Predicting prognosis in lung cancer: use of proliferation marker, Ki67 monoclonal antibody.

An investigation was carried out to assess the prognostic significance of proliferation marker Ki67 in a group of lung cancer patients treated by surgery (limited disease). Tissue was not available for Ki67 immunostaining in inoperable group. The diagnosis is established by bronchial biopsy which does not carry enough tissue for frozen section and counting. This study is supplemented by estimating the prognostic significance of histological sub-types in the operable group and in a group of inoperable patients with extensive disease. These are usually treated by radiotherapy and/or chemotherapy. In all, 267 patients were studied including 105 treated by surgery. These patients attended King's College and Brompton Hospital, UK, between 1986 and 1989. With regard to proliferation marker Ki67 done for the surgical group, only patients with Ki67 scores of less than 5% did survive significantly longer than the rest. Histology did not make any significant contribution in determining prognosis in both operable and inoperable groups. Although follow-up is limited (mean 20 months), Ki67 antibody seems promising in identifying low and high grade disease in the initial stage of lung cancer. It may prove useful for category of patients with high scores to be placed on chemotherapy/radiotherapy. Results suggest that in the case of lung tumour, proliferative activity is a better prognostic indicator than histological type.

A silicone rubber mould of the heart.

The macroscopical structure of the ventricular myocardium has been an unsolved problem since the XVIth century, when Anatomy started as an authentic science. Since then the spatial organization of the myocardial fibres has represented, as Pettigrew says, "an arrangement so unusual and perplexing, that it has long been considered as forming a kind of Gordian knot in Anatomy. Of the complexity of the arrangement I need not speak further than to say that Vesalius, Albinus, Haller and De Blainville, all confessed their-inability to unravel it". What is shown in the present paper is the result of an anatomical work, developed over 43 years, by means of which it has been shown that the ventricular myocardial mass consists of a band, curled in a helical way, which extends from the pulmonary artery to the aorta. This is illustrated by a silicone rubber model cast from an actual unrolled myocardial band.

The heart's fibre alignment assessed by comparing two digitizing systems. Methodological investigation into the inclination angle towards wall thickness.

Myocardial contractile pathways which are not aligned strictly parallel to the heart's epicardial surface, give rise to forces which also act in the ventricular dilating direction. We developed a method which allows us to assess any fibre orientation in the three-dimensional myocardial weave. Decollagenized hearts were prepared by peeling-off fibre strands, following their main fibre orientation down to near the endocardium. In the subepicardium the strands followed a course more or less parallel to the epicardium, whereas from the mid-wall on they tended to dive progressively deeper into the wall. The preparation displays more or less rugged surfaces rather than smooth layers. The grooves and crests on the exposed surfaces were sequentially digitized by two methods: (1) Using a magnet tablet (3 Draw Digitizer System, Polhemus, Cochester VTO 5446, USA) on a dilated pig heart we manually followed the crests using a stylus, handling each groove and crest as an individual contractile pathway. (2) A constricted cow heart was digitized using a contact-free optical system (opto TOP, Dr. Breuckmann, Meersburg, Germany), which is based on the principle of imaging triangulation. Using specially developed software the inclination angles of selected crests and grooves with respect to the epicardial surface were calculated. The two digitizing methods yield comparable results. We found a depth- and side-specific weave component inclined to the epi-endocardial direction. This oblique netting component was more pronounced in the inner 1/3 of the wall than in the subepicardium. The inclination angle probably increases with increasing wall thickness during the ejection period. Manual digitizing is an easy and fast method which delivers consistent results comparable with those obtained by the cumbersome high resolution optical method. The rationales for the assessment of transmural fibre inclination are (1) the putative existence of dilating forces inherent in the myocardial weave and (2) the possible overproportional increase in the oblique transmural weave component during myocardial hypertrophy, which would entail a reduction in efficiency of ventricular performance in terms of haemodynamic work.

Inhomogeneities in wall stress measured by microergometry in the heart muscle in situ.

Microergometry is a method which we have developed as a tool to measure local mesh-tension within the myocardial weave at any measuring site of both ventricles and the septum on the beating heart in situ. In a mapping procedure on pig and dog hearts, both in control conditions and in the hypertrophied state after aortic banding, local mesh-tension was measured in several areas and in up to eight depths proceeding from the epicardium to the endocardium: Probe-to-fibre coupling is definitely more stable in the canine myocardium than in the porcine heart muscle, probably due to a more effective connective tissue fettering of the canine myocardial weave. The observed longitudinal gradient, with the highest tension in the base, of control dog hearts was levelled out in the hypertrophied hearts. Furthermore, in control dog hearts mesh-tension in the subepi- and subendocardial layers was higher than in the midlayers. This pronounced midlayerhypotension was smoothed in the hypertrophied hearts. Further studies will be dedicated to the question of whether the impact of ventricular size and shape on intersegmental stress transmission is determined by the Frank-Starling mechanism alone or whether protracted remodelling processes on the level of the local fibre weave cause slow coupling alterations.

Standardized mitotic counts in breast cancer. Evaluation of the method.

Twenty-one pathologists and technicians participated in a study evaluating the variation present in mitotic counts for prognostication of breast cancer. The participants counted the mitotic figures in 20 breast cancer samples from ten high power fields (mitotic activity index, MAI, giving the results in mitotic figures per 10 fields) and also made a correction for field size and area fraction of the neoplastic epithelium to get the standardized mitotic index (volume fraction corrected mitotic index, or M/VV index, giving the result in mitotic figures per square mm of neoplastic epithelium). The difference in variation between the two methods was not big, but the standardized mitotic index (SMI) showed consistently smaller variation among all participants and different subgroups. Experienced pathologists had the highest variation in mitotic counts, and specially trained technicians, the lowest. The efficiency of the mitotic counts in grading (the grading efficiency) was used to evaluate the mitotic counts. In groups without special training for mitotic counts the mean grading efficiency was lower (experienced and training pathologists both on average had the potential to grade 88% of the cases correctly) than in the group specially trained for the purpose (trained technicians had the potential to grade 95% of the cases correctly). Among the specially trained technicians, the grading efficiency was of the same magnitude as the grading efficiency achieved in determining the S-Phase fraction of cells from paraffin embedded breast cancers by flow cytometry in different laboratories. The results suggest that special training is helpful in making mitotic counts more reproducible, and that in trained hands, the mitotic counts give results comparable to more sophisticated methods of determining proliferative activity in breast cancer.

Reproducibility of Bayesian belief network assessment of breast fine needle aspirates.

OBJECTIVE: To assess the consistency of diagnosis of fine needle aspiration biopsies of breast lesions by three experienced and five less experienced pathologists using conventional means and applying a Bayesian belief network (BBN) to 10 diagnostic features to support diagnostic decision making. STUDY DESIGN: Forty fine needle aspiration biopsies, previously assessed by one of the experienced pathologists both conventionally and using a BBN, were assessed by two further experienced pathologists and five less experienced pathologists. RESULTS: Using the BBN, the experienced pathologists arrived at diagnoses in agreement with an established consensus at a slightly lower rate than by conventional means. The less experienced pathologists arrived at the correct diagnoses no more frequently with the help of the BBN than conventionally. CONCLUSION: As used in this study, the BBN did not help less experienced pathologists to interpret their observations but did not enable less experienced pathologists to identify how their observations differed and affected their diagnoses. The prototype system used in this study has since been upgraded by providing computer graphic displays of the features to be observed so that a more uniform mental image can be held by the participating pathologists. This will be tested with the same study design.

3D reconstruction and quantitation of pathological tissues.

3DR techniques are under continuous development, as is the technology which supports them. Systems developed for engineering and other applications are coming within the price range of many users. Recently publications have started to appear using standard high-quality graphics workstations. As these are coming down in price, more applications will be possible. The advent of new computer technology, notably possible. The advent of new computer technology, notably highly parallel systems and chips such as the Intel i860, offer the prospect of very fast reconstructions and the software to make 3DR a routine technique. Most significant however is the continuing increase in type and numbers of scanning systems, both macroscopic and microscopic. Prior to these systems becoming available, the critical limiting factor inhibiting the wide application of 3DR was generating adequately registered, undistorted, complete sets of serial section data for the reconstruction process. The fact that scanning optical microscopes can produce such datasets easily means that the everyday use of 3DR for studies of pathology is now feasible.

High-frequency oscillation in an adult porcine model.

OBJECTIVE: Controversy exists as to whether high-frequency oscillatory ventilation can be used on babies and small laboratory animals only, or whether high-frequency oscillatory ventilation can also be efficient in the adult patient and large (> 65 kg body weight) laboratory animals. Moreover, controversy exists as to whether limitations in high-frequency oscillation efficiency are caused by the size and shape of the bronchial system, by the lack of low impedant intersegmental gas flow in lung parenchyma, or by inappropriate high-frequency ventilators and ancillary hardware. Therefore, our objective in this study using the adult pig as a model of the adult patient was to test whether the adult airway system is suited to the use of high-frequency oscillatory ventilation or whether there are geometrical, structural, or functional limitations to efficient ventilation by high-frequency oscillation. DESIGN: Prospective, controlled, randomized comparison over 8 to 16 hrs of ventilatory management. SETTING: Experimental thoracovascular surgery laboratory in a university hospital. SUBJECTS: Fifteen adult, female, house swine (weight 90 to 140 kg). INTERVENTIONS: We evaluated the ventilatory effect of a wide range of oscillation frequencies (10-15 to 35-45 Hz), tidal volumes (0.5 to 2.2 mL/kg), and bias flow volumes (10 to 70 L/min) at a mean airway pressure of 12 +/- 1 cm H2O in anesthetized and relaxed pigs who did not have lung injury. MEASUREMENTS AND MAIN RESULTS: Arterial blood gases are mainly dependent on tidal volume, frequency, and mean airway pressure. A threshold bias flow volume of 35 +/- 5 L/min is required to prevent CO2 rebreathing. In the group of lightweight animals (65 to 99 kg), the most efficient frequency band for CO2 elimination was approximately 25 Hz. The most efficient frequency band for arterial oxygenation was found to vary between individuals more than the most efficient frequency band for CO2 elimination. In the group of heavy animals (100 to 140 kg), no most efficient mean frequency could be assessed, probably because the excitation system was limited. We confirmed that tidal volume on its own had an effect on CO2 elimination ("tidal-volume effect"), although CO2 elimination was mainly determined by the product of tidal volume and oscillation frequency (oscillated minute volume), at least up to a critical frequency. Beyond that frequency, CO2 elimination could not be enhanced. The most efficient mean airway pressure in unimpaired lungs was assessed at 12 +/- 1 cm H2O. CONCLUSIONS: Adult pigs with a body weight in the range of the weight of clinical adult patients can be ventilated by high-frequency oscillation at tidal volumes smaller than, equal to, or slightly more than anatomical deadspace. The most efficient frequency for gas exchange varied between individuals. Tidal volume had an enhancing effect on CO2 elimination. The frequency dependency of PaO2 may have been related to a frequency-dependent structural remodeling of the airway system, which occurred even though the mean airway pressure was kept constant. These results demonstrate that failure of adequate ventilation by high-frequency oscillation is caused by a) CO2 rebreathing, b) the avoidance of an appropriate alveolar recruitment strategy, and c) an underpowered, high-frequency ventilatory system (oscillator) that is unable to deliver appropriate pressure oscillations. These limitations led to insufficient CO2 elimination and/or inadequate arterial oxygenation.

Distribution of alveolar wall per unit volume in the human lung.

Firstly, to assess the reproducibility of a computerised image analysis method of measuring alveolar size in terms of the alveolar wall surface area per unit volume (AWUV) in normal non-smoking adult human lungs fixed in inflation, one lung was fixed by simple manual prebronchial perfusion with formaldehyde (wet-fix), another by prolonged perfusion with formaldehyde followed by air until semi-dried (semi-dry). Blocks from both lungs were taken to assess the relative value of plastic and paraffin wax embedding. There was no statistical difference in AWUV between plastic and paraffin sections from each lung, but the wet-fix method gave statistically significantly (P < 0.05) higher AWUV readings than the semi-dry method. Secondly, when a whole semi-dry lung slice was divided into 66 blocks, the AWUV distribution was not random but largest in the centre and smallest in the periphery anteriorly and superiorly. This is obviously important in sampling of lung tissue for morphometry and other purposes.

The difficult access to morphology of the heart: clinical implications.

The literature on the morphology of the heart is reviewed within the context of recent histological findings. There is strong evidence for a dualistic myocardial function, whereby both ventricular constricting and expanding forces are supposed to act synchronously although with variable effect over the heart cycle.The morphological basis of this dualistic myocardial function is the contorted rope-like structure worn into the bulk of the heart muscle. Opinions are divided about the invasiveness of blunt preparation on the heat denatured heart by which the fascicular architecture is carved out of the muscle. Histology confirms the existence of a fascicular substructure. It results from an inhomogeneous repartition of myocardial fibre branchings and the arrangement of the delicate connective tissue netting by which myocardial strands are bundled and wrapped. One important feature of the fascicular structure of the heart muscle is an oblique transmurally arranged element which yields a force vector opposing systolic wall thickening. This structural element which acts in the direction of ventricular dilation probably gains pathological import in some cases of architectural remodelling, namely in myocardial fibrosis and hypertrophy.

How reliable is the diagnosis of lung cancer using small biopsy specimens? Report of a UKCCCR Lung Cancer Working Party.

BACKGROUND: A study was undertaken to investigate the accuracy of typing of a series of bronchial carcinomas by experienced pathologists with an interest in lung cancer from the examination of bronchoscopic biopsy specimens. METHODS: Eighty bronchial biopsy specimens showing positive results for bronchial carcinoma were circulated to five pathologists, who recorded diagnostic criteria and diagnosis for each. Diagnoses were then compared with the diagnosis agreed from the resection specimen corresponding to each biopsy specimen. A "non-small cell carcinoma, not further specified" classification group was introduced for small biopsy specimens. RESULTS: A diagnostic accuracy of 75% was achieved for squamous cell carcinomas, 66% for small cell carcinomas, and 50% for adenocarcinomas. There was diagnostic confusion between small cell and non-small cell carcinoma in less than 10% of cases. The introduction of a non-specific non-small cell classification improved diagnostic accuracy by 10-15% for each non-small cell tumour group. CONCLUSIONS: There are appreciable inaccuracies in applying the World Health Organisation's 1981 classification of lung cancer to the diagnosis of bronchial carcinoma from small biopsy specimens and these inaccuracies have been measured. They can be diminished by introducing a less specific "non-small cell" category for use with this sort of biopsy material. Care should be taken not to overinterpret small biopsy specimens in lung cancer.

Three-dimensional reconstruction of human embryonic notochords: clue to the pathogenesis of chordoma.

Three-dimensional reconstruction experiments performed on serial sections of human embryos showed that the anatomy of the caudal and rostral ends of the notochord was complex. Forking of the ends, with separate fragments of chordal tissue, was demonstrated and these provide a way by which notochordal cell rests could be left behind in the basicranial and sacral regions when the notochord involutes elsewhere. Assuming the histogenesis of chordomas from notochordal cell rests, this would furnish an explanation for the observed skeletal distribution of chordomas.

Three-dimensional reconstruction: methods of improving image registration and interpretation.

(i) Image registration. The use of serial images for computerised three-dimensional reconstruction necessitates the inclusion of three separate sources of information at the stage of data input. These are (i) artificial registration points or fiducials, (ii) a calibration scale and (iii) an outline of each slab of the section to be included in the reconstruction. Most traditional methods rely on the production of drawings of the contours of the structure under investigation which also include both registration points and a calibration scale. We report on a method which considerably reduces the time involved at this labour intensive stage of reconstruction and in addition allows subsequent reconstructions of different structures to be performed without new drawings. Use is made of computerised alignment of tissue sections and the production of composite photomicrographs of both the tissue under investigation and an accurately registered stage micrometer scale. (ii) Improving image interpretation. Images derived from computerised three-dimensional reconstruction can be affected by the number of coordinates used to form the contour of each slice of a structure and by the number of slices that are used to construct the final model. Too little or too much data can considerably reduce the ability of the observer to interpret accurately the image generated by the computer. We report on a feature-based method which enables the experimenter to assess objectively the amount of data required in the two-dimensional plane, i.e. the number of data points per slice, and the three-dimensional plane, i.e. number of slices per structure, so that optimal reconstructions are generated.

The microanatomy of the alveolar duct of the human lung imaged by confocal microscopy and visualised with computer-based 3D reconstruction.

The most widely accepted model of human lung alveolar duct systems is that they are constructed from central helical fibres between the turns of which lie alveolar opening. Experimental difficulties of handling and sectioning lung tissue have made it difficult to confirm this. Confocal laser scanning microscopy (CLSM) was therefore used to generate optical serial sections of the lung that were reconstructed in three dimensions and displayed using volume rendering techniques. From images of the reconstructions, a new structure is proposed in which alveolar ducts consist of collections of connected oval, twisted loop structures with eccentric openings.

Cardiodynamic effects of dopamine and dobutamine.

On 11 patients undergoing coronary surgery, at the end of the surgical intervention, the inotropic responses to 0.4 and 0.8 microgram x kg-1 x min-1 dopamine and dobutamine given via the aorto-coronary bypass directly into the coronary artery were compared. These dosages correspond to ones 10 times greater applied intravenously. The measurements were made using needle force probes which were implanted into the myocardial offstream area in the left ventricular wall. Bypass flow was measured simultaneously by an electromagnetic flow probe. There is a significant increase in coronary bypass flow induced by both rates of 0.4 and 0.8 microgram x kg-1 x min-1 dobutamine, but there was no significant effect on bypass flow induced by dopamine. Developed myocardial force is raised more by dobutamine medication than by dopamine. However, the rate of contraction increases significantly and relaxation is significantly accelerated by dopamine at both dosages. A significant increase in rate of contraction and relaxation was only induced by the higher dosage of 0.8 microgram x kg-1 x min-1 dobutamine.

Distribution and estimation of nucleolar organizer regions in various human lung tumours.

A quantitative study of nucleolar organizer regions in human lung carcinomas was carried out on routinely processed paraffin embedded tissue sections. We examined 104 lung carcinomas including 38 squamous cell carcinomas, 36 adenocarcinomas, 18 large cell anaplastic carcinomas, 6 small cell carcinomas and 6 carcinoids. No significant differences were found in mean number of NORs between squamous, adenocarcinoma and undifferentiated carcinomas including large cell and small cell carcinomas. Carcinoids had comparatively lower means except for one typical carcinoid. Considering the high incidence of overlap between ranges of NOR counts in these groups of tumours and in agreement with the only other study of lung tumours (which comprised only carcinoids and small cell carcinomas), we conclude that this technique cannot be reliably used to discriminate between various histologic types of lung cancers. However, long term follow up of these patients is needed to establish the value of the AgNOR technique for prognostic guidance.