Survival analysis of the CEAwatch multicentre clustered randomized trial.

BACKGROUND: The CEAwatch randomized trial showed that follow-up with intensive carcinoembryonic antigen (CEA) monitoring (CEAwatch protocol) was better than care as usual (CAU) for early postoperative detection of colorectal cancer recurrence. The aim of this study was to calculate overall survival (OS) and disease-specific survival (DSS). METHODS: For all patients with recurrence, OS and DSS were compared between patients detected by the CEAwatch protocol versus CAU, and by the method of detection of recurrence, using Cox regression models. RESULTS: Some 238 patients with recurrence were analysed (7·5 per cent); a total of 108 recurrences were detected by CEA blood test, 64 (55·2 per cent) within the CEAwatch protocol and 44 (41·9 per cent) in the CAU group (P = 0·007). Only 16 recurrences (13·8 per cent) were detected by patient self-report in the CEAwatch group, compared with 33 (31·4 per cent) in the CAU group. There was no significant improvement in either OS or DSS with the CEAwatch protocol compared with CAU: hazard ratio 0·73 (95 per cent 0·46 to 1·17) and 0·78 (0·48 to 1·28) respectively. There were no differences in survival when recurrence was detected by CT versus CEA measurement, but both of these methods yielded better survival outcomes than detection by patient self-report. CONCLUSION: There was no direct survival benefit in favour of the intensive programme, but the CEAwatch protocol led to a higher proportion of recurrences being detected by CEA-based blood test and reduced the number detected by patient self-report. This is important because detection of recurrence by blood test was associated with significantly better survival than patient self-report, indirectly supporting use of the CEAwatch protocol.

Cost-effectiveness of a carcinoembryonic antigen (CEA) based follow-up programme for colorectal cancer (the CEA Watch trial).

AIM: The study CEA Watch (Netherlands Trial Register 2182) has shown that an intensified follow-up schedule with more frequent carcinoembryonic antigen (CEA) measurements but fewer outpatient visits detects more curable recurrences compared with the usual follow-up protocol in colorectal cancer (CRC) patients. The aim of the study was to compare the cost and cost-effectiveness between various follow-up programmes. METHOD: In total, 3223 patients with stage I-III CRC were followed between October 2010 and October 2012. Direct medical costs were calculated per patient adding the costs for all visits, CEA measurements and imaging. Productivity losses and travel expenses were calculated using answers from questionnaires. The cost-effectiveness displayed the additional costs per additional patient with recurrent disease and used an incremental cost-effectiveness ratio (ICER) to compare them. RESULTS: The mean yearly cost per patient was €548 in the intensified protocol and €497 in the control protocol. The ICER was €94 (95% CI €76-€157) per cent; to detect one additional patient with a recurrence in the intervention protocol compared with the control protocol would require an additional €9400. For curable recurrences, the ICER was €607 (95% CI €5695-€5728). Annual patient-reported costs were €509 per year in the intervention protocol and €488 in the control protocol. CONCLUSION: The current study demonstrates that the direct medical and patient-reported cost of a newly introduced, safe and effective way of CRC follow-up was comparable to that of standard care. The ICER per curable recurrence was considered acceptably low.

Palliative resection of the primary tumour in stage IV rectal cancer.

AIM: The aim of this study was to investigate the use of resection in a cohort of palliatively treated patients with stage IV rectal cancer. To avoid selection bias, particular attention was paid to correction for comorbidity and extent of disease. METHOD: Patients with stage IV rectal cancer in two hospitals in Groningen were consecutively included over a 5-year period. Comorbidity was defined as major (dementia, cardiac failure or left ventricle ejection fraction <30%, or severe chronic obstructive pulmonary disease), minor (diabetes, hypertension, mild renal disease or mild pulmonary disease) or none. The effect of patient and disease characteristics on survival was assessed using Kaplan-Meier and Cox regression analyses. RESULTS: Of 88 patients, 11 (13%) underwent elective surgical resection without chemotherapy, 15 (17%) received both elective resection and chemotherapy, 21 (24%) underwent palliative chemotherapy only and 41 (47%) had supportive care only. The extent of disease (P<0.01), hospital (P=0.02) and comorbidity (P=0.04) were correlated with worse survival. Patients treated surgically survived for longer than patients treated nonsurgically, when the data were corrected for age, comorbidity, extent of disease and hospital [hazard ratio (HR)=0.4 (95% CI=0.2-0.7)]. Perioperative morbidity was seen in 38% of the patients, and 30-day mortality was 0%. CONCLUSION: In this retrospective cohort, resection was associated with longer survival independently of the extent of distant metastases, age and comorbidity.

Isolation of alveolar type II cells from fetal rat lung by differential adherence in monolayer culture.

Type II alveolar epithelial cells were isolated from fetal rat lung by differential adherence in monolayer culture. The preparation had a high degree of purity, as assessed by phase contrast microscopy and immunocytochemistry. Purity, based on reactivity with specific anti-adult lung serum (SAALS), which recognizes only type II cells, was 91% for cells isolated from 19-day fetal lungs and 79% for cells isolated from 21-day fetal lungs. The lower purity of type II cells in cultures derived from 1-day postnatal rat lungs (51% cells reactive with SAALS) is probably due to a lower tendency of the type II cells from neonatal rats to adhere to culture dishes than of type II cells from fetal rats. Type II cells isolated from 21-day fetal lungs contained a higher percentage phosphatidylglycerol and incorporated [Me-3H]choline faster into phosphatidylcholine (PC) than type II cells isolated from 19-day fetal lungs. Moreover, in cell preparations derived from lungs at fetal day 21, a higher percentage of epithelial cells contained lamellar bodies than in preparations derived from lungs at fetal day 19. The observation of these differences in the stage of maturation indicates that these differences, which are typical features of the original material, are not obliterated by differentiation during the culture. Type II cells isolated according to the present procedure were capable of synthesizing PC with a high percentage of the disaturated species. This method for the isolation of fetal type II cells may be a useful tool in studies concerning surfactant synthesis and its regulation in the fetal lung.

Intensified follow-up in colorectal cancer patients using frequent Carcino-Embryonic Antigen (CEA) measurements and CEA-triggered imaging: Results of the randomized "CEAwatch" trial.

AIM: The value of frequent Carcino-Embryonic Antigen (CEA) measurements and CEA-triggered imaging for detecting recurrent disease in colorectal cancer (CRC) patients was investigated in search for an evidence-based follow-up protocol. METHODS: This is a randomized-controlled multicenter prospective study using a stepped-wedge cluster design. From October 2010 to October 2012, surgically treated non-metastasized CRC patients in follow-up were followed in eleven hospitals. Clusters of hospitals sequentially changed their usual follow-up care into an intensified follow-up schedule consisting of CEA measurements every two months, with imaging in case of two CEA rises. The primary outcome measures were the proportion of recurrences that could be treated with curative intent, recurrences with definitive curative treatment outcome, and the time to detection of recurrent disease. RESULTS: 3223 patients were included; 243 recurrences were detected (7.5%). A higher proportion of recurrences was detected in the intervention protocol compared to the control protocol (OR = 1.80; 95%-CI: 1.33-2.50; p = 0.0004). The proportion of recurrences that could be treated with curative intent was higher in the intervention protocol (OR = 2.84; 95%-CI: 1.38-5.86; p = 0.0048) and the proportion of recurrences with definitive curative treatment outcome was also higher (OR = 3.12, 95%-CI: 1.25-6.02, p-value: 0.0145). The time to detection of recurrent disease was significantly shorter in the intensified follow-up protocol (HR = 1.45; 95%-CI: 1.08-1.95; p = 0.013). CONCLUSION: The CEAwatch protocol detects recurrent disease after colorectal cancer earlier, in a phase that a significantly higher proportion of recurrences can be treated with curative intent.

Expression of the major surfactant-associated protein, SP-A, in type II cells of human lung before 20 weeks of gestation.

In a previous paper (Otto-Verberne et al., Anat. Embryol. 178, 29-39 (1988) we reported that the type II alveolar epithelial cell can be identified in fetal human lung on the basis of morphological and immunological characteristics from 10 to 12 weeks after conception (a.c.) onward. For immunological recognition we used a lung-specific antibody, called SALS-Hu (specific anti-lavage serum, rabbit antihuman). The present immunoblotting experiments, after one-and two-dimensional electrophoresis, showed that SALS-Hu-reactive proteins in lavage fractions obtained from alveolar proteinosis patients exhibited molecular masses of mainly 29, 31 to 36, and 62 to 66 kDa. All SALS-Hu-reactive proteins migrated in the same acidic isoelectric point range (pI 4.4-5.1) and were almost undetectable when we used SALS-Hu preabsorbed with recombinant surfactant-associated protein A. We concluded that SALS-Hu recognizes exclusively isoforms of the major surfactant-associated protein, SP-A. In vitro translation assays in which we used mRNA isolated from adult human lung confirmed that SALS-Hu recognized the 29 to 31 kDa SP-A precursor proteins. These SALS-Hu-immunoreactive precursors for SP-A were already detectable (though in much lower amounts) in human fetuses aged 17 to 18 weeks, indicating that mRNA coding for SP-A is present at that time. We concluded that the cytoplasmic staining of fetal (from 10-12 weeks a.c. onward) and adult human type II cells by SALS-Hu is due to the presence of SP-A.

Development of the pulmonary acinus in fetal rat lung: a study based on an antiserum recognizing surfactant-associated proteins.

In this study on the development of the pulmonary acinus in fetal rat lung use was made of an antiserum, rabbit anti-mouse, that recognizes the type II alveolar epithelial cell or its precursor (a cuboidal cell lacking multilamellar bodies) by the presence of a cell-specific antigen. This serum had already been used in studies on mouse-lung development in our laboratory. Immunoblotting experiments showed that this serum reacts with surfactant-associated proteins in the pellet fraction of rat-lung lavage fluid having molecular weights of about 26,000, 32,000, and 38,000 daltons. In adult and fetal rat-lung homogenates the antiserum reacts with proteins with apparent molecular weights of about 40,000 and 42,000 daltons, probably also surfactant-associated proteins. No reaction with serum proteins was seen. Use of this antiserum in immuno-incubations of frozen sections of lungs of 15- to 21-day-old rat embryos showed that the type II epithelial cell or its precursor first appears on day 16 in embryos weighing 349-398 mg. Our results indicate that in the rat - as in the mouse - the bronchial and respiratory portions develop from morphologically and immunologically different parts of the tubular system in the fetal lung. The basic structure in the genesis of the pulmonary acinus is a tubule, called the acinar tubule, which is lined by the type II epithelial cell or its precursor.

Detection of the type II cell or its precursor before week 20 of human gestation, using antibodies against surfactant-associated proteins.

The present study was performed to find out whether the type II alveolar epithelial cell or its precursor (an approximately cuboidal cell lacking multilamellar bodies) is present before the twentieth week of human gestation. For this purpose we used an antibody, SALS-Hu(E), which recognizes the human type II cell on the basis of surfactant-associated proteins. Application of SALS-HuE (by indirect immunofluorescence) to acetone-fixed frozen sections of fetal lung tissue gave a distinct staining of the cuboidal or low columnar epithelial cells lining the end-pieces of the tubular system of fetal lung (initially only a few): this staining started around weeks 10 to 12 after conception. Around week 16 some of the labeled epithelial cells appeared to be rather flat and by week 19 a combined cellular and linear fluorescence pattern was seen. Columnar epithelial cells of the prospective bronchial portion did not show this specific staining. Our results indicate that the type II cell or its precursor cell is indeed present in the pseudoglandular period of human lung development, i.e., starting around the tenth to twelfth week. This cell type lines the acinar tubule, the basic structure of the pulmonary acinus. Transformation of this cell type into the type I alveolar epithelial cell seems to start in week 16.

Ultrastructural characteristics of inclusion bodies of type II cells in late embryonic mouse lung.

As we reported earlier, type II alveolar epithelial cells make their appearance in the early embryonic mouse lung around day 14.2, and show distinctive ultrastructural features. The present study focuses on the ultrastructural characteristics of the inclusion bodies by investigating embryos aged 17-19 days (birth on day 19), using transmission electron microscopy. Late embryonic type II cells appear also as low-columnar or cuboid cells having large, approximately round nuclei and cytoplasm displaying typical features of a differentiated cell. The inclusion bodies show a widespread distribution and are extremely variable in appearance. Schematically we discern five main types, namely cytoplasmic, granular/flocculent, multivesicular, dense, and (multi)lamellar, which occur with intermediate and composite forms. All these inclusion bodies frequently contain glycogen particles, and show a structural relation to profiles of endoplasmic reticulum which are wrapped around them. Other distinctive properties are the osmiophily of multivesicular inclusion bodies, and the presence of vesicles in many dense inclusion bodies. The possible interrelationship, and the differences in various aspects of electron density, suggest that the five main types of inclusion bodies may represent different stages in the formation of mature multilamellar bodies.

Human tonsil B lymphocyte function. I. The proliferative response to Staphylococcus aureus and pokeweed mitogen in relation to surface heavy chains mu and delta.

Human tonsil non-T cells were separated into surface IgD positive (sIgD+), surface IgD negative (sIgD-), sIgM+ and sIgM- fractions by rosetting and gradient centrifugation with ox red blood cells, coated with immunosorbent purified goat anti-human heavy chain antibodies. No activation or suppression was found as a result of the separation technique. The sIg patterns of the isolated B cell fractions showed the effectiveness of the separations; sIgD+ and sIgM+ fractions were to a great extent overlapping populations, but there was a definite population of s mu + delta- cells (about 20% of the sIgD- fraction). The isolated fractions were tested for proliferative responses to Staphylococcus aureus (Sta) and pokeweed mitogen (PWM). The sIgM+ fraction showed the best response to Sta, eight times better than the sIgM- fraction, while the sIgD+ and sIgD- fraction showed equal responses to Sta. The sIgM- fraction responded best to PWM but all fractions responded quite well. Our results indicate that PWM is a mitogen to a whole variety of rather mature B cells while the Sta target B cell is restricted to the relatively immature sIgM+D- and sIgM+D+ cells.

Human tonsil B lymphocyte function. II. Pokeweed mitogen-induced plasma cell differentiation of B cell subpopulations expressing multiple heavy chain isotypes on their surface.

Human tonsil non-T cells were successfully separated into surface IgM+G-ve (sIgM+G-), sIgM+G+, sIgM-G-, and sIgM-G+ B cell fractions. The two-step separation technique used did not affect the pokeweed mitogen- (PWM) induced plasma cell differentiation of the B cells. The highest percentages of plasma cells after PWM stimulation were found in the sIgM- fractions, and the lowest percentage was in the sIgM+G- fraction (20.8 +/- 2.3%), the latter predominantly cytoplasmic mu-chain-positive (c mu +) (84.1 +/- 3.5%) plasma cells. In this fraction, almost no c gamma + plasma cells were found. The sIgM+G+ produced c mu + (47.5 +/- 7.5%), c gamma + (35.7 +/- 5.8%), and c alpha + (16.8 +/- 4.0%) plasma cells. All three isotypes were found on the surface of the B cells in this fraction before PWM stimulation. The sIgM-G- fraction contained about 10% plasma cells before stimulation, which were predominantly c gamma +. After PWM stimulation, primarily c alpha + (64.2 +/- 4.3%) plasma cells were found. The sIgM-G+ fraction produced both c gamma + (75.0 +/- 2.3%) and c alpha + (24.1 +/- 2.5) plasma cells. A mu to gamma class switch did not occur in vitro in the sIgM+G- fraction on PWM stimulation, and the sIgM+G+ fraction did not complete in vitro the mu to gamma switch it had started in vivo.

Ultrastructural features of type II alveolar epithelial cells in early embryonic mouse lung.

Immunofluorescence studies of type II alveolar epithelial cells indicate that they first appear in the pseudoglandular period of mouse lung development (around day 14.2). They are the only cell type to line the prospective pulmonary acinus at this time. The ultrastructural characteristics of this cell are defined by investigating embryos aged 13-16 days with transmission and scanning electron microscopy. Early embryonic type II cells appear as low-columnar or cuboid cells having large, approximately round nuclei and distinct ultrastructural features, including a well-developed Golgi apparatus with many associated vesicles, multivesicular bodies, dense bodies, and large apical and basal glycogen fields. These fields represent a distinctive property of the cell. Frequently, they show compartmentalization due to the presence of membrane systems, and association with dense bodies of various sizes.

Histochemical characterization of an antigen specific for the great alveolar cell in the mouse lung.

Previous papers reported on a specific antigenic marker for the great alveolar (type-II) cell of the mouse lung and described its recognition by a specific rabbit anti-adult mouse lung serum. In the present study light- and electron-microscopical immunohistochemistry on fixed mouse lung sections showed the presence of the marker on the alveolar surface. The antigenic determinants recognized by the antibody were further characterized by immunoblotting and immunoprecipitation studies after in vitro translation of mouse lung messenger RNA. Immunoblots of a surfactant-enriched pellet of a bronchoalveolar lavage fraction of mouse lung showed that the antibody reacted with surfactant-associated proteins having apparent molecular weights of about 27,000, 32,000, and 38,000 daltons in SDS gels. Immunoblots of mouse-lung homogenate revealed the presence of 27,000, 30,000, 39,000, and 41,000 daltons proteins, presumably also surfactant-associated proteins. Immunoprecipitation after in vitro translation of mouse-lung mRNA showed specific reactivity only with a 12,000 dalton polypeptide, a component of the cell marker we were unable to relate to surfactant. Our findings indicate that the 12,000 dalton component of the antigenic marker for the great alveolar cell is a polypeptide whose synthesis is a lung-specific process and that the immunoreaction of the larger and surfactant-associated components is due to post-translational modifications.

The proximal border of the human respiratory unit, as shown by scanning and transmission electron microscopy and light microscopical cytochemistry.

The epithelial cell types present in respiratory (= distal alveolarized) and terminal (= distal nonalveolarized) bronchioles in adult human lung were characterized with scanning and transmission electron microscopy (SEM, TEM) and light microscopic cytochemistry, using specific antibodies against surfactant protein SP-A and mucins, and Alcian blue/periodic acid-Schiff (AB/PAS) staining. In the respiratory bronchiole, two epithelial cell populations share the same basal lamina: one pseudostratified columnar with ciliated, secretory, and basal cells and the other predominantly simple cuboid with some interspersed flat (type I) cells. The columnar secretory cells show the ultrastructure of mucous cells. Light microscopically, they react with mucin antibodies and contain primarily periodate-reactive acid mucins. The mucous cells are the distal secretory cells described by Clara (1937). The cuboid cells are identified as type II (precursor) cells based on ultrastructural criteria for embryonic type II cells (Ten Have-Opbroek et al., 1988a, 1990a), including a cuboid cell shape, a large and roundish nucleus, rough and smooth endoplasmic reticulum (ER), osmiophilic multivesicular bodies, and dense bodies. These dense bodies in turn frequently exhibit--like those in embryonic type II cells--internal vesicles or lamellae, variability in size and shape, a specific relationship to ER and a widespread cytoplasmic distribution. Finally, the cuboid cells show a cytoplasmic staining pattern for SP-A. The terminal bronchiole is lined by the columnar cell population. In the respiratory bronchiole, the columnar (bronchial) and cuboid (alveolar) cell populations occupy distinctly different zones (pulmonary artery zone versus remaining wall). The alveolar part of the respiratory bronchiole (called alveolar tubule) defines the proximal border of a true respiratory unit.

Detection of antileukoprotease in connective tissue of the lung.

An indirect immunofluorescence technique was applied to frozen sections of central and peripheral human lung tissue to search for extracellular localizations of antileukoprotease (ALP). Two monoclonal anti-ALP antibodies recognizing different epitopes and polyclonal anti-ALP antibodies were used. ALP was found to be localized along elastic fibers in alveolar septa, and also along elastic fibers in the walls of bronchi, bronchioles and blood vessels. Serous cells of bronchial submucosal glands showed labelling as well. In frozen sections of liver and spleen no label was found. Cells and elastic fibers were not labelled when lung tissue sections were processed with polyclonal or monoclonal anti-ALP antibodies, that were blocked with purified ALP before the immunostaining. The association of ALP with elastic fibers of human pulmonary connective tissue is of importance in understanding the role of the inhibitor in the defense of the lung parenchyma against the action of proteolytic enzymes, which is thought to result in emphysema.

Protective effect of pulmonary surfactant on elastase-induced emphysema in mice.

The aim of this study was to obtain some evidence of a protective role for pulmonary surfactant in the pathogenesis of emphysema. Firstly, we developed a quick and easy method to treat mice with a series of intratracheal instillations. Subsequently, three groups of mice were treated as follows: two groups received intratracheal instillations with pancreatic elastase (1.8 mg.kg-1 BW) followed after 3, 48 and 96 h in one group (El/Surf group) by intratracheal administration of surfactant (100 mg phospholipid.kg-1 BW), and in the other group by instillations with saline (El/s group). The third group of control mice was treated with saline followed by three doses of surfactant (s/Surf group). After eight weeks, the mice were killed and emphysema was measured by calculating the mean linear intercepts (Lm) of airspaces. The Lm values in the different groups were statistically tested for differences by the Mann-Whitney test. Instillation of pancreatic elastase (El/s group) resulted in an evenly distributed increase in Lm compared with the control group. Administration of surfactant in elastase-treated mice (El/Surf group) resulted in a statistically significant inhibition of airspace enlargement. Although the Lm in the El/Surf group was still higher than in the control group, analysis of histograms of Lm values per field of examination revealed that the Lm distribution in the former group was similar to that of the s/Surf group. The El/s group, on the contrary, showed the presence of many fields with enlarged air spaces. Repeated instillations with saline and/or surfactant had no effect on the Lm.(ABSTRACT TRUNCATED AT 250 WORDS)

Lack of type II cells and emphysema in human lungs.

Ten surgically removed human lungs or lobes were studied, to assess the relationship between the abundance of type II alveolar epithelial cells and the degree of emphysema. Type II cell abundance (total number as well as percentage of the total parenchymal cell population) was determined in sections of randomly selected tissue samples of these lungs or lobes by using a type II cell specific antibody specific anti-lavage serum (SALS-Hu), which recognizes surfactant-associated proteins. In these tissue samples we also determined the degree of emphysema with the aid of a number of morphometric parameters, destructive index (DI), mean linear intercept (Lm in mm), and the number of normal alveolar attachments on (pre)terminal bronchioles (normal AA.mm.1). We subsequently calculated the Spearman rank correlation coefficients (rs) between the abundance of type II cells and parameters for emphysema. We found a significant negative correlation between the percentage of type II cells and DI at tissue sample level (rs = 0.55; p = 0.02). We also calculated correlation coefficients between the abundance of type II cells and the degree of small airways disease in (pre)terminal and respiratory bronchioles (SADscore), lung function, age and smoking habits. The results suggest a role for type II cells in the pathogenesis of emphysema.