measurement as a second independent variable. RESULTS: This study demonstrated that CHOL levels in PF are related to S cholesterol levels and to the permeability of the pleura (r = 0.88; p < 0.001). However, the percentage of CHOL associated with LDL and HDL (56%) in the PF was much lower than that associated with LDL and HDL in S (93%), suggesting that lipoproteins are modified once they enter the pleural space. The PF TRIG was not closely related to its S level or to the PF/S protein ratio (r = 0.49). CONCLUSION: PF cholesterol levels can be closely predicted from the S cholesterol levels and the permeability of the pleura, as reflected by the ratio of PF protein to S protein. Therefore, the CHOL ratio should not provide additional information to that provided by the protein ratio when trying to differentiate transudates from exudates. PF lipoproteins (LDL and HDL) undergo metabolic alterations once they enter the pleural space. PF TRIG levels are not closely related to S levels or to the permeability of the pleura.

Impact of BAL in the management of pneumonia with treatment failure: positivity of BAL culture under antibiotic therapy.

BACKGROUND: Pneumonia is responsible for 50% of antibiotics prescribed in ICUs. Treatment failure, ie, absence of improvement or clinical deterioration under antibiotic therapy, presents a dilemma to physicians. BAL is an invasive method validated for etiologic diagnosis in pneumonia. STUDY OBJECTIVE: To evaluate in ICU patients the impact of BAL in the etiologic diagnosis, treatment, and outcome of pneumonia with treatment failure. DESIGN: Prospective clinical study. SETTING: Nonsurgical, medical ICU of a university hospital in Brazil. PATIENTS AND PARTICIPANTS: Sixty-two episodes of pneumonia treated for at least 72 h without clinical improvement in 53 patients hospitalized for diverse clinical emergencies. Mean duration of hospitalization was 14.2 days. Mean duration of previous antibiotic therapy was 11.4 days. INTERVENTIONS: Bronchoscopy and BAL were performed in each episode. BAL fluid was cultivated for aerobic and anaerobic bacteria; the cutoff considered positive was 10(4) cfu/mL; 10(3) cfu/mL was also analyzed if under treatment. Pneumocystis carinii, fungi, Legionella spp, and Mycobacterium spp were also researched. MEASUREMENTS AND RESULTS: Fifty-eight of 62 BAL were performed under antibiotics. The results showed positivity in 45 of 62 (72.6%); 42 of the 45 positive episodes (93.3%) had > 10(4) cfu/mL. The three cases with between 10(3) and 10(4) cfu/mL were considered positive and were treated according to BAL cultures. The main agents were Acinetobacter baumannii (37.1%), Pseudomonas aeruginosa (17.7%), and methicillin-resistant Staphylococcus aureus (MRSA; 16.1%); 46.7% of the episodes (21 of 45) were polymicrobial. BAL results directed a change of therapy in 34 episodes (54.8%). Overall mortality was 43.5%. There was no difference in mortality among positives, negatives, and patients who changed therapy guided by BAL culture. CONCLUSIONS: (1) BAL fluid examination was positive in 45 of 62 episodes (72.6%), with 58 of 62 BAL performed under antibiotics. This suggests that BAL may be a sensitive diagnostic method for treatment failures of clinically diagnosed pneumonias, even if performed under antibiotics; (2) the main pathogens in our study were A baumannii, P aeruginosa, and MRSA, and approximately 45% of infections were polymicrobial; (3) BAL culture results directed a change of therapy in 75.6% of positive episodes (34 of 45) and in 54.8% of all episodes of treatment failure (34 of 62); and (4) there was no difference in mortality among positives, negatives, and patients who changed therapy guided by BAL culture.

Useful prognostic panel markers to express the biological tumor status in resected lung adenocarcinomas.

BACKGROUND: Tumor stage and its histological subtype remain the most important predictors of clinical behavior in current pulmonary practice of lung cancer. However, many investigators agree that these parameters are not sufficient to predict which tumor will recur, even after radical curative surgery. Therefore, it is necessary to evaluate the significance of other morphological, biological and molecular parameters beyond TNM classification. METHODS: Pathological specimens were collected from 45 patients after resection for stage IA (five), stage IB (10), stage IIB (10), stage IIIA (14) and stage IV (six) lung adenocarcinomas. A panel of two morphological (proportion of stroma within the tumor and degree of tumor differentiation), two biological [DNA ploidy and argyrophilic nucleolar organizer region (AgNOR)] and three molecular (immunohistochemical expression of Ki-67, p53 and bcl-2) markers was chosen for analysis of the primary tumor. Life Tables for Survival were used to analyze the individual impact of each variable on survival. Cox proportional hazards model analysis was used to construct an independent tumor status model for cancer recurrence and death. Chi-squared analyses were used to determine the statistically significant relationship among all the variables present in the study. RESULTS: Multivariate analysis demonstrated statistically significant risk for the following markers: AgNOR, p53 and bcl-2, controlled for stages and surgical resection. CONCLUSIONS: The immunohistochemical expression of p53 and bcl-2 oncogenes and the expression of AgNOR cell proliferation index are critical values in the progression of lung adenocarcinomas. They can express the biological tumor status and indicate a more accurate prognosis.

Acute and chronic pleural changes after the intrapleural instillation of mitoxantrone in rabbits.

A previous study demonstrated that the intrapleural injection of 2 mg/kg mitoxantrone in rabbits resulted in a degree of pleurodesis which is comparable to that seen after 35 mg/kg tetracycline but that the animals had a high mortality rate after this dose of mitoxantrone. The objective of the present study was to assess the acute pleural fluid findings, the acute gross and microscopic pleural findings, and the chronic gross and microscopic findings in rabbits that received mitoxantrone. Mitoxantrone, 1.5 mg/kg, was instilled intrapleurally in 70 lightly anesthetized male rabbits. Groups of rabbits were sacrificed 1, 2, 4, 7, 15, 28, and 60 days after the injection. The intrapleural injection of mitoxantrone resulted in an exudative effusion on day 1. The pleural fluid contained predominantly neutrophils and had a mean lactate dehydrogenase (LDH) level that exceeded 4,000 IU/liter. Over the following week the volume of fluid diminished, the predominant cell became the macrophage, and the LDH levels decreased to less than 400 IU/liter. Macroscopic examination of the pleural space revealed that the mean degree of pleurodesis increased progressively over the 60-day observation period. With microscopy, the mean degree of pleural fibrosis also increased progressively. There were also substantial fibrosis and inflammation of the underlying lung and the contralateral lung. The mortality rates were low in the first 28 days (3/70) but subsequently increased and exceeded 80% in the period between 60 and 120 days. This experimental model of pleurodesis should be useful in future studies directed toward uncovering the mechanisms of pleurodesis.

Prognostic significance of argyrophilic nucleolar organizer region (AgNOR) in resected non-small cell lung cancer (NSCLC).

The expression of the nucleolar organizer regions (NORs) was quantified in paraffin sections of tumors and lymph node metastasis, by means of digital image analysis, in 75 patients with resected non-small cell lung cancer (NSCLC). Patients were divided in two groups: early stage (stages I and II) and advanced stage (stages IIIa, IIIb and IV). The prognostic significance of AgNOR expression was tested by Cox regression analysis in models controlled for age, sex, vital status, stage and histological type. Tumors at early stages had a lower expression of AgNOR than those at more advanced diseases. The mean values obtained for NORs in advanced disease were almost the same as those in the primary tumors when compared with the corresponding lymph node metastasis (r = 0.90; p < 0.01; linear regression). The prognostic role of AgNOR was significant only for tumors at stages I and II and not for advanced neoplasms (stages IIIa, IIIb and IV). These results encourage the inclusion of AgNOR quantitation in routine material, especially in early lung cancer.